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Gene Therapy &

Molecular Biology

FROM BASIC MECHANISMS TO CLINICAL APPLICATIONS

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Gene Therapy and Molecular Biology Vol 5

Table of contents

Gene Therapy and Molecular Biology

Vol 5, December 2003

Article title Authors (corresponding author is in

boldface)

A novel expression vector induced by

heat, !-radiation and chemotherapy

Misregulation of pre-mRNA splicing

that causes human diseases. Concepts

and therapeutic strategies

Th2-type immune response induced by

a phage clone displaying a CTLA4binding

domain mimic-motif

Segregation of partly melted molecules

and its application to the isolation of

methylated CpG islands in human

cancer cells

PNA (peptide nucleic acid) antigene/antisense

can access intact viable

cells and downregulate target genes

Delivery of plasmid DNA by in vivo

electroporation

Potential roles of p53 in

recombination

Characterisation of the p53 gene in the

rat CC531 colon carcinoma

Recombinant adenoviruses as

expression vectors and as probes for

DNA repair in human cells

Chromatin remodeling and

developmental gene regulation by

thyroid hormone receptor

Farha H. Vasanawala, Tom Tsang, Abdul

Fellah, Peter Yorgin, and David T.

Harris

Oliver Stoss, Peter Stoilov, Rosette

Daoud, Annette M. Hartmann, Manuela

Olbrich, and Stefan Stamm

Yasuhiro Kajihara, Shuhei Hashiguchi,

Yuji Ito, and Kazuhisa Sugimura

Masahiko Shiraishi, Leonard S.

Lerman, Adam J. Oates, Xu Li,,Ying H.

Chuu, Azumi Sekiguchi, and Takao

Sekiya

Lidia C Boffa, Elisabetta M.Carpaneto,

Benedetta Granelli, Maria R. Mariani

Loree Heller and M. Lee Lucas

Nuray Akyüz, Gisa S. Boehden,

Christine Janz, Silke Süße, and Lisa

Wiesmüller

Sacha B Geutskens, Diana JM van den

Wollenberg, Marjolijin M van der Eb,,

Hans van Ormondt, Aart G Jochemsen,

Rob C Hoeben

Andrew J. Rainbow, Bruce C. McKay,

and Murray A. Francis

Laurent M. Sachs, Peter L. Jones, Victor

Shaochung Hsia, and Yun-Bo Shi


111-120

121-130

131-145

147-156

157-162

Review

Article

Review

Article

Review

Article

Research

Article

Research

Article

Gene Therapy and Molecular Biology Vol 5

Signal transduction pathways in

cancer cells; novel targets for

therapeutic intervention

Control of pre-mRNA processing by

extracellular signals: emerging

molecular mechanisms

Herpes Simplex Virus vector-based

gene therapy for malignant glioma

Viral vectors carrying a markersuicide

fusion gene (TK-GFP) as tools

for TK/GCV –mediated cancer gene

therapy

Aberrant DNA methylation of p16

onco-suppressor gene in human

cervical carcinoma

Christos A. Tsatsanis and Demetrios A.

Spandidos

Rossette Daoud, Peter Stoilov, Oliver

Stoss, Mark Hübener, Maria da Penha

Berzaghi, Annette M. Hartmann,

Manuela Olbrich, and Stefan Stamm

Edward A. Burton and Joseph C.

Glorioso

Sami Loimas, Tiina Pasanen, Andreia

Gomes, Susana Bizarro, Richard A.

Morgan, Juhani Jänne and Jarmo

Wahlfors

Luciano Mariani, Giuseppe Zardo,

Cesare Rapone, Anna Reale, Giuseppe

Netri, Serena Buontempo, Adriana de

Capoa and, Paola Caiafa


Gene Therapy and Molecular Biology Vol 5, page 1

1

Gene Ther Mol Biol Vol 5, 1-8, 2000

A novel expression vector induced by heat, !radiation

and chemotherapy

Research Article

Farha H. Vasanawala 1 , Tom Tsang 1 , Abdul Fellah 2 , Peter Yorgin 2 and David T.

Harris 1*

1 Department of Microbiology & Immunology and 2 Department of Pediatrics, University of Arizona, Tucson, AZ. USA.

_________________________________________________________________________________________________

* Correspondence: David Harris, Ph.D, Dept. Microbiology and Immunology, Bldg # 90, Main Campus, University of Arizona,

Tucson, AZ 85721 USA. Tel: (520) 621-5127; Fax: (520) 621-6703; E-mail: davidh@u.arizona.edu

Key words: expression vector, inducible promoter, heat, radiation, chemotherapy

Received: 19 April 2000; accepted: 29 April 2000

Summary

In many gene therapy applications and molecular biology manipulations it is desirable to be able to control the

expression of the therapeutic gene. In this study a new expression vector, pHOT-MCS, was constructed using a 451

bp fragment from the human heat shock protein 70B (HSP 70) promoter. The vector has a large multiple cloning

site and the neomycin selectable marker, making it more user-friendly. Using the human Interleukin-2 (IL-2) gene

as a marker, it was demonstrated that heat can induce the pHOT-IL-2 plasmid to express 2 fold more IL-2 than

levels obtained with the human cytomegalovirus (CMV) promoter.

Using the Enhanced Green Fluorescence Protein (EGFP) gene as a reporter gene, a human breast carcinoma cell

line (MCF-7) was transfected with pHOT-EGFP and stably transfected cells selected with neomycin. The stable

transfectants were subjected to three different experimental conditions; heat treatment at 42°C for one hour,

treatment with geldanamycin at 1µg/ml (an anti-leukemic drug) or 3000 rads of !-radiation. EGFP expression was

measured for up to 72 hours by flow cytometry. The non-treated cells expressed a basal level of EGFP that

increased 387% above background at 24 hours after heat-shock. Cells treated with Geldanamycin had a 208%

increase in EGFP intensity at 24 hours which was maintained up to 72 hours as compared to the non-treated cells.

Exposure of cells to 3000 rads of !-radiation had a 150% increase in EGFP expression at 48 hours post-treatment as

compared to the non-treated cells. Induction of heat shock proteins by heat, radiation and geldanamycin was

confirmed by Western blot analysis. This inducible gene expression system may be applicable to clinical use in

synergy with other types of standard therapy (e.g, hyperthermia, radiation and chemotherapy).

I. Introduction

The major therapies currently used to treat cancer

include radiotherapy, chemotherapy, hyperthermia and

immunotherapy. Gene therapy is emerging as a new

treatment for cancer, with encouraging clinical results.

With the emerging view of combinatorial therapy as an

approach to cancer treatment (Feyerabend et al, 1997;

Otte, 1988), there is a need to integrate gene therapy with

conventional therapies. Most gene therapy approaches

have utilized constitutive expression of therapeutic genes

(e.g. co-receptors such as B7.1 and B7.2, cytokines such

as IL-2 and GM-CSF) by viral promoters (e.g,

cytomegalovirus and Rous sarcoma virus). An alternative

method is the use of expression vectors that can be

induced to express therapeutic genes by one or more of the

aforementioned conventional therapies. Thus, vectors with

inducible gene expression could be advantageous in some

gene therapy protocols.

The human HSP70B promoter is a well characterized

promoter (Schiller et al, 1988) known to be induced by a

family of heat shock factors that respond to diverse forms

of physiological and environmental stress including high

temperatures, heavy metals, oxidative stress and antiinflammatory

drugs (Morimoto et al, 1997). The HSP70B


promoter contains heat shock regulatory sequences that

bind to the heat shock transcription factor, thus "turning

on" any gene downstream of the promoter. The HSP70B

promoter has previously been incorporated in heterologous

systems to express foreign genes in an inducible manner

(Dreano et al, 1986). These vectors are difficult to use due

to their large size (>10kb), small multiple cloning sites and

lack of a selectable marker, such as neomycin.

In the present study a new user-friendly expression

vector was constructed using a fragment of the HSP70B

promoter and was tested for inducible gene expression.

This promoter was compared to the CMV promoter, which

has been shown in several studies to be one of the

strongest promoters available (Boshart et al, 1985).

Results showed that when heat-induced, the heat shock

promoter fragment was twice as strong as the CMV

promoter. Our results also demonstrated that this

expression vector was inducible by !-radiation and the

anti-leukemic drug, geldanamycin (a benzoquinoid

ansamycin antibiotic and a potent inhibitor of a protein

kinase (Uehora et al, 1986) known to induce heat shock

proteins (Conde eet al, 1997)). The enhanced green

florescence protein (EGFP) was used as a reporter gene to

detect induction of the expression vector by the different

treatments.

II. Results

A. Construction of vectors

The HOT-MCS vector (Figure 1) was generated by

replacing the cytomegalovirus (CMV) promoter of

pcDNA3 (Invitrogen, San Diego, CA) by a BamHI- Hind

III fragment of the human HSP70B promoter from the

p173OR plasmid (StressGen, Victoria, BC). The fulllength

HSP70B promoter is 2.3 kilo base pairs (kbp) in

size, while the BamHI-HindIII fragment of the promoter is

451 bp in size. This adaptation resulted in a smaller vector,

which was advantageous for further cloning. The pHOT-

MCS vector also has a neomycin selectable marker that

can be useful for in vitro research work. The pHOT-MCS

vector has a large multiple cloning site which facilitates

the cloning of genes into the vector. Thus, this newly

constructed pHOT-MCS vector has many advantages over

the p173OR plasmid.

B. Characterization and comparison of

the inducible promoter expression vector

The pHOT-MCS plasmid containing a betagalactosidase

reporter gene ("-gal) was evaluated for its

ability to be induced by heat shock treatment. SW480 cells

were transfected with the plasmid by the lipofection

technique as described in Materials and Methods.

Vasanawala et al: A novel, inducible expression vector

2

Figure 1: Map of the HOT-MCS expression plasmid. The

plasmid was generated by replacing the CMV promoter of the

pcDNA3 expression plasmid with a 400 bp BamH1/Hind III

fragment of the human HSP70B promoter derived from the

p173OR plasmid.

Percent "-gal positive cells at:

PLASMID 37°C 42°C

p173OR 0% 27%

pHOT-"-gal 0% 23%

Table 1: Inducible gene expression by a fragment of the

human HSP promoter. SW480 human colon carcinoma cells

were transfected with either the HOT-"-gal or the p173OR

expression plasmid using the lipid DMRIE/DOPE as described in

Materials and Methods. Transiently transfected cells were

exposed for 1 hour to either 37°C or 42°C as described. Fortyeight

hours after transfection the cells were harvested and stained

for "-gal using standard staining procedures (Invitrogen, San

Diego, CA). Data are expressed as the percentage of cells

staining positive for each treatment.


Figure 2: Promoter strength comparison by IL-2 production.

MCF-7 cells were plated overnight at a density of 0.5x10 6 cells

per well in a six well plate. The cells were transfected with the

HOT-IL-2 plasmid using the lipid Novafector. The cells were

heated to 42°C for 60 min and returned to 37°C. Twenty-four

hours later the supernatants were harvested and analyzed by

ELISA for IL-2 levels. Results are the mean of triplicate

experiments.

Twenty-four hours after transfection the cells were heated

to 42°C for one hour. Twenty-four hours after heat shock

treatment the cells were stained for "-gal gene expression.

The p173OR plasmid expressing "-gal under the control

of the full-length 2.3kb HSP promoter was used as a

positive control. The analyses (Table 1) revealed that "gal

gene expression was not induced at 37°C, whereas the

pHOT- "-gal plasmid transfected cells stained positive 24

hours after heat treatment. Results were comparable with

both vectors. These results confirmed that the 451 bp

BamHI /Hind III fragment of HSP70B promoter

maintained it's ability to be induced by heat shock.

The promoter strength of the inducible expression

vector was also tested against the CMV promoter. The IL-

2 gene was cloned into the EcoRI site of the multiple

cloning site of the pHOT-MCS vector. The HOT-IL-2

plasmid and the pcDNA3-IL-2 plasmid were transfected

into MCF-7 cells with the lipid Novafector. Twenty-four

hours after transfection the cells were heated and 24 hours

after heat treatment supernatants were collected and

assayed for IL-2. Results (Figure 2) showed that the

pHOT-MCS vector promoter was twice as strong as the

CMV promoter when induced by heat treatment (67 units

v/s 31 units of IL-2).

Gene Therapy and Molecular Biology Vol 5, page 3

3

Figure 3: Increase in EGFP expression. Mean

fluorescence channel (MFC) of EGFP expression following

treatment with heat, radiation or chemotherapy. MCF-7 cells

were treated as described in Materials and Methods, harvested at

the indicated time points and analyzed by flow cytometry for

EGFP intensity.

C. Induction of the transgene by heat

shock, !-radiation and geldanamycin

Due to the difficulty in quantitating and comparing

"-gal assays and the expense of IL-2 assays, the enhanced

green fluorescent protein (EGFP) reporter gene was

cloned into the HOT-MCS plasmid yielding the HOT-

EGFP plasmid. This plasmid can easily be detected by

flow cytometry and was used for further experiments.

MCF-7 cells were transfected with HOT-EGFP by the

calcium phosphate method and stable transfectants were

selected and maintained with the antibiotic G418. Stable,

as compared to transient, transfections were chosen in

order to eliminate differences in transfection efficiencies

between experiments thereby allowing direct comparisons

of increases in gene expression.

Stably transfected MCF-7 cells were plated overnight

into 35mm, 6 well tissue culture plates at a density of

1x10 6 cells/plate. The next day adherent cells were treated

with either heat (42°C, 1hour), !-radiation (3000 rads, a

previously determined optimal level) or geldanamycin

(1µg/ml). Synergy of gene induction was also analyzed for

by using !-radiation and geldanamycin treatments

together. Four, 24, 48, and 72 hours after treatment the

cells were harvested and assayed for EGFP expression by

flow cytometry. All transfected cells displayed a basal

level of EGFP expression. Thus, results from the cell

treatments are shown as the mean florescence channel

(Figure 3) and as the percent increase in EGFP intensity


(Table 2) over the basal level. Flow cytometric analyses

indicated that heat shock treatment induced the highest

level of gene expression. As early as 4 hours posttreatment

there was a 243% increase in EGFP expression

above the basal level. Maximal EGFP expression was

observed at 24 hours after heat treatment (387% increase

above background) after which it started to decline.

Geldanamycin was also observed to induce the HSP

promoter, with EGFP intensity increasing to 208% above

background at 24 hours post-treatment. !-radiation also

induced the HSP promoter, although to a weaker extent

than geldanamycin treatment. The highest level of gene

expression was seen at 48 hours post-radiation treatment,

with an increase of 150% over background levels. Finally,

a combination of geldanamycin and radiation treatments

together was not observed to increase gene expression (i.e.

act synergistically) above that seen with either treatment

alone (data not shown). These results were significant in

that it demonstrated that the HOT-EGFP vector could be

induced not only by heat shock, but also by !-radiation and

chemotherapy.

D. Analysis of heat shock proteins by

western blots

As the time course for induction of EGFP expression

by both geldanamycin and !-radiation treatments were

different from that of heat-shock treatment, Western blots

were performed on the treated cells at these time points to

quantitate HSP72/73 production. As each of the treatments

theoretically induced EGFP expression via the HSP

promoter, the treatments should have induced an increase

in cellular heat shock proteins. Western blot analyses

(Figure 4) for HSP72/73 expression indicated that all

three treatments (heat, radiation and geldanamycin)

induced the HSP 72/73 proteins. The highest level of HSP

72/73 production was induced by heat at 4 hours posttreatment.

However, at 24 hours post-treatment and

thereafter, treatment with geldanamycin induced higher

levels of HSP72/73 expression. !-radiation also induced

Vasanawala et al: A novel, inducible expression vector

4 hours 24 hours 48 hours 72 hours

Heat 243 387 376 327

Geldanamycin 120 208 201 201

Radiation 113 138 150 154

4

HSP72/73 protein expression at 4 hours post-treatment,

but the levels were not significant at the later timepoints.

III. Discussion

We have constructed a novel, user-friendly inducible

expression vector, that possesses a large multiple cloning

site and the neomycin gene as a selectable marker. This

vector has the useful property of being inducible by heat,

chemotherapy, and radiation. Using pcDNA3 as the

backbone plasmid, pHOT-MCS was derived by replacing

the CMV promoter with a heat inducible promoter, the

human HSP70B promoter.

As the size of a plasmid may affect transfection

efficiency, such that smaller plasmids transfect with a

higher efficiency than larger ones, size was an important

factor in our plasmid design. Thus, a 451 bp fragment of

the human HSP70B promoter was used rather than the

entire 2.3kbp promoter. From previously published work

(Schiller et al, 1988), the 451 bp fragment was expected to

be as heat inducible as the parental promoter since it

contains the heat shock element (HSE) sequences and the

TATA box.

Results using "-gal as the reporter gene indicated

that the 451bp fragment of the human HSP70B promoter

was indeed sufficient for heat inducible gene expression.

Currently, the most commonly used promoter is the CMV

promoter and it was imperative to compare the pHOT-

MCS plasmid with this promoter. The results showed that

the 451 bp fragment of the human HSP70B promoter upon

treatment with heat was twice as strong as the CMV

promoter. Thus, promoter strength was not compromised

in the construction of the HOT-MCS plasmid.

In further experiments, EGFP was used as the

reporter gene rather than "-gal due to the ease of assaying

EGFP gene expression by flow cytometry. Thus, the

EGFP gene was cloned into the HOT-MCS plasmid,

yielding the HOT-EGFP plasmid. The reporter gene was

easily detected by flow cytometry (Figure 3).

Table 2: Percent Increase in EGFP Intensity. Mean

Fluorescence Channel units from Figure 3 were converted into

percent intensity increases above background. An increase in 75

units of MFC = a doubling in intensity. The above formula was

used to calculate the percent increase in EGFP intensity over

background, which was considered to be 100%.


Gene Therapy and Molecular Biology Vol 5, page 5

Figure 4. HSP72/72 protein expression after treatment with either heat, radiation or chemotherapy. MCF-7 cells were treated as described in

Figure 2 and heat shock protein expression quantitated by western blotting and densitometry at the indicated time points. Data are presented

optical density for each measurement.

MCF-7 cells stably transfected with HOT-EGFP were

used instead of transient transfections to eliminate

differences in transfection efficiencies between

experiments. Stably transfected cells allowed for direct

comparisons in increased gene expression by the different

experimental treatments. Since stable transfectants were

used, EGFP gene expression was also observed in nontreated

(but transfected) cells. This background expression

was considered to be the background level and increases

in gene expression were calculated as the fold-increase

over the basal level.

It was a novel finding that the HSP70B promoter

fragment was not only inducible by heat but also by !radiation

and geldanamycin. These treatments represent

radiation therapy and chemotherapy, and thus present an

opportunity to combine gene therapy with existing cancer

therapies such as hyperthermia therapy, chemotherapy and

radiation therapy. Although, the fluorescence intensities

with geldanamycin and !-radiation weren’t equivalent to

that observed with heat shock, the levels were still

significantly increased above basal levels.

There was a difference in the induction of gene

expression between the three treatments. Heat shock

induced gene expression rapidly by 4 hours, while

maximal gene induction was seen at 24 hours with

geldanamycin and !-radiation treatments. Thus, different

mechanisms of heat induction occurred with geldanamycin

5

and !-radiation. As these various treatments turned on the

reporter gene, each of the treatments must have induced

heat shock proteins in the cell since the heat shock

promoter was activated. Thus, western blots detecting heat

shock proteins were performed to possibly elaborate the

mechanisms of gene induction. The western blot

experiments indicated that heat shock increased HSP72/73

production at 4 hours and a similar effect was also seen

with geldanamycin treatment. This increase in HSP72/73

levels was however, not reflected in an increase in EGFP

intensity with geldanamycin treatment. Thus, it can be

concluded that different mechanisms were affecting gene

expression, which needs to be further characterized.

In conclusion, we have constructed a vector that

opens up the possibility of a combinatorial type of therapy

using gene therapy and chemotherapy, hyperthermia or

radiation. Synergistic effects between these therapies may

be more beneficial than any one therapy alone. Further, it

may be possible to utilize lower doses of chemotherapy or

radiation in combination with the above gene therapy

vector expressing a biologically active gene (e.g, HSV-tk

or cytokines) to achieve less toxic clinical results.

IV. Materials and Methods

A. Vector construction

Three reporter constructs were made from pHOT-MCS

(see results for further details). One construct contained beta-


galactosidase ("-gal) as the reporter gene (from the pCMVB

plasmid, Clontech, Palo Alto, CA) cloned into the Not I site of

pHOT-MCS.

For the IL-2 construct, the human IL-2 gene (a gift from

Dr. Evan Hersh, University of Arizona) was first adapted for

EcoR1 site. Briefly, a 0.5kb BamHI-PstI DNA fragment

containing the IL-2 gene was inserted into the Sac-KiSS-#

(Tsang et al, 1996) following a complete digestion with BamHI

and a partial digestion with PstI to create the plasmid pSac-KiSS-

IL-2. The IL-2 gene was then excised from the pSac-KiSS-IL-2

as an EcoRI fragment and inserted into the EcoRI site of pHOT-

MCS to generate pHOT-IL-2. The third reporter construct

contained EGFP (Clontech, Palo Alto, CA) as a reporter gene,

which was inserted into the Kpn I – Not I multiple cloning site of

pHOT-MCS.

B. Cell lines and transfections

MCF-7, a human breast carcinoma cell line, was

transfected with the pHOT-EGFP vector by standard calcium

phosphate methodologies (Sambrook et al, 1989). Stable

transfectants were obtained by selection with G418 (400 µg/ml)

and were maintained in complete RPMI medium, (GIBCO,

Gaithersburg, MD) supplemented with 10% fetal bovine serum

(JRH Biosciences, Lenaxa, KS). The cells were maintained at

37°C in 5% CO 2.

For IL-2 studies, MCF-7 cells were seeded in 35mm plates

(Falcon, Franklin Lakes, NJ) at 0.5x10 6 cells/plate. Cells were

transfected with the plasmids using the lipid Novafector

(VennNova, Pompano Beach, FL) at a ratio of 1µg DNA to 4 µl

of lipid for 6 hours. SW480, a human colon carcinoma cell line,

was transfected using DMRIE-DOPE (Vical Inc, San Diego, CA)

(Parker et al, 1996).

For "-gal studies, SW480 cells were plated out at 4x10 5

cells/well in a six well tissue culture plate (Falcon, Franklin

Lakes, NJ). A lipid to DNA ratio of 4:1 was used for

transfections. Transfections were performed in reduced serum

media OPTI-MEM (GIBCO, Gaithersburg, MD). Four hours

after transfections, 0.5 ml of 30% FBS in OPTI-MEM was added

to each well. The next day an additional 1ml of 10% FBS in

OPTI-MEM was added. Forty-eight hours after transfection the

cells were trypsinized and stained for "-gal using the Invitrogen

"-gal staining kit (Invitrogen, Carlsbad, CA).

C. Cell treatments

Transfected cells were plated overnight in a 35mm 2 tissue

culture dish (Falcon, Franklin Lakes, NJ) at a density of 1x10 6

cells in 5ml of RPMI medium. The following day the cells were

treated as follows. Heat shock treatment was performed by

sealing the plate with parafilm and immersing it in a 42°C water

bath for 60 min. Geldanamycin (Sigma, St. Louis, MO) was

added to the cell cultures at a concentration of 1µg/ml. !radiation

treatment was performed using a 60 Co !- irradiation

unit. The cells were exposed to a total of 3000 rads (225

rads/min) of radiation in a single dose. All treated cells were

trypsinized and assayed by flow cytometry for EGFP expression

at 4, 24, 48 and 72 hours post-treatment.

D. IL-2 assay

Cell supernatants were harvested 24 hours after heat

treatments. The supernatants were assayed by ELISA for IL-2

Vasanawala et al: A novel, inducible expression vector

6

levels using MEDGENIX IL-2 EASIA Kit (Biosource Europa,

Belgium).

E. Flow cytometry

Flow cytometric analysis was performed using a FACStar

PLUS flow cytometer (Becton Dickinson Immunocytometry

Systems, Mountain View, CA). Data was acquired utilizing a

COHERENT (Palo Alto, CA) 90-5 5W argon ion water-cooled

laser tuned to 488 nm at 100mW power for excitation. Emitted

fluorescence was collected with a standard 530/30 band pass

filter. A minimum of 10,000 events were collected in a 'live' gate.

Data was acquired and analyzed on an HP340 computer with

Lysys II (Becton Dickinson Immunocytometry Systems

Mountain View, CA) software. Data was collected as mean

florescence channel of EGFP. An increase in 75 units of mean

florescence channel indicates a doubling in fluorescence intensity

(as per personal communications from Becton Dickinson,

according to the formula:

where x= log channel

channel value

= 10x

channel per decade

Data is represented as the percent increase in EGFP

intensity over background intensity.

F. Western blots

Western immunoblots were performed to estimate

production of heat shock proteins.

Cells to be assayed were washed with phosphate buffered

saline and resuspended in cell lysis buffer. Total protein was

estimated by the BCA protein assay (Pierce, Rockford, IL).

Protein samples (30mg each) were fractionated for Western blot

analysis by separation on denaturing SDS-PAGE and transferred

onto nitrocellulose filters. Filters were blocked by soaking the

membrane in buffer containing 3% milk in TTBS (Tris-buffered

saline containing 1% Tween-20) to minimize non-specific

binding. After three washes in TTBS the membranes were

incubated with anti-HSP 72/73 antibody (StressGen, Victoria,

BC). Goat anti-mouse IgG-horse radish peroxidase (Pierce,

Rockford, IL) was used as a secondary/developing antibody.

Both incubations were performed at room temperature for 1

hour. The membrane was washed three times with TTBS and

incubated in substrate reagent containing peroxide for 5 min.

Heat shock proteins were detected by exposure to ELC hyperfilm

and developed for autoradiogram. Heat shock protein expression

was quantitated by scanning densitometry.

Acknowledgements

The authors would like to thank Dr. Ashok Gupta for

his technical assistance. The authors would also like to

thank Barb Carolus for all her help with flow cytometry.

For plasmid requests please contact Dr. David Harris

at davidh@u.arizona.edu or write to Dr. David T. Harris.

Dept. of Microbiology and Immunology, Building # 90,

University of Arizona, Tucson, AZ 85721.


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Gene Therapy and Molecular Biology Vol 5, page 7

7

benzoquinonoid ansamycins accompanies inactivation of

p60src in rat kidney cells infected with Rous sarcoma virus.

Mol Cell Biol 6, 2198-2205.


Vasanawala et al: A novel, inducible expression vector

(From left): Jean Boyer, David Harris, Tom Tsang and Farha Vasanawala (Crete 1998)

8


Gene Therapy and Molecular Biology Vol 5, page 9

9

Gene Ther Mol Biol Vol 5, 9-30, 2000

Misregulation of pre-mRNA splicing that causes

human diseases. Concepts and therapeutic strategies

Review Article

Oliver Stoss 1 , Peter Stoilov 1 , Rosette Daoud, Annette M. Hartmann, Manuela

Olbrich, and Stefan Stamm*

Max-Planck Institute of Neurobiology, Am Klopferspitz 18a, D-82152 Martinsried, Germany

__________________________________________________________________________________

*Correspondence: Stefan Stamm, Ph.D., Phone: +49 89 8578 3625, Fax: +49 89 8578 3749, E-mail: stefan@stamms-lab.net

Key words: alternative splicing, kinases, tau, FTDP-17, signal transduction

1 authors contributed equally to the work

Received: 27 August 2000; accepted: 4 October 2000

Summary

About one third of all human genes are subject to alternative splicing. The molecular mechanisms

that regulate alternative splice site usage are beginning to emerge and show that transcription and

pre-mRNA processing are integrated processes that can be modified by cellular signals. Several

diseases are caused by mutations in sequences that regulate pre-mRNA processing. Their molecular

characterization indicates that contributions of pre-mRNA splicing defects to human diseases have

been underestimated and could account for pleiotropic phenotypes. The understanding of the

molecular mechanisms allows the development of therapeutic strategies.

Abbreviations: ACTH: adrenocorticotrophic hormone;

CFTR: cystic fibrosis transmembrane conductance regulator;

CKI: casein kinase I; Clk: Cdc2-like kinase; ConA:

Concanavalin A; EGF: epidermal growth factor; ESE: exonic

splicing enhancer; ESS: exonic splicing silencer; FGF:

fibroblast growth factor; hnRNP: heterogeneous nuclear

protein; IFN: interferon; IL: interleukin; ISE: intronic

splicing enhancer; ISS: intronic splicing silencer; MKK:

mitogen-activated protein kinase kinase; NGF: nerve growth

factor; PDGF: platelet-derived growth factor; PI3-K:

Phosphatidylinositide-3-kinase; PKC: protein kinase C;

I. Introduction

A. Splicing and basal splicing

machinery

In eukaryotes, the primary transcript generated by

polymerase II undergoes an extensive maturation process

that involves capping, polyadenylation, editing, and premRNA

splicing. Pre-mRNA splicing removes intervening

sequences (introns) and joins the remaining sequences

(exons) to form mature mRNA that is finally exported into

the cytosol. With a few exceptions, all human polII

transcripts are spliced, and it is estimated that about 30%

of all pre-mRNA transcripts are subject to alternative

PKG-I: protein kinase G-I; polII: polymerase II; PTP-1B:

protein tyrosine phosphatase 1B; SAM68: src associated in

mitosis; SELEX: systematic evolution of ligands by

exponential enrichment; snRNP: small nuclear riboprotein;

SR: protein: protein with serine-arginine-rich domain;

SRPK: SR protein kinase; STAR: signal transduction and

activation of RNA; TCR: T cell receptor; TNF: tumor

necrosis factor; U2AF: U2 auxiliary factor; UTR:

untranslated region.

splicing (Mironov et al, 1999; Brett et al, 2000).

Alternative splicing is a mechanism where parts of the premRNA

are either excluded from, or included in the mature

mRNA. This process can be regulated in a cell-type or

developmental-specific way (Stamm et al, 1994, 2000),

that can be used to regulate gene expression at the level of

pre-mRNA processing. Furthermore, it allows different

protein isoforms to be created from a single gene. In many

cases, stop codons are introduced by alternative splicing

(Stamm et al, 1994, 2000), which usually changes the

carboxy terminus of proteins. This can affect the

physiological function of a protein, as shown by several

examples:


(i) creation of soluble instead of membrane-bound

receptors (Baumbach et al, 1989; Eipper et al, 1992;

Toksoz et al, 1992; Zhang et al, 1994; Hughes and Crispe,

1995; Tabiti et al, 1996);

(ii) altered ligand affinity (Sugimoto et al, 1993;

Xing et al, 1994; Suzuki et al, 1995);

(iii) protein truncations producing inactive variants

(Swaroop et al, 1992; van der Logt et al, 1992; Duncan et

al, 1995; Sharma et al, 1995; Eissa et al, 1996); and

(iv) changes of endocytotic pathways (Wang and

Ross, 1995). In addition, inclusion or skipping of

alternative exons can

(v) add or delete protein modules that change the

affinity towards ligands (Danoff et al, 1991; Giros et al,

1991; Guiramand et al, 1995; Strohmaier et al, 1996);

(vi) modulate enzymatic activity (O'Malley et al,

1995);

(vii) create different hormones (Amara et al, 1982;

Courty et al, 1995); and

(viii) change properties of ion channels (Sommer et

al, 1990; Kuhse et al, 1991; Xie and McCobb, 1998).

Finally,

(ix) numerous transcription factors are subject to

alternative splicing, which contributes to control of gene

expression (reviewed in Lopez, 1995). A recent

compilation and statistical analysis of alternative exons

(Stamm et al, 2000) is available on the world wide web

under www.stamms-lab.net.

Proper splicing regulation is important for an

organism, since it has been estimated that up to 15% of

genetic defects caused by point mutations in humans

manifest themselves as pre-mRNA splicing defects caused

by changing splice site sequences (Krawczak et al, 1992;

Nakai and Sakamoto, 1994). These mutations can be

viewed as new sources of variation in human evolution

that was probably accelerated by alternative splicing

mechanisms, allowing the combination of different RNA

processing events to generate appropriate mRNAs as a

result of changing cellular needs (Herbert and Rich, 1999).

Significant progress has been made in understanding

the mechanism of constitutive splicing. Three major ciselements

on the RNA define an exon, the 5' and 3' splice

sites and the branch point (Berget, 1995). These elements

are recognized by the spliceosome, a 60S complex

containing small nuclear RNAs (U1, U2, U4, U5, U6) and

over 50 different proteins (Neubauer et al, 1998). In the

spliceosome, U1 snRNP, U2AF, SF1, U6 snRNP, and

U2 snRNP are the trans-acting factors that ultimately

recognize the 5', 3' splice sites and the branch point

(reviewed in Green, 1991; Krämer, 1996; Elliot, 2000;

Moore, 2000). Sequence compilations of the 5', 3' splice

sites and the branch point revealed that they follow only a

loose consensus sequence (Breitbart et al, 1987). Only the

GT and AG nucleotides directly flanking the exon, together

with the branch point adenosine (Figure 1A), are always

conserved, whereas in all other positions nucleotides can

deviate from the consensus. However, some positions in

Stoss et al: RNA splicing and disease

10

splice sites adhere better to a consensus than others

(Breitbart et al, 1987; Stamm et al, 1994; Stamm et al,

2000). Interestingly, a new class of exons has been

discovered that uses AT and AC instead of the GT/AG

flanking nucleotides (Tarn and Steitz, 1997). Due to the

degenerate nature of splice sites, it is difficult to predict

exons in genomic sequences, and current computer

programs cannot accurately predict exons from genomic

DNA (Thanaraj, 2000). This contrasts the high accuracy

and fidelity characteristic for splice-site selection in vivo.

One reason for the specificity observed in vivo are

additional regulatory elements known as exonic or intronic

enhancers or silencers (Figure 1, Table 1). These

elements are again characterized by loose consensus

sequences. The enhancers can be subdivided into purinerich

(GAR-type), pyrimidine-rich and AC-rich (ACE)

enhancers (Cooper and Mattox, 1997). Enhancers bind to

proteins that are able to recruit components of the basal

splicing machinery, which results in recognition of splicesites

located near an enhancer (Hertel et al, 1997). The

degeneracy of splicing enhancers is most likely necessary

to allow for the amino acid usage needed. The importance

of splice-site enhancers becomes apparent when they are

changed by mutation, which can alter their interaction with

trans-acting factors.

Interestingly, some of these mutations are silent, e.g.

they do not change the amino acid usage, but generate an

aberrant gene product by causing an abnormal splicing

product. It remains to be determined whether all missense

mutations cause a pathological state by an amino acid

exchange or are actually unrecognized splicing mutations.

Such an analysis could be made by analyzing the premRNA

processing of the mutation by RT-PCR or RNAse

protection. An overview of diseases caused by splicing

enhancers/silencers is shown in Table 2.

Proteins binding to enhancer or silencer sequences

can be subdivided into two major groups: members of the

SR family of proteins (Manley and Tacke, 1996) and

hnRNPs (Weighardt et al, 1996). Binding of individual

proteins to enhancer sequences is intrinsically weak and

not highly specific. However, multiple proteins bind to all

known exon enhancers forming a complicated

RNA:protein complex. This binding involves

protein:protein as well as protein:RNA interactions, and

results in the specific recognition of an exon (Figure 1).

As a result, proper splice-site recognition is governed by

the ratio of various proteins involved, as well as the

enhancer and silencer sequences.

B. Splice-site recognition is influenced

by the relative concentration of proteins that

form a complex to recognize exon-intron

borders

In contrast to constitutive splicing, the mechanisms

regulating alternative exon usage are less well understood.

It is clear that the relative concentration of splicingassociated

proteins is responsible for alternative splice-site


Gene Therapy and Molecular Biology Vol 5, page 11

Figure 1: cis and trans factors involved in pre-mRNA splicing.

A) Elements involved in alternative splicing of pre-mRNA. Exons are indicated as boxes, introns as thin lines.

Splicing regulator elements (enhancers or silencers) are shown as gray boxes in exons or as thin boxes in introns. The 5' splicesite

(CAGguaagu) and 3' splice-site (y)10ncagG, as well as the branch point (ynyyray), are indicated (y=c or u, n=a, g, c or u).

Upper-case letters refer to nucleotides that remain in the mature mRNA. Two major groups of proteins, hnRNPs (yellow) and SR or

SR related proteins (orange), bind to splicing regulator elements; the protein:RNA interaction is shown in green. This protein

complex assembling around an exon enhancer stabilizes binding of the U1 snRNP close to the 5' splice-site, due to

protein:protein interaction between an SR protein and the RS domain of U170K (shown in red). This allows hybridization (thick

red line with stripes) of the U1 snRNA (red) with the 5' splice-site. The formation of the multi-protein:RNA complex allows

discrimination between proper splice-site (bold letters) and cryptic splice-sites (small gt ag) that are frequent in pre-mRNA

sequences. Factors at the 3' splice-site include U2AF which recognizes pyrimidine rich regions of the 3' splice-sites, and is

antagonized by binding of several hnRNPs (e.g hnRNP I) to elements of the 3' splice-site. orange: SR and SR related proteins;

yellow: hnRNPs; green: protein:RNA interaction; red: protein:protein interaction; thick red line with stripes: RNA:RNA

interaction

B) The RNA factory. RNA is generated after genes are recognized by transcription factors (TF) by RNA polymerase II (polII)

(dark blue). Exons present on the RNA are recognized by SR proteins and hnRNPs that interact with exonic or intronic sequence

elements. SR proteins interact with factors assembled around the promoter and can form protein networks across exons. SR

proteins directly interact with the carboxy terminal domain of RNA polII (polII-CTD), which assembles proteins near active sites

of transcription. Among polII interacting proteins is scaffold attachment factor B (SAF-B) that can couple SR proteins and RNA

polII to chromatin organizing elements (S/MAR, thick green line). The processed RNA is coated with hnRNPs and transported into

the cytoplasm, where it is translated into protein. SR proteins and hnRNPs are recruited from storage sites (speckles) through

phosphorylation. Some SR proteins and hnRNPs shuttle between nucleus and cytoplasm. Protein shuttling can be regulated by

phosphorylation or arginine methylation.

11


Stoss et al: RNA splicing and disease

12


Gene Therapy and Molecular Biology Vol 5, page 13

Table 1. Compilation of RNA elements that influence splice-site selection (previous two pages)

The first column shows the gene and exon that contains the RNA element, which is characterized in the next columns according to

its type (ESE: exonic sequence element; ISE: intronic sequence element) and sequence. Trans-acting factors are indicated in bold

under the sequence if they were identified experimentally. Most RNA elements will work in combination with additional RNA

regulatory sequences that are not shown. Meth. indicates the experimental method used: 1: deletion analysis; 2: in vivo splicing

assay; 3: in vitro splicing assay; 4: gel mobility shifts; 5: mutagenisis; 6: in vitro binding; 7: UV-crosslink; 8: competition

experiments; 9: SELEX; 10: immunoprecipitation; 11: spliceosomal complex formation; 12: nuclease protection

R= G or A; W=A or U.

13


selection (Black, 1995; Grabowski, 1998; Varani and

Nagai, 1998) . It has been shown experimentally, that the

relative concentration of SR proteins and hnRNPs can

dictate splice- site selection, both in vivo and in vitro

(Mayeda and Krainer, 1992; Cáceres et al, 1994; Screaton

et al, 1995; Wang and Manley, 1995). The expression

levels of various SR proteins (Ayane et al, 1991; Mayeda

and Krainer, 1992; Zahler et al, 1993; Screaton et al,

1995) and hnRNPs (Kamma et al, 1995) vary amongst

tissues and could therefore account for differences in splicesite

selection. Several examples of antagonistic splicing

factors have been described (Cáceres et al, 1994; Mayeda et

al, 1993; Gallego et al, 1997; Jumaa and Nielsen, 1997;

Polydorides et al, 2000).

Here, one factor promotes inclusion of an exon and

the other factor promotes its skipping. In most of these

cases, it remains to be determined whether this

antagonistic effect is achieved by (i) an actual competition

of the factors for an RNA binding site, (ii) through

sequestration of the factors by protein:protein interaction

and, (iii) by changes in the composition of protein

complexes recognizing the splicing enhancer.In addition,

cell-type specific splicing factors have been detected. In

Drosophila, for example, the expression of the SR protein

transformer is female-specific (Boggs et al, 1987) and

determines the sex by directing alternative splicing

decisions. Other tissue-specific factors include the male

germline specific transformer-2 variant in D. melanogaster

(Mattox et al, 1990) and D. virilis (Chandler et al, 1997),

an isoform of its mammalian homologue htra2-beta3 that

is expressed only in some tissues (Nayler et al, 1998a), the

muscle specific protein Nop30 (Stoss et al, 1999a) , the

neuron-specific factor NOVA-1 (Jensen et al, 2000) as well

as testis and brain enriched factor rSLM-2 (Stoss et al,

submitted) and NSSR (Komatsu et al, 1999). For most of

these factors, the tissue-specific target genes remain to be

determined. However, a combination of knockout

experiments and biochemical analysis allowed the

identification of doublesex, fruitless, and transformer-2 as a

target of the transformer-2/transformer complex in

Drosophila ( Hoshijima et al, 1991; Mattox and Baker,

1991; Heinrichs et al, 1998) and glycine receptor alpha2

and GABA(A) pre-mRNA as a target for NOVA-1 (Jensen

et al, 2000). Although this analysis is currently limited, it

is likely that a given splicing factor will influence several

pre-mRNAs.

C. Coupling of splicing and

transcription

Transcription and splicing can be separated by

biochemical means. Especially when RNA splicing is

studied in vitro, the two processes are uncoupled, since the

RNA is made synthetically. However, when transcription

and splicing factors were analyzed by microscopy methods

(Misteli, 2000) and in yeast two-hybrid screens, an

intimate association became apparent. A number of studies

used the carboxyl terminal domain of the RNA polII and

found several interacting proteins, some of which, e.g.

snRNPs and SR like proteins, were most likely involved

Stoss et al: RNA splicing and disease

14

in pre-mRNA processing (Du and Warren, 1996; Kim et

al, 1996; Yuryev et al, 1996; Bourquin et al, 1997; Corden

and Patturajan, 1997). Furthermore, these complexes are

most likely associated with S/MAR elements (Bode et al,

2000) via SAF-B, an hnRNP-like protein that can link

factors involved in pre-mRNA processing, and the CTD to

chromatin-organizing elements (Nayler et al, 1998c).

Finally, it was shown directly that RNA polymerase II can

stimulate splicing reaction in vivo (Hirose et al, 1999) and

targets splicing factors to sites of active transcription

(Misteli and Spector, 1999). These close interactions most

likely influence splice-site usage and it has been shown

that the particular promoter usage influences splice-site

selection (Cramer et al, 1999). Together, these data

indicate that pre-mRNA is generated and processed by a

large complex that was termed 'RNA factory' (McCracken

et al, 1997). It is most likely that 5' capping (Cho et al,

1997), polyadenylation (McCracken et al, 1997), and

editing (Higuchi et al, 2000) activities are also part of this

complex.

When cells are stained with antibodies against

splicing factors, the proteins are concentrated in 20-40

large nuclear structures that are called speckles. Using

imaging techniques, it has been shown that splicing and

transcription take place concomitantly in the vicinity of

speckles (Jiménez-Garcia and Spector, 1993; Huang and

Spector, 1996). Within speckles, no transcriptional

activity could be detected (Fay et al, 1997), indicating that

these structures serve as storage particles. Speckles are

dynamic (Misteli et al, 1997) structures that can release

splicing components when these are phosphorylated

(Nayler et al, 1998b). A new subnuclear structure, the YT

bodies, was discovered that forms around speckles and

often partially overlaps with them (Nayler et al, 2000). YT

bodies contain YT521-B, a protein that binds to factors

implicated in pre-mRNA processing and is subject to

tyrosine phosphorylation through src kinases (Hartmann et

al, 1999). YT bodies change in response to the tyrosine

phosphorylation status of the cell (Nayler et al, 2000 and

our unpublished results) and harbor sites of transcription

(Nayler et al, 2000), suggesting that the RNA factory can

be modulated by tyrosine phosphorylation in YT bodies.

Finally, proteins implicated in splicing shuttle between

the nucleus and the cytosol. After a stress-induced change

of the cellular phosphorylation status they accumulate in

the cytosol (van Oordt et al, 2000), which affects premRNA

processing patterns in the nucleus, because the

nuclear concentration of the splicing factors is changed.

Together, these data suggest that the concentration of

factors involved in splice-site selection, which dictates

exon usage, can be controlled by several ways: (i) a

specific amount of the factor expressed in a tissue, (ii)

release from storage sites by phosphorylation, (iii) export

from the nucleus, (iv) sequestration by protein-binding

partners that, e.g., assemble at an active promoter, and (v)

local concentration at different sites of the nucleus.

Together, these mechanisms allow the cell to process a

given pre-mRNA with a specific set of splicing regulatory

proteins, such as SR proteins and hnRNPs.


Gene Therapy and Molecular Biology Vol 5, page 15

Table 2: Mutation in RNA regulatory elements that cause disease

Mutations that disrupt RNA regulatory elements and cause a disease are listed. The name of the gene is under the disease and is

underlined. Point mutations that change splice-sites (Krawczak et al, 1992; Nakai and Sakamoto, 1994) are not added to the table,

if they are not part of exonic elements. ESE: exonic sequence element; ISE: intronic sequence element, IVS: intron.

15


II. Change of splice-site selection in

response to an external stimulus

A. Overview

Alternative splicing pathways are not static, since the use

of alternative exons can change during development (for a

summary see: Stamm et al, 1994, 2000), or in response to

outside stimuli. For example, insulin administration

influences the incorporation of the alternative exon 11 of

the insulin receptor (Sell et al, 1994) and activates exon

ßII inclusion in the PKC gene (Chalfant et al, 1998);

serum deprivation alters usage of the serine/arginine-rich

protein 20 (SRp20) exon 4 (Jumaa and Nielsen, 1997); and

neuronal activity changes the alternative splicing pattern of

clathrin light chain B, the NMDAR1 receptor, and c-fos

(Daoud et al, 1999). In the brain, stress changes splicing

patterns of potassium channels (Xie and McCobb, 1998)

and of acetylcholin esterase (Kaufer et al, 1998). ConA has

been shown to change splicing patterns of the class 1b

major histocompatibility complex molecule Qa-2

(Tabaczewski et al, 1994) and the splicing patterns of

tumor necrosis factor ß are regulated by src kinases

(Gondran and Dautry, 1999; Neel et al, 1995). Finally,

programmed cell death is concomitant with a change in the

alternative splicing patterns of several cell death regulatory

proteins (reviewed in Jiang and Wu, 1999).

As can be seen in Table 3, numerous extracellular

stimuli, such as growth factors, cytokines, calcium

concentration, and extracellular pH can change alternative

exon usage. Since these alternative exons are in mRNAs

of diverse biological functions, it is likely that the change

of alternative splicing in response to an extracellular signal

is a general regulatory mechanism in higher eukaryotes.

Although for some cases the signal transduction pathways

have been established, the molecular mechanism that

transduces the signal to the spliceosome remains largely

obscure.

For several systems, it was demonstrated that

changes in alternative splicing do not require de novo

protein biosynthesis. Examples include the splicing of

exon v5 of the CD44 gene in response to TPA (König et

al, 1998) or the differential splicing of the Ca-ATPase

transcript upon a rise in intracellular calcium (Zacharias

and Strehler, 1996). It is likely that these changes in

splicing patterns are the result of posttranscriptional

modifications of regulatory proteins, e.g. phosphorylation,

methylation, and glycosylation. However, it is largely

unknown which factors are affected. In the following, we

summarize several protein groups that are likely endpoints

of signal transduction pathways in the spliceosome.

B. SF1

Some signal transduction pathways to spliceosomal

components have been investigated in detail. One paradigm

is SF1 (Berglund et al, 1998; Rain et al, 1998), a factor

that recognizes the branch point and is therefore important

for the formation of the spliceosomal A complex. SF1 has

recently been identified as a target of PKG-I (Wang et al,

1999). This kinase is activated by cGMP. The cGMP level

Stoss et al: RNA splicing and disease

16

itself can be regulated by a membrane bound guanylyl

cyclase receptor that is activated by natriuretic peptides or

by a cytoplasmic guanylyl cyclase which is activated by

nitric oxide (NO). Phosphorylation of SF1 on Ser20

inhibits the SF1-U2AF65 interaction, leading to a block

of pre-spliceosome assembly.

C. hnRNPA1

hnRNP A1 has also been implicated as a mediator of

signal transduction. This protein antagonizes the action of

SR proteins that promote distal 5' splice-site usage in E1A

and ß-globin pre-mRNAs bearing thalassemia mutations

(Cáceres et al, 1994; Mayeda and Krainer, 1992). In

addition, hnRNPA1 controls inclusion of exon 7b of its

own transcript (Blanchette and Chabot, 1999) and of exon

2 of the HIV Tat-pre-mRNA (Caputi et al, 1999). Two

signal transduction pathways have been described to change

the RNA binding properties and the intracellular

localization of hnRNPA1. Stimulation of the PDGF

receptor causes phosphorylation of hnRNP A1 by

PKCzeta (Municio et al, 1995). This phosphorylation

impairs the RNA binding and strand annealing activity of

hnRNPA1.

Furthermore, hnRNP A1 is phosphorylated by the

MKK3/6-p38 signaling cascade after cellular stress induced

by osmotic stress or UV irradiation, but the direct kinase

remains to be determined (van Oordt et al, 2000). Stress

induced phosphorylation leads to the cytoplasmic

accumulation of hnRNP A1 and results in a change of the

alternative splicing pattern of the adenovirus E1A premRNA

splicing reporter.

D. STAR proteins

Other likely candidates for proteins that can transduce

a signal to the spliceosome are STAR proteins. STAR is

an abbreviation for signal transduction and activation of

RNA (Jones and Schedl, 1995; Vernet and Artzt, 1997).

This protein family is also called GSG for GRP33,

Sam68, GLD-1 (Jones and Schedl, 1995). Its members

belong to an expanding group of proteins that share an

amino-terminal maxi-KH-RNA binding domain as well as

proline and tyrosine-rich regions present in many adapter

proteins involved in signal transduction (Richard et al,

1995). The binding properties of STAR proteins suggest

their involvement in splice-site selection. For example,

Sam68 has been found to crosslink to a splicing regulator

region on the rat tropomyosin pre-mRNA (Grossman et al,

1998), and also binds to FBP21, a protein implicated in

splicing (Bredford et al, 2000). A protein related to

SAM68 is SLM-2, for Sam68 like molecule (Di Fruscio

et al, 1999). In humans, the protein is called T-STAR and

was shown to interact with RBM, an hnRNP G like

protein previously implicated in splice-site regulation

(Venables et al, 1999, 2000). We identified the rat

homolog and demonstrated that it regulates alternative

splicing of CD44, htra2-beta, and tau pre-mRNAs by


Gene Therapy and Molecular Biology Vol 5, page 17

Table 3: Stimuli that change alternative splicing patterns

Signals known to influence alternative splicing patterns are shown in the first column. Known proteins involved in signal

propagation to the splicing machinery are indicated in the second column and the gene which changes its alternative splicing

pattern is shown in the fourth column. In some cases, it has also been determined whether de novo protein biosynthesis is

necessary for the observed change of a given splicing pattern, which is shown in the fourth column.

17


inding to purine-rich enhancer sequences (Stoss et al,

submitted). The physiological importance of STAR

proteins becomes apparent in mutations of the quaking

locus. The molecular defect is a mutation in the STAR

family member QKI, which results in severe defects in

myelination in the nervous system (Ebersole et al, 1996).

SAM68 is phosphorylated by the tyrosine kinases Src or

Fyn during mitosis (Fumagalli et al, 1994). SAM68

tyrosine phosphorylation is inducible by insulin in

fibroblasts, or after TCR stimulation (Fusaki et al, 1997;

Lang et al, 1997; Sanchez-Margalet and Najib, 1999). The

tyrosine phosphorylation results in a decrease of the RNA

binding affinity and leads to the dissociation of Sam68

multimers which could have a direct influence on the

regulation of alternative splicing (Chen et al, 1997; Wang

et al, 1995). We were able to show that rSam68, rSLM-1,

and rSLM-2 bind to the scaffold attachment factor B (SAF-

B), a component that binds to DNA-nuclear matrix

attachment regions as well as to RNA polymerase II and

various SR proteins (Stoss et al, submitted). This

association again emphasizes the intimate connection

between pre-mRNA processing, transcription, and

chromatin structure. The exact change of protein:protein

interaction caused by tyrosine phosphorylation of complex

components and its influence on alternative splicing

remain to be determined, but will most likely influence

splice-site selection.

E. SR proteins and their kinases

Finally, in humans, SR proteins are phosphorylated by

four different Cdc2-like kinases of the LAMMER family

(Clks) and two structurally related kinases called SRPK1

and SRPK2 (Gui et al, 1993; Koizumi et al, 1999; Nayler

et al, 1997; Prasad et al, 1999; Stojdl and Bell, 1999;

Wang et al, 1998). These kinases all contain an RS

domain; they interact with SR proteins and their

overexpression leads to the disassembly of speckles

(Nayler et al, 1998b). A direct involvement of Cdc2-like

kinases in the regulation of alternative splicing has

recently been shown for the Clk1-, E1A- and SRp20-, and

tau pre-mRNAs (Duncan et al, 1997; Hartmann et al,

2000; Stoss et al, 1999b). However, there are important

differences among these kinases. First, the Cdc2-like

kinases are ubiquitously expressed, whereas the SRPKs

show a more differentiated expression pattern with SRPK1

predominantly expressed in testis and SRPK2 in brain

(Papoutsopoulou et al, 1999a; Wang et al, 1998). In

addition, SRPK1 shows higher specificity towards

ASF/SF2 in comparison with Clk/STY, since Clk/STY

phosphorylates Ser-Arg, Ser-Lys, or Ser-Pro sites, wheras

SRPK1 shows a strong preference for Ser-Arg sites

(Colwill et al, 1996a). There is also evidence that these

kinases phosphorylate SR proteins at different sites.

Finally, there are differences in the intracellular

localization, since the Clks are predominantly nuclear and

colocalize with speckles, whereas SRPK1 was primarily

found in the cytoplasm and is believed to phosphorylate

the cytosolic SR protein fraction.

Stoss et al: RNA splicing and disease

18

In addition, there are three other kinases that are able

to phosphorylate SR proteins: CKIa, the Lamin B receptor

and topoisomerase I (Gross et al, 1999; Nikolakaki et al,

1996; Rossi et al, 1996). In contrast to the Cdc2-like

kinases, the substrates of CKIa must already be

phosphorylated, suggesting a phosphorylation hierarchy in

the regulation of SR proteins. The lamin B receptor is part

of a nuclear envelope complex that also phosphorylates

lamin A and B. This kinase phosphorylates RS domains

and seems to have substrate specificities identical to

SRPK1 (Papoutsopoulou et al, 1999b).

Phosphorylation of SR proteins has numerous effects

on protein:RNA and protein:protein interactions. For

example, phosphorylation of SRp40 enhances its RNA

binding affinity (Tacke et al, 1997) and phosphorylation of

ASF/SF2 stimulates its binding to U170K, a component

of the U1snRNP (Xiao and Manley, 1998). This type of

covalent modification of SR proteins has a direct effect on

their activity in the splicing reaction (Prasad et al, 1999;

Tazi et al, 1993).

An important consequence of SR protein

phosphorylation is the release of these proteins from their

storage compartments, the speckles (Colwill et al, 1996b;

Gui et al, 1993; Koizumi et al, 1999), and their

recruitment to sites of transcription (Misteli, 2000;

Misteli et al, 1998). In addition, phosphorylation

influences the ability of some SR proteins to shuttle

between the nucleus and the cytoplasm (Cáceres et al,

1998; Yeakley et al, 1999). In a manner that is similar to

hnRNP A1 (van Oordt et al, 2000), this could result in a

rapid change of the nuclear concentration of SR proteins,

which changes alternative splice-site selection.

Together, these data show that pre-mRNA splicing

can be regulated by external stimuli. Some of the signal

transduction pathways to the spliceosome are beginning to

emerge and result in regulated serine and tyrosine

phosphorylation of splicing proteins.

III. Diseases caused by splicing defects

A. Overview

Most of the diseases associated with defects in premRNA

processing result from a loss of function due to

mutations in regulatory elements of a single gene. These

mutations have previously been compiled (Krawczak et al,

1992; Nakai and Sakamoto, 1994) and are available on the

web (cookie.imcb.osaka-u.ac.jp/nakai/asdb.html). A few

diseases have been attributed to a change in trans-acting

factors. Knockout experiments of essential splicing factors

have proven lethal (Hirsch et al, 2000; Wang et al, 1996).

However, the knockout of an enzyme involved in premRNA

editing (Higuchi et al, 2000) and overexpression of

mutated SR proteins in Drosophila (Kraus and Lis, 1994)

causes phenotypes that cannot be linked to a single

mRNA, which shows that defects in pre-mRNA

processing factors can result in a complex pathological

state. The combinatorial nature of trans-acting factors

raises the interesting possibility that pleiotropic diseases

with a variable phenotype might be caused by alterations


of trans-acting factors. Using minigenes of CFTR

mutations, it has been shown that the splicing patterns of

mutated alleles strongly depend on the cell type (Nissim-

Rafinia et al, 2000), indicating that variations in transacting

factors could be the reason for a variable penetrance

of mutations among individuals with different ethnic

backgrounds (McInnes et al, 1992; Rave-Harel et al,

1997). Similarly, a contribution of pre-mRNA processing

could explain why natural mutations in human genes

frequently have tissue- or cell-type specific effects. In these

cases, the mutated gene is similarly expressed in all cells,

but is processed in a tissue specific manner, since the

relative concentrations of splicing factors vary among

tissues (Hanamura et al, 1998).

Since diseases caused by splicing defects have been

recently reviewed (Philips and Cooper, 2000), we will

concentrate on three diseases, FTDP-17, spinal muscular

atrophy, and !-thalassemia to illustrate the complex

relationships between trans-acting factors and their

corresponding cis-elements, and to outline possible

therapeutic approaches.

B. FTDP-17: frontotemporal dementia

with parkinsonism linked to chromosome 17

Frontotemporal dementias (Figure 2A) represent a

rare form of presenile dementias that are clinically defined

by behavioral and personality changes, psychomotor

stereotypes, as well as loss of judgment and insight. The

neuropathological findings include an asymmetric

frontotemporal atrophy and the presence of filamentous tau

deposits. The disease was mapped to the tau locus on

chromosome 17. Tau is a microtubule-associated protein.

Knockout experiments revealed that tau is not essential for

brain formation (Harada et al, 1994), although it is

involved in the pathology of several neurodegenerative

diseases (Spillantini and Goedert, 1998). Tau transcripts

undergo complex regulated splicing in the mammalian

nervous system. The alternative splicing of one of its

exons, exon 10, is species-specific. This exon is

alternatively spliced in adult humans, but is constitutively

used in the adult rodent brain. In addition, the usage of

exon 10 is regulated during development and increases

when neuronal development proceeds. In the protein, it

encodes one of the four microtubuli binding sites of tau.

Some of these tau mutations that activate exon 10 usages

were shown to cause an accelerated aggregation of tau into

filaments (Nacharaju et al, 1999), which is a hallmark of

several neurodegenerative diseases, e.g. Alzheimer's

disease.

A disruption of the proper balance of tau isoforms

with three and four microtuble binding sites is observed in

the pathology of several tauopathies, including FTDP,

Picks disease, corticobasal degeneration, Guam amytrophic

lateral sclerosis/parkinsonism dementia complex.

Secondary structure predictions suggest a stem loop

structure at the 5' splice-site of exon 10 that contributes to

its regulation (Grover et al, 1999; Jiang et al, 2000);

however the in vivo relevance of this structure remains to

be proven. In addition, mapping of elements in tau exon

Gene Therapy and Molecular Biology Vol 5, page 19

19

10 revealed a complicated set of cis-acting elements that is

disrupted by natural mutations (D'Souza et al, 1999).

Some of these mutations, such as L284L, are silent, but

lead to disease by interrupting an exonic element that

causes missplicing.

Since alternative splice-site usage can be regulated by

the relative concentration of SR proteins or hnRNPs,

several such trans-acting factors were tested in vivo, and it

was found that some SR or SR related proteins (SF2/ASF,

SRp75 and U2AF65) stimulate exon 10 skipping (Gao et

al, 2000). It was shown that SR proteins are released from

their storage compartments, the speckles, by Cdc2-like

kinases (clk1-4). Those kinases were found to strongly

inhibit missplicing of exon 10, even in several mutations

that activate exon 10 usage (Hartmann et al, submitted).

These examples show that missplicing can be

reversed in vivo by activating regulatory proteins through

their kinases. It is possible that lower molecular weight

substances can be isolated that cause the release of specific

regulatory factors, by either activating the appropriate

kinase or blocking the corresponding phosphatase. In

addition, recent results show that drugs such as

aminoglycoside antibiotics which can directly interact with

regulatory RNA structures, may also have a therapeutic

potential (Varani et al, 2000).

C. Spinal muscular atrophy (SMA)

Proximal spinal muscular atrophy (SMA, Figure

2B) is a neurodegenerative disorder with progressive

paralysis caused by the loss of alpha-motor neurons in the

spinal cord. With an incidence of 1 in 10,000 live births

and a carrier frequency of 1 in 50, SMA is the second-most

common autosomal recessive disorder and the most

frequent genetic cause of infantile death (Pearn, 1980). The

gene responsible for the disease was identified as SMN1

(survival of motor neurons, Lefebver, 1995) and the

disease is caused by loss of (96.4%) or mutations in

(3.6%) the SMN1 gene (Wirth, 2000). A nearly identical

copy of the SMN1 gene exists but cannot compensate for

the absence of SMN1, because it is processed differently.

Due to a single nucleotide difference in exon 7, this exon

is skipped in SMN2. Therefore the proteins generated by

both genes differ in their carboxy terminus, which is most

likely crucial for the function. The protein generated by

SMN2 encodes a truncated, less stable protein with reduced

self-oligomerization activity ( Lefebvre et al, 1995, 1997;

Coovert et al, 1997). The exon enhancer containing the

single nucleotide difference has been characterized (Lorson

et al, 1999) and was found to be of the GAR type. A

systematic search for trans-acting factors identified human

transformer2-beta, a member of the SR related family of

proteins (Hofmann et al, 2000). An increase of the

concentration of htra2-beta1 results in stimulation of exon

7 increase. A mRNA generated by this pathway would

encode for a protein that can complement for the loss of

SMN1. This example demonstrates that pre-mRNA

processing can be maniplulated in vivo to complement the

loss of a gene product.


Stoss et al: RNA splicing and disease

Figure 2: Examples of diseases that are caused by errors in pre-mRNA splicing

A) Frontotemporal dementia with Parkinsonism (FTDP-17) as an example of mutation in splicing

enhancers and silencers. FTDP-17 is caused by a misregulation of tau exon 10 usage. Exon 10 splicing is tightly regulated

by exonic enhancer and silencer sequences in exon 10. It is unclear whether a secondary structure that masks the 5' splice-site is

important in vivo. Mutations close to exon 10, which are observed in FTDP-17 patients (shown in red), favor exon 10 inclusion.

The Cdc2-like kinases clk1-4 are able to revert the increase in exon 10 inclusion. This opens new therapeutic possibilities to treat

FTDP-17 and related diseases by screening for factors or substances that selectively affect the regulation of alternative splicing.

B) Spinal muscular atrophy (SMA): compensation of a non-functional gene. Positional cloning strategies led to

the identification of the survival of motorneuron (SMN) genes as one of the genes affected in SMA. Two non-equal copies of SMN

exist on chromosome 5q13. Full-length SMN can only be generated from the telomeric copy (SMN-1). A silent C/T transition of

the sixth nucleotide of the centromeric copy (SMN-2) (in red) disturbs splicing of exon 7, leading to the generation of a truncated

SMN protein. SMA is caused by failure to express the telomeric SMN-1 gene. One possibility to compensate for the loss of SMN-1

expression is overexpression of htra2-beta1. This SR like protein can switch the splicing pattern of the SMN-2 pre-mRNA

towards the inclusion of exon 7, leading to a functional SMN-2 copy.

C) !-thalassemia: A point mutation activates a cryptic splice-site. The second intron of the thalassemic !-globin

gene harbors a C to T mutation at nucleotide 654. This creates an additional 5' splice-site that activates a cryptic splice-site at

nucleotide 579 of the !-globin pre-mRNA, leading to the retention of an intronic region. Antisense oligonucleotides can be used

to mask the aberrant splice-sites, resulting in the formation of the desired gene product.

20


D. !-thalassemia

!-thalassemias are autosomal recessive diseases

(Figure 2C), in which the amount of !-globin is

reduced. Individuals carrying a single mutated gene are less

prone to malaria infection. About 3% of the world

population, mostly people living in regions endemic with

malaria, or their descendants, are carriers. !-thalassemia

causes a hypochromic, microcytic and hemolytic anemia.

The imbalance of globin synthesis causes the "-chains to

precipitate and damages the red blood cells. More than 100

mutations of the !-globin gene leading to thalassemia have

been described (Wetherall and Clegg, 1981), among them

are at least 51 point mutations, which mostly affect premRNA

processing (Kazzazin and Boehm, 1988; Krawczak

et al, 1992). These mutations either destroy the 5' or 3'

splice-sites or generate cryptic splice-sites that are usually

located in the introns (Figure 2C). As a result, no

functional !-globin protein is produced. Since point

mutations often manifest themselves as defects in premRNA

splicing, missplicing of !-thalassemias is a model

for a large number of mutations (Krawczak et al, 1992;

Nakai and Sakamoto, 1994). Cryptic splice-sites of

thalassemic !-globin can be changed in vivo by

overexpression of the SR protein SF2/ASF (Cáceres et al,

1994). Furthermore, the mutated cryptic sites can be

blocked by a complementary oligonucleotide (Schmajuk et

al, 1999; Sierakowska et al, 2000), that enhances the

formation of the desired !-globin product in cell-culture

systems. This example illustrates that cryptic splice-sites

can be masked in vivo, which promotes the formation of

the desired gene product.

IV. Detection and treatment of splicing

defects

A. Alternative splicing as an indicator of

disease

Since pre-RNA pathways can adapt according to

environmental signals, the splicing pattern of pre-mRNAs

is most likely a reflection of the cellular state. There are

numerous examples in which a change of exon usage is

associated with a pathological state. The most prominent

example is cancer. Some of the changes found in splicesite

selection that are associated with cancer are shown in

Table 4. One of the best described genes that shows the

importance of pre-RNA processing for tumor progression

and metastasis is CD44. In this gene, at least 12 exons are

alternatively spliced (Screaton et al, 1992) and their usage

relates to metastatic potential.

With the completion of the human gene project and

the progress in array techniques, it will be possible to

detect differences in splicing patterns between a normal and

pathological state. Arrays with exon-specific

oligonucleotides will make it possible to discover changes

in alternative splicing patterns. Given the numerous

examples in which changes in exon usage are associated

with disease, the development of such a DNA exon chip

might help in the diagnosis of cancer and the elucidation of

Gene Therapy and Molecular Biology Vol 5, page 21

21

the underling molecular pathology. Furthermore, such a

tool might be used to detect the effect of trans-acting

factors in vivo and could ultimately be used to unveil

misregulation of trans-acting factors in complex diseases.

B. Suppression of point mutations by

oligonucleotides

The majority of known pathological states associated with

splicing are generated by point mutations that either

destroy splice-sites or generate new, cryptic sites in the

vicinity of normally used exons (Krawczak et al, 1992). It

has been demonstrated that antisense nucleic acids binding

to the aberrant splice-sites can inhibit the usage of the

wrong sites and promote the formation of the normal gene

product. Among nucleic acids, modified RNA

oligonucleotides have been used (Sierakowska et al, 2000).

Blocking the aberrant splice-sites forces the splicing

machinery to reselect the original splice-site and can

restore the correct gene product. Currently, 2'-O-methyl

oligoribonucleoside phosphoro-thioates are the most

widely used nucleic acids, since they do not induce RNase

H mediated cleavage of targeted RNA and seem to have

only minor effects on cell viability, morphology, and

growth rates. Diseases targeted include ß-thalassemias

(Schmajuk et al, 1999), cystic fibrosis (Friedman et al,

1999), muscular dystrophy mRNA (Wilton et al, 1999),

and eosinophilic diseases (Karras et al, 2000).

Furthermore, apoptosis can be influenced by

oligonucleotides directed against Bcl-x splice variants

(Taylor et al, 1999). The oligonucleotide approach offers a

high specificity to target a mutated gene. Most studies

have been performed in cell culture systems, where the

oligonucleotide approach works in multiple cellular

contexts, which argues for its broad applicability to

suppress an aberrant splice-site selection. It remains to be

seen whether this approach can also be used to modify

exon enhancer usage.

C. Modification of trans-acting factor

action

Since the selection of splice-sites is dependent on the

relative concentration of regulatory proteins, a change of

the concentration of a protein could possibly correct a

pathological ratio of exon inclusion to exon skipping. For

example, overexpression of SR protein and their kinases

clk1-4 can revert missplicing of tau exon 10 (Gao et al,

2000; Hartmann et al, submitted); overexpression of htra2beta1

can change the splicing pattern of SMN2 to

complement loss of SMN1 in spinal muscular atrophy

(Hofmann et al, 2000); and the levels of hnRNPA1 and

SF2/ASF regulate alternative splicing of mutated alleles of

the cystic fibrosis transmembrane conductance regulator

(Nissim-Rafinia et al, 2000) and mutated ß-globin genes

(Cáceres et al, 1994). Since most splicing factors are

released from nuclear storage compartments, a promising

strategy might be the identification of specific

antagonist/agonists for splicing factor kinases from a

chemical library.


Stoss et al: RNA splicing and disease

Table 4: Examples of genes that change their splicing pattern during cancer formation and progression.

Only a few examples are given

Furthermore, it is likely that release of splicing factors can

occur in response to a stimulation of a receptor and

specific agonists could be found to arrest this process.

Such molecules will be easier to deliver pharmacologically

because of their small size; however such agonists remain

to be identified, and their specificity proven.

V. Conclusions

The basic mechanisms regulating alternative splicesite

selection have been deciphered in recent years. The

abundant usage of alternative pre-mRNA processing has

most likely generated an evolutionary advantage for

vertebrates, since the formation of protein isoforms

required for specialized functions was greatly accelerated.

Disadvantages of this mechanism is the misregulation of

pre-mRNA processing that is apparent in several human

diseases. We hypothesize that the role of pre-mRNA

defects in human disease is just beginning to emerge and is

currently largely underestimated. Since splice-site selection

is regulated by extracellular signals, the analysis of these

signal transduction pathways might provide new insights

and novel chances for therapy. The connection of the

molecular mechanisms governing splice-site selection with

the detailed analysis of human diseases will provide

opportunities for diagnosis and treatment.

Acknowledgments

Work in the laboratory is supported by the Max-

Planck Society, the DFG (Sta 399 3-1), the HSFP (RG

562/96) and the EU (Bio4-98-0259).

22

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Stefan Stamm


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Gene Therapy and Molecular Biology Vol 5, page 31

31

Gene Ther Mol Biol Vol 5, 31-37, 2000

Th2-type immune response induced by a phage

clone displaying a CTLA4-binding domain mimicmotif

Research Article

Yasuhiro Kajihara, Shuhei Hashiguchi , Yuji Ito, and Kazuhisa Sugimura*

Department of Bioengineering, Faculty of Engineering, Kagoshima University, 1-21-40 Korimoto, Kagoshima 890-0065,

Japan

_____________________________________________________________________________________

*Correspondence: Kazuhisa Sugimura, Department of Bioengineering, Faculty of Engineering, Kagoshima University, 1-21-40

Korimoto, Kagoshima 890-0065, Japan, Phone: +81-99-285-8345; Fax: +81-99-258-4706, E-mail: kazu@be.kagoshima-u.ac.jp

Key words: CTLA-4, Th2, immune deviation, peptide mimic, phage library, molecular design, vaccine

Received: 4 May 2000; accepted: 21 September 2000

Summary

We have recently isolated a phage clone, F2, which displays the CTLA4-binding domain mimic from a

phage display library. To investigate the in vivo effects of an F2 motif on the regulation of immune

responses, we immunized Balb/c mice intraperitoneally with varying doses of an F2 phage in a

phosphate buffered saline and followed the resulting antibody and cytokine responses. It was shown

that the F2 phage enhanced the IgG antibody response to phage particles in comparison to control

phages that were randomly selected from the library. When the antigen specificity of the induced

antibody response was examined, the production of an anti-g3p antibody was preferentially increased

while an anti-g8p antibody was slightly down-regulated by the immunization of F2 in comparison to

control phage clones. The increase of an anti-g3p antibody response was found in the isotype of IgG1

but not in the IgM or IgG2a. When the cytokine production was examined by culturing spleen cells

from these mice under stimulation with anti-CD3 mAb, IL-4 production was approximately twice

higher in the F2-primed cells than in the L4-primed cells while IFN-! production was higher in L4primed

cells than in the F2-primed cells. Thus, these results suggested that the F2 phage clone

bearing g3p with the CTLA4-binding domain mimic-motif induced the Th2-type response when

compared to control phage clones.

I. Introduction

T-cell co-stimulatory receptors CD28 and CTLA4

deliver opposite signals on T-cell activation, mediating

augmentation and inhibition of T-cell responses,

respectively (Linsley, 1995; Thompson, 1995; Bluestone,

1997; Thompson and Allison, 1997). These two receptors

use the same ligands, CD80 (B7-1) and CD86 (B7-2),

expressed on the antigen-presenting cells (Azuma et al,

1993; Freeman et al, 1993). The kinetic study on the

expression of these molecules suggested that CD28 may

be responsible for CD86, and CTLA4 is responsible for

CD80, respectively, since CTLA4 and CD80 expression

reaches maximal levels 2-3 days after antigenic stimulation

(Hathcock et al, 1994; Schweitzer et al, 1997).

However, CTLA4 may have more of a role in

regulating T-cell responses at earlier stages in the process

than had initially been thought (Krummel and Allison,

1995).

In principle, the interactions of co-stimulatory

molecules were possible with four kinds of combinations:

CD28-CD80, CD28-CD86, CTLA4-CD80, and CTLA4-

CD86. However, the functional difference between CD80

and CD86 is still obscure although considerable interest

has focused on the possible role of the CD80 and CD86

co-stimulatory molecules expressed on antigen-presenting

cells (APC) in skewing CD4 + T cells to either the Th1 or

Th2 phenotype (Hathcock et al, 1994; Schweitzer et al,

1997; Manickasingham et al, 1998).

Recently, we selected phage clones from a phage

display library by employing a CTLA4-conformation

recognizing a monoclonal antibody (mAb) (Fukumoto et

al, 1998). A phage clone, F2, is specifically recognized

with the anti-CTLA4 mAb and able to bind to CD80. The

F2 motif consists of the unique 15-amino-acid sequence


with an internal disulfide bond and is inserted in gene 3

proteins (g3p), which display only three to five copies in

contrast to approximately 2700 copies of gene 8 proteins

(g8p) per fd phage. The HPLC-purified g3p (F2-g3p) is

also recognized with anti-CTLA4 mAb but not anti-CD28

mAb and binds to CD80 but not to CD86. When hen egg

lysozyme (HEL)-primed lymph node cells were stimulated

with HEL in the presence of the F2-g3p in vitro, cell

proliferation was highly potentiated (Fukumoto et al,

1998). In the absence of antigenic stimulation, the F2-g3p

induced no T-cell proliferation, indicating the costimulatory

nature of the F2-g3p. Thus, the F2 motif

represents a peptide mimic of the CTLA4-binding domain.

In this study, we examined the effect of F2 phage as

an immunogen on the anti-phage immune response in

vivo. When the phages were administered intraperitoneally

(i.p.) in mice in the form of a phosphate-buffered saline

(PBS) solution, F2 phage induced an anti-phage antibody

response two to three times higher than that of the control

phage. In this augmented response, the anti-g3p antibody

production was predominantly increased, whereas the antig8p

antibody production was rather decreased. The isotype

of the increased antibody was IgG1 but not IgM or IgG2a.

When the spleen cells were stimulated with the

immobilized anti-CD3 mAb in vitro, cells derived from

F2-immunized mice showed the augmented response in IL-

Kajihara et al: Th2-type immune response

32

4 production while a weaker response was shown in IFN-!

production in comparison to those of the control phageprimed

mice.

Thus, an F2 motif that interferes with the interaction

of CTLA4 with CD80 but not CD86 preferentially

generated the Th2-type immune response in vivo,

suggesting that the predominant interaction of CD86 with

CD28 in the absence of CTLA4/CD80 signaling may

skew the immune response to the Th2-type in vivo.

II. Results

A. In vivo antibody response of Balb/c

mice immunized with fd phage clones

Balb/c mice were administered i.p. with a PBS

solution containing varying doses of wild type (wt), F2 or

a control phage (L4), which was randomly selected from

the library. The anti-phage IgG antibody responses were

followed by using control phage (K7)-coated plastic plates

(K7-ELISA). As shown in Figure 1, 50 µg of F2 phage

induced a marked anti-phage antibody response when

compared to those of L4 and wt clones. In the case of an

L4 or wt clone, 50 and 5 µg of phage induced almost

comparable IgG antibody responses, while 0.5 µg barely

induced a response. In the next experiments, mice were

Figure 1. Antibody responses induced with intraperitoneal administration of PBS solution containing varying doses of phage

clones: Balb/c mice were administered i.p. with 0.5, 5, and 50 µg of either F2, L4, or a wild-type (wt) phage clone, in PBS. An antiphage

IgG antibody was measured by a control phage (K7)-coated ELISA every week after the immunization. K7 was randomly selected

from the phage library. Each line indicates a response pattern of a mouse. The serum was tested at a dilution of 1: 1080.


administered 5 µg of a wt, L4, or F2 phage clone, and the

antibody production was estimated by either the wt or K7

phage-coated ELISA plate. The wt-ELISA estimated the

amount of anti-phage antibodies except for the anti-g3p

antibody because the wt lacks g3p due to a frame shift,

while K7-ELISA measured the amount of whole antiphage

antibodies including the anti-g3p antibody. When

the antisera were assayed with K7-ELISA, F2 induced an

IgG response that was approximately four times higher in

comparison to those of the L4 or wt (Figure 2A). In

contrast, when the same antisera were estimated by wt-

ELISA, the magnitudes of antibody responses were almost

at the same levels among these groups (Figure 2B).

These results suggested that the amplified IgG response by

F2-immunization might be directed to the specificity

against g3p but not to the other constituents of phage in

comparison to the wt or L4-immunization.

B. F2 motif enhanced the antibody

response to g3p but not to g8p molecules

In order to determine the antigen specificity of

antibodies amplified by the F2 phage clone, the antisera

were assayed by using both phage clone- and HPLCpurified

g3p/g8p-coated plastic plates. The sera from the

L4-immunized mice showed a value by K7-ELISA that

was as high as the value of the wt-ELISA (Figure 3A).

Figure 2. F2 induced the augmented anti-phage IgG antibody

response relative to the control phage clones: Balb/c mice

were immunized i.p. with 5 µg of various phage clones (F2 !,

L4 ! ,WT ") in PBS. Antibodies were measured by a control

phage (K7)-coated ELISA (panel A) and wt phage-coated ELISA

(panel B). The serum was tested at the dilution of 1: 1080.

Gene Therapy and Molecular Biology Vol 5, page 33

33

Figure 3. Preferential enhancement of an anti-g3p antibody

response by the introduction of the F2 motif: Balb/c mice

immunized with 50 µg of various phage clones. Sera were

collected four weeks after immunization. The amount of antiphage

IgG antibodies was measured by phage (K7 " or wt )coated

ELISA (panel A: serum dilution:1/1080) or purified

g3p-( ) or g8p ( ! )-coated ELISA (panel B: serum

dilution:1/500) plates. Each value represents the mean of

three mice per group ± S.E.

In contrast, the sera of the F2-immunized mice

showed a value with the K7-ELISA that was

approximately twice as high as that shown by the wt-

ELISA. These sera were assayed by g3p or g8p-coated

plates (Figure 2B). The sera from the L4-immunized

mice reacted to both g3p and g8p with a slightly higher

value of the anti-g8p antibody, while the sera of the F2immunized

mice exhibited a value to g3p that was

approximately twice as high as that to g8p. The sera of the

wt-immunized mice predominantly reacted to g8p but not

to g3p (data not shown). Thus, the F2 motif influenced the

immune response to its carrier protein molecule but not to

other phage proteins associating with g3p.

C. IgG1 production was preferentially

augmented by the F2 motif

Next, we examined the immunoglobulin isotype on

the amplified anti-g3p antibody response induced by the

introduction of the F2 motif. As shown in Figure 4, L4

and F2 induced almost the same kinetic patterns of the

IgM antibody responses. However, the IgG1 isotype was

preferentially produced in the augmented anti-g3p antibody

response by the immunization of F2. The IgG2a isotype

was barely detected by the immunization of F2 or L4.

These results suggested that the F2 motif might skew the

immune response to the Th2-type. The F2 motif does not

appear to accelerate the time course of the IgG antibody

response because two weeks after the F2-immunization,

only the IgM and not the IgG1 was significantly produced.


D. Cytokine production affected by the

F2 motif in g3p

In order to examine the effect of the F2 motif on the

T-cell activation, spleen cells derived from L4- or F2immunized

mice were stimulated with anti-CD3 mAb in

vitro, and IL-4 and IFN-! that were produced in

supernatants were measured by ELISA. As shown in

Figure 5, the control phage of L4-primed cells showed a

significant amount of IFN-! production and a very weak

IL-4 production. However, in the case of the F2-primed

cells, the IFN-! production was rather decreased, and in

contrast, the IL-4 production was augmented in

comparison to the L4-primed cells. These results suggested

that the anti-g3p antibody response might be skewed

toward Th2-type immune responses by the insertion of the

F2 motif. We carried out the same kind of experiments

using L4 or F2 phage as an in vitro-stimulant instead of

anti-CD3 mAb. In these cases, we failed to detect a

significant production of IFN-! or IL-4 in culture

supernatants (data not shown).

III. Discussion

A phage clone, F2, was isolated from a phage display

library by using a CTLA4-conformation recognizing a

monoclonal antibody (Fukumoto et al, 1998). The HPLCpurified

F2-g3p exhibited the ability to bind to CD80 and

inhibited the interaction of CTLA4 and CD80. These

characteristics of F2-g3p appeared to result in the marked

augmentation of the antigen-stimulated T-cell proliferation

in vitro (Fukumoto et al, 1998).

In this study, we characterized the immune response

to fd phage by simply administering the F2 phage

intraperitoneally without an adjuvant to Balb/c mice. We

demonstrated here that the F2 motif augmented the antiphage

antibody responses approximately two to three

times higher than those of the control phage-primed mice,

although the F2-mediated augmentation on T-cell

Kajihara et al: Th2-type immune response

34

proliferation in vitro was much more remarkable as

described in previous studies (Fukumoto et al, 1998a,

1998b). The augmented response was shown in the

production of the anti-g3p but not in the anti-g8p antibody

(Figure 3). The anti-g8p antibody response was rather

decreased by the introduction of the F2 motif in the g3p

molecules. We have demonstrated in in vitro studies that

the addition of F2-g3p augments the proliferation of the

hen egg lysozyme (HEL)-primed lymphocytes when cells

are stimulated with HEL, indicating that the F2 motif is

not necessarilly fused with the antigen (Fukumoto et al,

1998). The F2-g3p inhibited the interaction of

CD80/CTLA4 and did not inhibit the interaction of

CD86/CTLA4 or CD86/CD28 (Fukumoto et al, 1998). It

is, therefore, conceivable that the fusion of the F2 motif

with the antigen, g3p, results in their efficient binding to

the g3p-specific T lymphocytes in vivo .

The enhancement of this response was markedly

demonstrated by the amount of IgG1 but not IgM or IgG2a

isotypes (Figure 4). A characteristic cytokine profile of

the F2-primed spleen cells was detected by stimulating the

cells with an anti-CD3 mAb, but it was not with the F2

or L4 phage. These results may be attributed to the very

small size of the F2-responding populations that were

generated. The IL-4 produced from these F2-responding T

cells may stimulate the generation of the bystander Th2type

T-cell populations. This amplifying process might

enable us to detect IL-4 or IFN-! in our cell culture

system when cells were stimulated with anti-CD3 mAb.

Thus, these results suggested that the inhibition of

the CTLA4/CD80 interaction with a peptide mimic of the

CTLA4-binding domain may have skewed the response to

the Th2 pathway, implying that the predominant

interaction of CD86 with CD28, in the absence of

CTLA4/CD80 signaling, preferentially induces the Th2

immune response in vivo.

Figure 4. The F2 motif augmented the anti g3p IgG1 antibody response: Balb/c mice immunized with 50 µg of F2 or L4 phage

clones. Anti-g3p (upper panel) or -g8p antibodies (lower panel) were measured on the immunoglobulin subclasses, which were detected

by AP-anti-mouse IgM (panel A), IgG1 (panel B), and IgG2a antibody (panel C). Sera were tested at a dilution of 1:500. Each value

represents the mean of five mice per group ± S.E.


Gene Therapy and Molecular Biology Vol 5, page 35

Figure 5. IL-4 production was augmented in F2-primed spleen cells relative to L4-primed spleen cells in vitro: Spleen cells were

obtained from Balb/c mice, which had been immunized i.p. with 50 µg F2 or L4 phage in PBS seven weeks before. The cells were

cultured in the anti-CD3 Ab-immobilized culture plates (10 µg/ well) for 48 hr (IL-4) or 72 hr (IFN-!). Murine IFN-! or IL-4 in

supernatants was measured using sandwich ELISA. Each value represents the mean of five mice per group ± S.E.

Regarding these results, studies by Freeman and

coworkers indicated that CD86, but not CD80, costimulation

during the anti-CD3 Ab-mediated CD4 + T-cell

activation skewed the cells to produce IL-4, suggesting

that CD80 and CD86 may provide distinct signals during

the development of CD4 + T-cell responses (Freeman et al,

1995). Consistent with this, studies by Kuchroo and

coworkers indicated that the administration of anti-CD86

Abs to mice during priming with proteolipid protein for

induction of experimental autoimmune encephalomyelitis

(EAE) skewed CD4 + T-cell development to the Th1

phenotype and exacerbated disease, whereas the

administration of anti-CD80 Ab skewed CD4 + T-cell

development to the Th2 phenotype and the induction of

disease was blocked or decreased (Kuchroo et al, 1995).

These results have suggested that CD4 + T-cell engagement

of CD80 may direct development to the Th1 phenotype

and engagement of CD86 may direct development to the

Th2 phenotype.

A similar finding was reported by Khoury and Gallon

et al. using a derivative of CTLA4, CTLA4IgY100F

(Khoury et al, 1996). This molecule binds to CD80 but

not to CD86, which is the same characteristic as F2-g3p.

These reagents are able to avoid the potential of signaling,

which is induced by the addition of anti-CTLA4

antibodies. Using the CTLA4IgY100F in the induction of

experimental autoimmune encephalomyelitis (EAE), they

showed that CD28-CD80 interaction may lead the response

35

to the Th1 pathway. However, other studies have reported

that the qualitative differences were not detected in the

capacities of murine CD80 and CD86 to induce IL-4

production (Natesan et al, 1996). In the case of Schweizer

et al, they showed that CD86 has a more important role

than CD80 in initiating antibody responses in the absence

of an adjuvant and that CD86, and to a lesser extent CD80,

makes significant contributions to the production of both

IL-4 and IFN-! .

CD80 and CD86 contribute to the magnitude of Tcell

activation, but they do not appear to selectively

regulate Th1 versus Th2 differentiation (Schweitzer et al,

1997; Schweitzer and Sharpe, 1998). A recent study by

Anderson et al. shows that a CTLA4 blockade can enhance

or inhibit the clonal expansion of different T cells that

respond to the same antigen, depending on both the T-cell

activation state and the strength of the T-cell receptor

signal delivered during T-cell stimulation (Anderson et al,

2000).

Thus, it is still unclear if there is any difference in

the role of CD80 as opposed to CD86 on the interaction

with CTLA4. In our case, we have shown here that the

selective inhibitor of CD80-binding, F2-g3p induced the

skewing toward a Th2-type response when administered in

mice in vivo. The influences of the F2-motif on the

generation of cytotoxic T cells or delayed-type

hypersensitivity-responsible T cells remains to be

investigated.


As functional motifs of co-stimulatory molecules

appear to manipulate the immune responses by introducing

it into the target molecule, this strategy may lead us to

novel approaches to gene therapy and DNA vaccine

developments.

IV. Materials and Methods

A. Mice and antibodies

Balb/c mice (female) were purchased from Nihon SLC

Co. (Fukuoka). Alkaline phosphatase (AP)-conjugated antimouse

IgG, !1, !2a, and antibody were obtained from ZYMED

(San Francisco, CA). AP-conjugated anti-µ antibody was

purchased from Southern Biotechnology Associates, Inc.

(Birmingham, AL). Anti-CD3 mAb (cat. # 01081D) was

purchased from PharMingen (San Diego, CA).

B. Phage proteins

The fd (fUSE5) phage clones were isolated and the phage

proteins were purified as described previously (Fukumoto et al,

1998; Nishi et al, 1996). Briefly, the fd phages (7mg/ml) were

incubated with 1% sodium dodecyl sulfate (SDS) at 37˚C for 20

min. The g3p and g8p were purified by size-exclusion

chromatography on a HiLoad superdex 200 (26/60) column

(Pharmacia, Uppsala, Sweden) as described. The F2 phage

displays the motif of GFVCSGIFAVGVGGRC at the fifth

position of the N-terminal of g3p molecule (Fukumoto et al,

1998).

C. ELISA

ELISA was performed as described previously (Fukumoto

et al, 1998; Fukumoto et al, 1998). Plastic plates (Nunc) were

coated with phages (4 x 10 9 transducing unit [TU]/ 40 µl/ well)

or phage proteins (30 ng / 40 µl/well) in 50 mM Tris HCl, pH

7.5 and 150 mM NaCl (TBS) containing 0.02% NaN 3.

Blocking was done by using 350 µl of 1% bovine serum

albumin (BSA, Sigma, St. Louis, MO). The plates were washed

five times with TBS containing 0.05% Tween 20 (TBS/Tween)

and once with TBS. After the incubation with varying

concentrations of mouse antiserum for 1 hr at 4˚C, APconjugated

anti mouse IgG, !1, !2a, or µ antibody was added at

a dilution of 1:250. The substrate (85 µl) consisted of 1 mg /

ml p-nitrophenolphosphate (Wako Co., Osaka), and 10%

diethanolamine (Wako Co., Osaka) in TBS. Absorbance was

read at 405 nm by a microplate photometer (InterMed NJ-

2300, Tokyo). All assays were carried out after the dilution

rates of sera were determined on their linearity for ELISA. The

cytokine ELISA was performed using the IL-4 plate (cat. #

M4000) and IFN-!-plate (cat. # MIF00) of R&D systems Co.

(Minneapolis, MN), according to the manufacturer's

description. Statistical analysis was carried out by a Student's

t-test.

D. Cytokine production assay

The cell culture was carried out as described (Fukumoto et

al, 1998; Fukumoto et al, 1998). Briefly, cells (2 x

10 6 /1ml/well of 24 well-plate) were stimulated with varying

doses of phage clones, anti-CD3 mAb (10 µg/ml). The

supernatants were harvested 72 hr later for the assay on the

cytokine production. In parallel with these cultures, T-cell

proliferation was monitored by culturing cells (1.5 x

Kajihara et al: Th2-type immune response

36

10 5 /0.2ml/well) in flat-bottomed 96-well plates (Iwaki Glass,

Tokyo) for three days and pulsing cells with 0.5 µCi 3 Hthymidine

(Amersham, St. Louis, MO) for the final 18 hr.

Acknowledgments

This work was partly supported by a Grant-in-Aid for

Scientific Research from the Japanese Ministry of

Education, Science, and Culture.

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Kajihara et al: Th2-type immune response

38


Gene Therapy and Molecular Biology Vol 5, page 39

39

Gene Ther Mol Biol Vol 5, 39-46, 2000

Segregation of partly melted molecules and its

application to the isolation of methylated CpG islands

in human cancer cells

Review Article

Masahiko Shiraishi 1* , Leonard S. Lerman 2 , Adam J. Oates 1 , Xu Li 1,3 , Ying H. Chuu 1 ,

Azumi Sekiguchi 1 , and Takao Sekiya 1

1 DNA Methylation and Genome Function Project, National Cancer Center Research Institute, 1-1 Tsukiji 5-chome, Chuo-ku

104-0045, Tokyo, Japan, and 2 Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue,

Cambridge, MA 02139, USA. 3 Present address: Department of Biochemistry, Nantong Medical College, Jiangsu 226001, P. R.

China

_____________________________________________________________________________________

* Correspondence: Masahiko Shiraishi, DNA Methylation and Genome Function Project, National Cancer Center Research

Institute, 1-1 Tsukiji 5-chome, Chuo-ku 104-0045, Tokyo, Japan; Tel: +81-3-3542-2511 (ext. 4809); Fax: +81-3-5565-9535; E-mail:

mshirais@ncc.go.jp

Key words: denaturing gradient gel electrophoresis, DNA methylation, methylated DNA binding column, adenocarcinomas of the

lung, epigenetics

Received: 12 Jume 2000; accepted: 26 Jume 2000

Summary

Segregation of partly melted molecules is a convenient and efficient method for isolation of DNA

fragments associated with CpG islands. DNA fragments digested with restriction endonucleases are

subjected to denaturing gradient gel electrophoresis. DNA fragments derived from CpG islands are

preferentially retained in the gel after prolonged field exposure because of their lower rate of strand

dissociation. An independent technique, methylated-DNA-binding column chromatography, permits

separation of DNA fragments on the basis of the number of methyl-CpG sequences in the fragment,

and it enables separation of methylated CpG islands from those that are not methylated. Segregation

of partly melted molecules and methylated-DNA-binding column chromatography were successfully

combined to isolate CpG islands methylated in human adenocarcinomas of the lung. The methylated

CpG island library will be valuable in order to elucidate epigenetic process in carcinogenesis.

I. Introduction

In the human genome, G+C content of which is

40%, the appearance of CpG dinucleotides would be

expected one every 25 base pair (bp), if all four bases are

evenly distributed throughout the genome, but the actual

frequency is about one every 150 bp. This indicates that

distribution of four bases is not uniform and that a CpG

dinucleotide appeared less frequently than the simple

assumption of random distribution predicts. Furthermore,

the 5-position of cytosine of most CpG’s is methylated in

the genome, but methylation is rare in certain regions.

Mosaic methylation is a characteristic feature of vertebrate

genomes.

There are regions in normal somatic cell DNA where

non-methylated CpGs are significantly clustered, called

CpG islands (Bird, 1986; Cross and Bird, 1995). A CpG

island is about 1–2 kilobase (kb) in size, and frequently

associated with 5’ region of many genes including

promoter elements. Although methylation often results in

gene silencing, CpG islands are not methylated in all

somatic tissues, even where associated genes are not

expressed.

A small fraction of CpG islands are methylated in

normal somatic cell DNA. So far as is known, CpG

islands on inactive X chromosome and those associated

with imprinted genes are methylated in normal DNA,

genes associated with these CpG islands are

transcriptionally silenced. Some tumor suppressor genes

are known to be inactivated by CpG island methylation

(for a recent review, see Baylin et al, 1998).

Denaturing gradient gel electrophoresis (DGGE) takes

advantage of the change in electrophoretic mobility of

DNA fragments accompanying partial melting, and

permits sequence-determined separation of DNA fragments

(Fischer and Lerman, 1979). The electrophoretic mobility

of partly melted DNA fragments, consisting of both


helical and dissociated portions, in polyacrylamide gel is

much lower than that of fully helical or fully dissociated

molecules, and the transition in each fragment to low

residual mobility results in stable band pattern. The

position corresponding to this transition is called the

retardation level. In some cases, two DNA fragments

which differ by only one base pair have different melting

temperature, and retard in a gradient gel at different levels.

DGGE is useful for detection of point mutations (Fischer

and Lerman, 1983).

DNA methylation affects Tm, and correspondingly

the retardation level, when it occurs in the domain having

the lowest Tm. Difference in methylation at one base, that

is, non-methylation, hemimethylation, or symmetrical

methylation, can be resolved by DGGE (Collins and

Myers, 1987). We have found that DGGE can be applied

to the study of DNA methylation associated with CpG

islands (Shiraishi et al, 1995), although DGGE itself does

not here depend on methylation. In this article, we describe

how DNA fragments associated with CpG islands are

isolated on the basis of reduced rate of strand dissociation

and its application to the isolation of methylated CpG

islands, with emphasis on methodology.

II. Segregation of partly melted

molecules

A. Background

DGGE is a technique to separate DNA fragments on

the basis of local variation in base composition within the

DNA fragments (Lerman et al, 1984). It is effective only

when the molecule is comprised of domains having

different melting temperatures. The Tm of the lowest

melting domain of sufficient length determines retardation

Shiraishi et al: Segregation in human cancer cells

40

level at which the mobility decreases abruptly in the gel,

while the residual regions determine the stability of the

partly melted structure. The separation depends on the

markedly reduced electrophoretic mobility, which occurs

when a part of DNA fragment melts, resulting in a

structure that is partly helical and partly random chain. On

prolonged field exposure, the retarded fragments will fade

away through strand dissociation. If the stability of the

helical part is appreciably higher than that of the melted

part, the dissociation rate will be low and the retarded

partly melted molecule remains in the gel for some hours.

It would be reasonable to speculate that DNA fragments

derived from the edges of CpG islands consist of at least

two different melting domains, because the G+C-rich

nature of CpG island sequence results in high Tm, while

flanking non-island sequences are not G+C-rich and would

be lower melting. Thus, preferential retention of DNA

fragments derived from the edges of CpG islands after

prolonged field exposure is strongly expected (Figure 1).

That DGGE can be used for the isolation of DNA

fragments associated with CpG islands was implicit when

Fischer and Lerman described detection of point mutations

by DGGE showing that it depends on partial melting

(Fischer and Lerman, 1983). Later on, Myers et al

proposed and showed that addition of 300-bp G+C-rich

sequence (GC-clamp) to DNA fragments lacking a high

Tm domain would ensure partial melting (Myers et al,

1985a, b). Sheffield et al also showed that addition of short

(40–45 bp) GC-clamp was sufficient to protect the DNA

fragment from strand dissociation and permit retention in a

gel (Sheffield et al, 1989). It can be expected that CpG

island sequences would serve as “natural” GC-clamps, as

demonstrated by attachment of one to the mouse ! major

globin promoter (Myers et al, 1985a, b).

Figure 1. Schematic model of separation. The polyacrylamide gel contains a linear gradient of chemical denaturant (urea and

formamide), low at the top and high at the bottom. Red and blue bars indicate DNA fragments having a G+C-rich region and those

without a G+C-rich region, respectively. Once part of a DNA molecule melts, pronounced drop in electrophoretic mobility occurs, and

low residual mobility restricts migration into more strongly denaturing regions. If the helical portion is stable enough, that fragment

persists in the gel, while all others become dissociated and run out of the gel.


B. Preferential isolation of DNA

fragments associated with CpG islands

Long cloned DNA fragments have to be fragmented

appropriately in order for island fragments to be enriched

by DGGE. It was shown that digestion of DNA fragments

with four restriction endonucleases, Tsp509 I (AATT),

Mse I (TTAA), Nla III (CATG), and Bfa I (CTAG),

yields DNA fragments of appropriate size for DGGE

(Shiraishi et al, 1995). Since occurrence of these sites are

rare in CpG islands but abundant in the remaining bulk

genomic DNA, this treatment keeps the integrity of CpG

island relatively intact, while the rest generally undergoes

severe fragmentation.

When cloned DNA molecules containing entire

region of some known genes with CpG island were

digested with four restriction endonucleases and subjected

to DGGE, DNA fragments associated with CpG islands

formed stable bands which persisted through continued

application of the field, but others did not (Shiraishi et al,

1995). DNA fragments that persisted in the gel were

recovered from all CpG islands that were analyzed.

Cosmid clones randomly selected from a human

genomic library were analyzed similarly (Figure 2).

Digestion yielded hundreds of fragments. When the digests

are subjected to DGGE, only a few selected fragments,

three per cosmid clone on the average, were retained in the

gel after 11 hours run. Nucleotide sequence analysis

revealed that about half of the retained fragments were

considered to be derived from CpG islands (Shiraishi et al,

1995). Thus, the method, named segregation of partly

melted molecules (SPM), provides a useful means to

isolate DNA fragments associated with CpG islands

(Shiraishi et al, 1995). A representative result is shown in

Gene Therapy and Molecular Biology Vol 5, page 41

41

Figure 3. A retained fragment, R16-2, contained the

sequence identical to the 5’-end of the prostacyclin

synthase cDNA and a putative promoter region (Shiraishi

and Sekiya, 1996). It is well known that the isolation of

5’ upstream region of a gene is notoriously difficult by the

conventional cDNA synthesis approach. Therefore, SPM

is also useful for isolation of promoter regions.

The melting map of the fragment, R16-2, is shown

in Figure 3. A region of high Tm is identified in about

position 400 to the end. It is expected that this G+C-rich

region is thermally stable and serves to tether the partly

melted molecule. This region is also inferred to be the 5’

edge of the island, since there is a clear boundary of CpG

distribution at about position 350.

C. Application to gene hunting

Since 56% of human genes are reported to be

associated with CpG islands (Larsen et al, 1992), detection

of CpG islands in unsequenced DNA fragments can provide

markers for unidentified genes. When P1 artificial

chromosome clones covering a 400-kb region of human

chromosomal region 11q13, a well known region enriched

with CpG islands (Craig and Bickmore, 1994), were

subjected to SPM analysis (Figure 4), the expected

numbers of CpG islands were isolated (Shiraishi et al,

1998). This result suggested that SPM is an efficient

method for isolation of bits of gene sequences from long

unsequenced DNA fragments.

The isolation of DNA fragments associated with

CpG islands by means of DGGE stands on the different

basis from that of current practice that makes use of

Figure 2. SPM analysis of cosmid clones. Cosmid clones were serially digested with four restriction endonucleases, Tsp509 I,

Mse I, Nla III, and Bfa I. Conventional polyacrylamide gel electrophoresis shows that numerous fragments were yielded after

digestion. After DGGE, only limited number of fragments was retained in the gel (Shiraishi et al, 1995). R16-2 is a fragment whose

profiles are shown in Figure 3 (Shiraishi and Sekiya, 1996).


The melting map of the fragment, R16-2, is shown

in Figure 3. A region of high Tm is identified in about

position 400 to the end. It is expected that this G+C-rich

region is thermally stable and serves to tether the partly

melted molecule. This region is also inferred to be the 5’

edge of the island, since there is a clear boundary of CpG

distribution at about position 350.

C. Application to gene hunting

Since 56% of human genes are reported to be

associated with CpG islands (Larsen et al, 1992), detection

of CpG islands in unsequenced DNA fragments can provide

markers for unidentified genes. When P1 artificial

chromosome clones covering a 400-kb region of human

chromosomal region 11q13, a well known region enriched

with CpG islands (Craig and Bickmore, 1994), were

subjected to SPM analysis (Figure 4), the expected

numbers of CpG islands were isolated (Shiraishi et al,

1998). This result suggested that SPM is an efficient

method for isolation of bits of gene sequences from long

unsequenced DNA fragments.

The isolation of DNA fragments associated with

CpG islands by means of DGGE stands on the different

basis from that of current practice that makes use of

clustering or presence of restriction sites characteristic for

CpG islands, some of which are BssH II (GCGCGC),

Eag I (CGGCCG), and Sac II (CCGCGG) (Lindsay and

Bird, 1987; Bickmore and Bird, 1992; Valdes et al, 1994).

The SPM method would be advantageous when unbiased

isolation of DNA fragments associated with CpG islands

is attempted since these restriction sites are not always

present in all CpG islands.

D. Rationalization

The system depends on both extensive digestion of

DNA fragments with restriction endonucleases and relative

rate of dissociation of partly melted molecules, which are

locally G+C-rich during DGGE. However, theory for

dissociation rates at temperature lower than that sufficient

for full dissociation is not established. Instead, a heuristic

approach was adopted to explain the molecular basis for

retention and examine the generality of the principle.

RHST is an index, which attempts to relate the rate of

dissociation to the length and sequence of the unmelted

portion of the molecule, where the index is the reciprocal

of the estimated relative rate (Shiraishi et al, 1995, 1998).

RHST would be large for retained fragments and small for

disappearing fragments.

{ ( ) "1}

RHST = # exp [ Tm(i) " Tret]/

q

where Tm is the calculated temperature for each pair at

which the unimolecular probability of helicity of each pair

in the stable segment falls to a chosen threshold. Tret

represents the equivalent temperature at the gradient level

of the retarded

Shiraishi et al: Segregation in human cancer cells

42

Figure 3. The melting map of fragment R16-2 and

corresponding gene structure. The contour shows the midpoint

of the melting equilibrium at each base pair (Lerman and

Silverstein, 1987). Arrows down and up indicate CpG

dinucleotides and GC-box sequences (GGGCGG or CCGCCC),

respectively.

Figure 5. Separation of methylated and non-methylated

CpG islands in genomic DNA. DNAs from male and female

tissues were digested with Tsp509 I, subjected to an MBD

column, and eluted with a stepwise gradient of salt

concentration as described (Shiraishi et al, 1999a). DNA from

each fraction was subjected to PCR-based detection of

fragments containing the CpG island of the human HPRT

gene.

fragment; q is an arbitrary constant that is uniform for all

fragments. The summation extends only over pairs

calculated by MELT (Lerman and Silverstein, 1987) to be

helical at the retardation level. It was shown that RHST

values could discriminate retention and non-retention when

the followings were assumed; Tm taken to be equivalent

(bath temperature + denaturant) gel temperature at which

melted domain is approximately 75% helical, the mobility

in the gel is reduced to about 28% of initial velocity,

equivalent to 95-bp melting, and q value of 1.546

(Shiraishi et al, 1998). All retained fragments showed

RHST values not less than 3.56x10 3 , while non-retained

ones showed RHST values not greater than 3.19x10 3 .

Fragments more or less uniformly dense in G+C are

not retained in the gel since they are already at the edge of

dissociation as partial melting begins. Their RHST values


are small. Both experimental results and calculation

showed that not the entire island but rather the edge of the

island can be generally isolated by the SPM method.

High RHST values are not only found for CpG island

fragments; some DNA fragments containing repetitive

sequences also have high RHST values. Alu repetitive

sequences are CpG-rich and contain poly(A) stretches (for a

recent review, see Schmid, 1996), and can have high

RHST values. When P1 clones from human chromosomal

region 11q13 was analyzed by SPM method, about 40% of

retained fragments contained Alu sequences, and RHST

values of these fragments were high (Shiraishi et al,

1998). These results reflects the observation that both

CpG islands and Alu sequences are enriched in R band

regions of chromosomes (Korenberg and Rykaowski,

1988; Craig and Bickmore, 1994), and strongly suggest

that RHST is a good indicator of retention.

III. Isolation of methylated CpG

islands in human cancer cells

A. Methods for analysis of DNA

methylation

It is well known that cancer is caused by

accumulation of genetic and epigenetic aberrations.

Epigenetic aberrations are those that can not be explained

by alteration of nucleotide sequence, and CpG island

methylation is representative. Epigenetic process in

carcinogenesis is less well understood compared with

genetic process, partly due to limitation of available

methods. Before describing application of SPM to the

isolation of methylated CpG islands, we briefly summarize

methods for analysis of DNA methylation in order to

know their features.

1. Sensitivity to digestion with some

restriction endonucleases

Until the appearance of bisulfite modification method

described in the next section, sensitivity to digestion with

some restriction endonucleases, first described in 1978

(Bird and Southern, 1978), was practically the only method

to analyze methylation status of a complex genome. The

most frequently used combination of restriction

endonucleases is that of Msp I and Hpa II. Both

endonucleases recognize CCGG sequence and cleave DNA

at that site. However, when the internal C is methylated,

the modified sequence becomes resistant to cleavage by

Hpa II, but not by Msp I. Subsequent Southern

hybridization or PCR permits discrimination of

methylation and non-methylation as presence or absence of

appropriate fragments. Restriction landmark genomic

scanning (RLGS) is a method involving two-dimensional

gel electrophoresis that permits genomic scan on the basis

of methylation status of Not I sites (Hatada et al, 1991).

The results of methods using restriction endonucleases

are strongly affected by incomplete digestion, which often

results in overestimation of methylation, especially in

PCR-based experiments. Moreover, the analysis is

restricted to methylation status of specific restriction sites,

which are not always a representative of the target region,

Gene Therapy and Molecular Biology Vol 5, page 43

43

and that non-methylation is negatively displayed as the

absence of a signal.

2. Modification by chemical reagents

Although some chemical reactions permit

discrimination of cytosine and methylcytosine (for recent

reviews, see Rein et al, 1998; Thomassin et al, 1999;

Oakeley 1999), their application to the analysis of

genomic methylation status was very limited due to

complexity of mammalian genomes.

The pyrimidine ring of cytosine is cleaved by

hydrazine treatment, but when 5-position of cytosine is

methylated, the ring is not cleaved. These properties were

applied to determine methylation status of the mouse IgM

heavy chain constant region gene Cµ in combination with

Southern hybridization (Church and Gilbert, 1984).

However, in addition to technical difficulties, indication of

the presence of methylcytosine as the absence of signal

makes interpretation difficult.

A major breakthrough was brought to analysis of

DNA methylation when chemical modification of cytosine

by sodium bisulfite, conversion of cytosine to uracil

(Hayatsu et al, 1970; Shapiro et al, 1970), was combined

with PCR to permit positive display of 5-methylcytosine

in individual DNA strand (Frommer et al, 1992).

Treatment of single-stranded DNA with sodium bisulfite,

followed by hydrolysis, converts nonmethylated cytosine

into uracil, while methylated cytosine remains intact.

Since adenine makes a base pair with uracil, subsequent

PCR converts original nonmethylated cytosines to

thymine. Consequently a non-methylated C-G pair

becomes converted to a T-A pair, while a methylated C-G

pair remains as a C-G pair. This reaction can be applied to

the analysis of any CpG sequence, and permits positive

display of methylcytosine.

Bisulfite modification method has now become

popular, and unraveled previously unknown features of

sequence. One of the unexpected findings revealed by

bisulfite modification method is that methylated CpG

islands, such as those on inactive X chromosomes, are not

uniformly or densely methylated, contrary to previous

thought. Hornstra and Yang reported that the CpG island

associated with the human HPRT gene on an inactive X

chromosome is not uniformly methylated (Hornstra and

Yang, 1994). CpG sites in the GC-box region were

methylation-free even in the CpG island on inactive X

chromosome. Although the interpretation of this finding

awaits further experiments, we are now closer to the

precise nature of methylation.

3. Methylated DNA binding column

chromatography

An approach that stands on a different principle in

terms of discrimination of methylation and nonmethylation

was developed recently. Rat nuclear protein

MeCP2 (Lewis et al, 1992) binds DNA at a mCpG site,

but not at a CpG site (Meehan et al, 1992) The DNA

binding domain of this protein is comprised of 85 amino

acids (Nan et al, 1993). A methylated DNA binding

column (MBD column) is an affinity matrix that contains


a polypeptide derived from the methyl-CpG binding

domain (Cross et al, 1994). Since the stoichiometry of

binding is one protein to one mCpG (Nan et al, 1993), it

is expected that highly methylated DNA fragments bind to

the column tightly, while poorly methylated DNA

fragments bind only weakly.

By MBD column chromatography, DNA fragments

with the same nucleotide sequence but different

methylation status can be separated (Cross et al, 1994;

Shiraishi et al, 1999a). A representative result of MBD

column chromatography experiment is shown in

Figure 5. The human HPRT gene is an X-liked gene

having a CpG island, and one allele is inactivated in

female. There is only one non-methylated HPRT-CpG

island in male DNA, while there are two HPRT-CpG

islands in female DNA, one is methylated and the other is

not methylated. Most part of the island is contained in a

0.9-kb Tsp509 I fragment and there are 86 CpG residues

in it (data not shown). When Tsp509 I digests of male

DNA was analyzed by MBD column chromatography,

DNA fragments containing the CpG island were detected

only in low salt fraction (fractions around number 16). In

contrast, corresponding fragment of female DNA were

detected both in lower (fractions around number 16) and

higher (fractions around 36) salt fractions. These results

show that DNA fragments from methylated CpG islands

and those from non-methylated CpG islands can be

separated by MBD column chromatography.

Not only number of mCpGs but also mCpG density

seems to be a factor that affects affinity (Shiraishi et al,

1999b; Brock et al, 1999), although possibility of

sequence-specific preferential binding can not be excluded.

Clearly this method is insensitive to discriminate

heterogeneity in methylation within the same DNA

fragment and small change in total number of mCpGs.

The nature of this method, which is not destructive and not

influenced by methylation status of specific restriction

sites, is advantageous for comprehensive isolation of

methylated CpG islands.

B. Isolation of methylated CpG islands

Isolation of CpG methylated islands in cancer cells

has been drawing growing interest since it provides

valuable information on cancer epigenetics, which is very

limited now. Several methods for this purpose have been

reported; arbitrary primed PCR method (Gonzalgo et al,

1997; Huang et al, 1997; Gonzalgo and Jones, 1998),

subtraction (Ushijima et al, 1997; Huang et al, 1999,

Toyota et al, 1999), and RLGS (Costello et al, 2000) are

those primarily dependent on the methylation status of

specific restriction sites.

In contrast, MBD column chromatography permits

separation of methylated DNA fragment independent of

methylation status of any internal restriction sites, and

seems to be excellent for unbiased, comprehensive

isolation of methylated CpG islands. Using MBD column

chromatography, highly methylated DNA fragments in

human adenocarcinomas of the lung was enriched and then

cloned. By SPM analysis of the clones, DNA fragments

Shiraishi et al: Segregation in human cancer cells

44

associated with CpG islands methylated in cancer were

isolated (Shiraishi et al, 1999a). Many CpG islands thus

obtained from cancer were also methylated in noncancerous

portion of the lung, possibly only in one allele. These

results suggest that number of CpG islands specifically

methylated in cancer is lower than that of normally

differentially methylated ones.

IV Perspective

In this article, we introduced approaches to study

CpG island and DNA methylation standing on novel

principles; these may play an important role in cancer

research (Terada, 1999). There are many issues yet to be

clarified in the field of DNA methylation, such as

molecular mechanism of gene silencing by methylation

and comprehensive identification of genes that are

inactivated by methylation and involved in carcinogenesis.

Development of new experimental techniques and their

application will be a key to solve these problems.

Acknowledgments

We thank the Ministry of Education, Science,

Sports, and Culture and the Ministry of Health and Welfare

of Japan for research support.

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46


Gene Therapy and Molecular Biology Vol 5, page 47

47

Gene Ther Mol Biol Vol 5, 47-53, 2000

PNA (peptide nucleic acid) anti-gene/antisense can

access intact viable cells and downregulate target

genes

Review Article

Lidia C Boffa*, Elisabetta M.Carpaneto, Benedetta Granelli, Maria R. Mariani

National Cancer Institute, IST, L.go R. Benzi 10, 16132 Genoa, Italy

_____________________________________________________________________________________

* Correspondence: Lidia Boffa, National Cancer Institute, IST, L.go R. Benzi 10, 16132 Genoa, Italy, Tel: -39-010-5600214; Fax:

-39-010-5600217; E-mail: boffa@hp380.ist.unige.it

Key words: Peptide Nucleic Acid (PNA), anti-gene, antisense, cell /nuclear localization vectors, gene downregulation

Received: 27 June 2000; accepted: July 2000

Summary

In this paper we summarize our recent data on the anti-gene properties of PNA constructs both with a

classical Nuclear Localization Signal peptide, that appears to be effective in all the cell lines tested,

and with other vectors designed to be specific for cells carrying their receptors on the nuclear

membrane. We discuss the cellular localization pattern of PNA constructs, the consequent regulatory

effects on the target gene and their influence in the cellular metabolism. The data are discussed from

the perspective of the very recent literature on the access of PNAs antisense/anti-gene in intact viable

cells and their consequent regulatory effect in the form of: (i) PNA linked to cellular/nuclear peptidic

localization vectors; (ii) PNA linked to non peptidic vectors; (iii) unmodified PNA.

I. Introduction

Peptide Nucleic Acids (PNA) are a recent

development in the field of oligonucleotides analogues.

PNAs were originally conceived as oligonucleotide

homologues that could be used in the sequence-specific

targeting of double-stranded DNA (Nielsen et al, 1991;

Egholm et al, 1992). They are constructed in such a way

to have a neutral charge, achiral, pseudo peptide backbone

of N-(2-aminoethyl) glycine polymer. Each unit is linked

to a purine or pyrimidine base to create the specific

sequence required for hybridization to the targeted

polynucleotide. Therefore PNA is chemically more closely

related to peptides and proteins than to nucleic acids.

In vitro PNA/DNA and PNA/RNA duplexes are, in

general, thermally more stable than the corresponding

DNA/DNA or RNA/RNA duplexes and PNA2/DNA

triplexes formed between homopyrimidine PNAs and

sequence complementary homopurine DNA show even

higher stability (Egholm et al, 1993; Nielsen et al, 1994).

Although PNAs may bind complementary oligonucleotide

or PNA targets in both orientations (parallel and

antiparallel) with significant efficiency, the most stable

duplexes are formed with an antiparallel Watson–Crick

orientation (the PNA N-terminus facing the 3' end of the

oligonucleotide) and, in case of triplex helix formation, the

Hoogsteen PNA strand should be parallel to the DNA or

RNA target. Structural information on the four possible

PNA complexes has been obtained by NMR spectroscopy

for PNA/RNA (Brown et al, 1994) and for PNA/DNA

duplexes (Eriksson and Nielsen, 1996) and by X-ray

crystallography for PNA2/DNA triplex (Betts et al, 1995)

and for PNA/PNA duplex (Rasmussen et al, 1997).

The overall conclusion from these studies is that the

rather flexible PNA oligomer is able to a large extent to

adapt its conformation to its rigid complementary

oligonucleotide. In terms of sugar conformation, in

PNA/RNA duplexes the RNA strand is basically A-form

and in PNA/DNA duplexes the DNA strand is close to Bform.

The advantages of using PNA oligomers over the

conventional antisense oligonucleotides, that have been

used for some time to try to downregulate target gene

expression, are numerous partially due to the high

flexibility and absence of charge of the artificial backbone:

they are resistant to nucleases and proteases (Demidov et

al, 1994) and consequently have a longer life span in the

cellular environment than any other oligonucleotide; they

can invade duplex DNA and hybridize with complementary

sequences with such a superior thermal stability (Giesen et

al, 1998), resulting from the decrease in electrostatic

repulsion, so that they can successfully compete and

eventually displace the natural complementary strand; they


Boffa et al: A new and innovative approach to the in “vivo” antisense and anti-gene therapy

have a higher mismatch discrimination therefore forming

strong, selective duplexes upon binding to complementary

DNA or RNA sequences (Almarsson et al, 1993; Egholm

et al, 1993). In cell chromatin the PNA/DNA hydrogenbonded

double is further stabilized by three factors: in the

cellular environment PNA/DNA hybrids are more stable

than their homologues DNA/DNA since they are ionic

strength independent (Jensen et al, 1997); PNA/DNA

binding is more tight if DNA is supercoiled than if it is

linear; PNA/DNA hybridization is favored in

transcriptionally active, open chromatin (Bentin and

Nielsen, 1996; Boffa et al, 1997). The greater stability of

PNA hybrids with the complementary DNA of

transcriptionally active chromatin was used as a tool for

selection and characterization of active chromatin fragment

as large as 23 kb (Boffa et al, 1995).

Experiments with permeabilized cells and isolated

nuclei (Boffa et al, 1996; Boffa et al, 1997) have shown

that complex sequence PNAs are highly effective in

blocking transcription of the targeted gene without

inhibiting RNA synthesis in unrelated genes.

Unfortunately at 37ºC PNAs easily enter the cells by

endocytosis but are readily sequestered by cytoplasmic

vesicles (endosomes and lysosomes) before they can enter

the nucleus (Bonham et al, 1995).

II. Review

A. PNA linked to cellular/nuclear

peptidic localization vectors

Cellular and nuclear localization signal peptides have

successfully been used for some time to carry bulky

uncharged molecules into live cells. Several recent reports

have demonstrated that PNAs conjugated to such vectors

are quite efficiently taken up by some eukaryotic cells.

1. SV 40 Nuclear Localization Signal

peptide (NLS)

PKKKRKV is a basic Nuclear Localization Signal

peptide (NLS) that was shown first to mediate the transfer

of SV40 large T antigen across the nuclear membrane

(Kalderon et al, 1984) and later to facilitate the nuclear

delivery of large proteins (Gorlich and Mattaj, 1996).

Our Laboratory first described the construct of this

NLS with a PNA (Boffa et al, 1997) and observed that it

was capable of remarkably decreasing the time of access of

PNA to nuclei of permeabilized cells in vitro without

altering their anti-gene effects. Permeabilized cells were

exposed for increasing time to a 17mer anti-gene PNA (+/-

NLS) complementary to a unique sequence at the

beginning of the second exon of c-myc oncogene. In

human adenocarcinoma derived cell line (COLO320-DM)

at short time of exposure, we described a selective c-myc

transcriptional inhibition that was significantly higher

48

upon exposure to the anti-gene PNA-NLS than to the

unmodified matching PNA (Figure 1).

In particular, all cells were subjected to run on

transcription assay in the presence of [!- 32 P]UTP. Total

RNA was purified and the newly synthesized, radioactively

labeled mRNA was analyzed by hybridization for its

content in specific sequences, located not only at the

PNA/DNA binding site but also upstream and downstream

from it, that were previously blotted on an appropriate

membrane. We showed that the PNA binding to its target

sequence in the c-myc gene strongly inhibited the sense

transcription of 4 sequences downstream from the

PNA/DNA hybridization site and that the extent of this

inhibition depended on the distance of the sequences from

the PNA block.

Recently (Cutrona et al, 2000) we have described the

anti-gene effect of the above described construct in live

cultured cells. When Burkitt’s lymphoma derived cell lines

(BL) were exposed to the c-myc anti-gene PNA-NLS this

molecule was localized predominantly in the cell nuclei.

The PNA nuclear localization was not only due to the

basic nature of the peptide, but also to the specific amino

acids sequence. In particular previous studies have

determined that the nuclear localization function of the

peptide was strictly dependent on the presence of lysine as

the third amino acid (termed Lys 128 , as from the original

sequence of SV40 NLS) (Colledge et al, 1986). In order to

demonstrate that only the correct original NLS sequence

can specifically confer a cellular/nuclear localization to the

bound PNA, we designed a scrambled NLS (KKVKPKR)

mutated at the third amino acid to be used as a negative

control (NLS scr). BL cells were exposed in culture to PNAmyc

+/-NLS or NLS scr tagged with a fluorophore

Rhodamine (Rho) ( Rho-PNA-myc, Rho-PNA-myc-NLS

or Rho-PNA-myc-NLS scr) and analyzed by confocal

microscopy. Maximum cellular fluorescence intensity was

obtained at 24 h (Figure 2). Rho-PNA-myc (a) and Rho-

PNA-myc-NLS scr (b) were localized in the cytoplasm,

while Rho-PNA-myc-NLS (c) was clearly detectable in the

cell nuclei.

The PNA myc-NLS access to the cell nuclei caused a

rapid consequent downregulation of c-myc transcription as

from the time course of MYC expression determined by

Western blot and Northern blot analysis of c-myc mRNA

(Figure 3).

Figure 1. c-myc and its specific anti-gene PNA


Gene Therapy and Molecular Biology Vol 5, page 49

Figure 2 Delivery of PNA-NLS to intact nuclei of BL cells. BL cells were exposed to Rho- PNA-myc (a), to Rho- PNA-myc-NLS (b) or

to Rho- PNA-myc-NLS scr (c) and analyzed by confocal microscopy. The pictures show the section crossing the middle of the nuclei with

the phase contrast and fluorescence images superimposed.

Figure 2. Inhibition of c-myc expression by PNA-myc-NLS.

a. MYC expression determined by Western blot following exposure of BL cells to the indicated PNAs for 18h.

b. Northern blot analysis of c-myc mRNA expression in BL cells exposed to the indicated PNAs for 18h.

PNA-myc-NLS-treated BL cells displayed a largely

impaired growth capacity and a decrease in 3 H-thymidine

incorporation compared to those treated with control PNAmyc

(not shown). Concomitantly, there was a substantial

reduction in the proportion of cells in the S or G 2M phases

of the cell cycle as determined after 36 h following PNA

treatment (Figure 4a). The presence of PNA-myc-NLS

caused a decrease in viability of the cells in culture that

became particularly evident by 72 h. Cell death, however,

was unrelated to apoptosis that was not above the control

values, as measured by Annexin-V staining (Figure 4b).

A PNA-NLS construct was also shown to increase

the transfection efficacy of plasmids containing the PNA

complementary sequences (Branden et al, 1999).

2. Antennapedia peptide

A 16-amino acid peptide corresponding to the third

helix of DNA binding domain of Antennapedia was shown

49

to be able to translocate bulky or uncharged molecules

through biological membranes and such internalization

does not depend on classical endocytosis (Derossi et al,

1996).

This peptide coupled to PNA (Simmons et al, 1997)

was shown to confer permeability through cellular

membranes. In particular the intracellular delivery property

of this peptide was verified when conjugate with few

different PNAs:

i) antisense PNAs targeting the galanin receptor

were shown to downregulate its expression by receptor

activity assays and by Western blot (Pooga et al, 1998) in

Bowers cells. Furthermore intrathecal injection of such a

PNA conjugate into the brain of living rats reduced

receptor activity in the brain, implicating in vivo antisense

effects. Also, the behavioral response of the rats was

compatible with a decreased galanin receptor level;


Boffa et al: A new and innovative approach to the in “vivo” antisense and anti-gene therapy

Figure 4 . PNA-myc-NLS effects on completion of a

productive cell cycle and apoptosis

a Flow-cytometric analysis of the cell cycle upon 36-h

exposure to the indicated PNAs.

b. BL cells were incubated with the indicated PNAs for

different times. Apoptotic cells and dead cells were detected by

Annexin-V binding and PI incorporation.

ii) antisense PNA to the prepro-oxytocin mRNA

selectively and significantly decreased mRNA and protein

product in neuronal cells in culture (Herrada et al, 1998);

50

Figure 5 Chemical structure of PNA-dihydrotestoterone

construct. Dihydrotestoterone was bound through a flexible

linker to the N terminal position of PNA in order to confer the

construct a better accessibility to nucleic acid in chromatin.

iii) two antisense PNAs to telomerase mRNA were

selectively effective in downregulating the telomerase

activity in human melanoma cells (Villa et al, 2000).

In all cases the antisense was not only efficiently

delivered to the cells but eventually migrates into the

nuclei.

3. D-peptide analog of insulin growth factor

(IGF1)

IGF1 PNA conjugates displayed a much higher

cellular uptake than unmodified PNAs but the uptake was

in correlation with the level of expression of the IGF1

gene in the cells. In fact, in Jurkat cells that do not express

the gene there is no PNA uptake, while in p6 cells, where

the gene expression is high, a relevant uptake is detectable

(Basu and Wickstrom, 1997) suggesting for the first time a

possible cell-specific, tissue specific application of PNAs

as gene-regulatory agents in vivo.

4. Hydrophobic tetrapeptide (FLFL)

FLFL linked to PNA caused not only PNA

internalization, but also remarkable stability of the

complex in the cellular environment in Namalwa cells

(Scarfì et al, 1997). This peptide could also internalize

inducible Nitric Oxide synthase (iNOs) cDNA

complementary DNA and significantly reduce the level of

the enzyme in Macrophages in culture (Scarfì et al, 1999).

B. PNA linked to non peptidic vectors

1. Dihydrotestosterone (T)

T covalently linked to PNA acts as a vector (Figure

5) for targeting a c-myc anti-gene PNA selectively to cell

nuclei of prostatic cancer LNCaP cells, which express

Androgen Receptor (AR) gene, but not to DU145 cells, in

which the AR gene is silent (Boffa et al, 2000).

T vector was covalently linked to the N-terminal

position of a PNA complementary to a unique sequence of

c-myc oncogene (PNAmyc-T). A fluorophore (Rho) was

also attached at the C-terminal position to localize the

vector-free PNA and the PNA myc-T conjugate (PNAmyc-

Rho, PNAmyc-T-Rho) within the cells. The cellular

uptake was


Gene Therapy and Molecular Biology Vol 5, page 51

Figure 6. Effect of the different specific PNA constructs on c-myc expression.

LNCaP and DU145 cells were treated for 24h with PNA-myc, PNA-myc-T, and PNA-myc-NLS. In this picture is shown a Western blot

analysis of MYC .These findings suggest a strategy for targeting of cell-specific anti-gene therapy in prostatic carcinoma.

monitored by confocal fluorescence microscopy. PNAmyc-

Rho was detected in the cytoplasm of both prostatic cell

lines, whereas PNAmyc-T-Rho was present only in nuclei

of LNCaP (AR+) cells. The effects of the complete set of

PNAs on expression of the c-myc gene in LNCaP and

DU145 cells was monitored by Western blots of the MYC

protein content of cell lysates: The results of a series of

these analyses proved that in LNCaP cells only PNAmyc-

T induced a significant and persistent decrease of MYC

expression (Figure 6).

2. OX26 murine monoclonal antibody to the

rat transferrin receptor

OX26 linked to the antisense PNA for the rev gene

mRNA of the human HIV1 virus, not only was able to

cross the blood-brain barrier (BBB) but also retained the

capacity to bind the target mRNA, if injected in rats

(Pardridge et al, 1995). This model system has recently

been proposed also in the treatment of Alzheimer’s disease

(Pardridge et al, 1998).

3. Spermine

Covalent conjugation of Spermine to PNAs was

shown to increase PNA solubility with consequent

increase in cellular accessibility and a 2-fold acceleration of

the rate of molecular association with complementary

DNA (Gangamani et al, 1997).

51

4. Adamantyl group

This group is probably the most innovative PNA

vector. Adamantyl is a lipophilic group that when

covalently attached to PNA (Ardhammar et al, 1999)

shows at least a 3-fold improvement in PNA cellular

uptake in a variety of cell lines in culture (Ljungstrom et

al, 1999).

C. Unmodified PNA

PNAs have been shown to enter to a small extent in

cultured cells, for example neurons, probably through a

mechanism of endocytosis (Aldrian-Herrada et al, 1998).

Antisense PNAs uptake by cultured human myoblasts was

shown to cause a specific inhibition of replication of

mutant mitochondrial DNA (Taylor et al, 1997). In the

literature there are recent reports of antisense effects of

unmodified PNAs uptake in live mice brain. The first

study (Tyler et al, 1998) was on a 14-mer PNA directed

against the neurotensin, NTR1, (position +103) and mu

opioid (position -70) receptors mRNAs. PNAs were

injected into the periaqueductal gray (PAG) of rats.

Neurotensin as well as opioids are well known to exert an

antinociceptive effect. In addition, neurotensin induces

hypothermia. Behavioral studies of anti-NTR1 or antiopioid

mu receptors PNA treated animals showed

dramatically reduced responses to neurotensin and

morphine, respectively. Furthermore, hypothermia induced


Boffa et al: A new and innovative approach to the in “vivo” antisense and anti-gene therapy

by neurotensin was substantially reduced. These effects

where reversible and specific. A similar pattern of results

was obtained in a subsequent study (Tyler et al, 1999) with

NTR1 antisense (injected intraperitoneally) and sense

(injected directly into the PAG of rats) PNA. The PNA

uptake into the brain was detect by a gel shift assay. A

50% decrease of NTR1 mRNA level in brain induced only

by the specific sense PNA, determined by quantitative

PCR, suggested an anti-gene mechanism.

Moreover a recent study described the non toxicity of

an antisense PNA targeted toward mRNA of the opioid

receptor gene injected repeatedly in mice (Fraser et al,

2000).

III. Conclusion

We hope to have been able to clearly illustrate the

recent body of evidence in support of the novel

anti-gene/antisense PNAs-cellular/nuclear vector constructs

capacity to access intact viable cells and consequently to

downregulate target genes. These new modified PNAs

could really provide an innovative and effective approach to

the in “vivo” antisense and anti-gene therapy.

Acknowledgments

We acknowledge the support of the Italian

Association for Cancer research (AIRC) and National

Research Council (CNR “Biotechnologies”).

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Boffa et al: A new and innovative approach to the in “vivo” antisense and anti-gene therapy

54


Gene Therapy and Molecular Biology Vol 5, page 55

55

Gene Ther Mol Biol Vol 5, 55-61, 2000

Delivery of plasmid DNA by in vivo electroporation

Review Article

Loree Heller 1* and M. Lee Lucas 2

1 Center for Molecular Delivery, University of South Florida, Tampa, FL 33612, 2 Department of Medical Microbiology,

University of South Florida, Tampa, FL 33612

__________________________________________________________________________________

* Correspondence: Loree Heller, University of South Florida, Center for Molecular Delivery, MDC16, 12901 Bruce B. Downs

Blvd., Tampa, FL 33612; Telephone: 813-974-8184; Fax: 813-974-2669; Email: lheller@com1.med.usf.edu

Key words: gene therapy, electroporation

Received: 13 June 2000; accepted: 13 July 2000

Summary

For gene therapy, in vivo delivery of plasmid DNA offers an alternative to viral delivery methods.

Since the efficiency of plasmid delivery to tissues is generally lower than viral delivery, several

methods have been introduced to augment in vivo plasmid delivery including liposome

conjugation, particle mediated delivery, and electroporation. In vivo electroporation has reached

clinical trials for the delivery of chemotherapeutic agents to cancers, and a number of preclinical

studies have been performed demonstrating that this technique also enhances plasmid delivery and

expression of both reporter and therapeutic genes or cDNAs. This delivery has been performed to a

number of tissues including skin, muscle, liver, testes and tumors employing a wide range of

electrical conditions and electrodes. While this preclinical research is promising, further

optimization of electrical conditions and electrodes may be necessary for clinical use. With the

availability of a variety of therapeutic gene delivery techniques, it will be possible to tailor gene

therapies to individual diseases.

I. Introduction

Gene therapy has the potential to play a role in the

effective treatment of a variety of diseases. Biological gene

therapy employs genetically engineered viruses to deliver

the desired gene or cDNA. Several types of viral vectors

including recombinant retroviruses, adenoviruses, and

adeno-associated viruses have been described or employed

in gene delivery (Kay, 1997). While gene therapy with

these vectors may result in high protein levels and long

term expression, short term, lower levels of expression of

molecules such as immune modifiers may also be

desirable. Ideally, multiple gene delivery techniques may

be necessary to fit multiple treatment regimens.

The delivery of plasmid DNA encoding the gene or

cDNA of interest may also be used for gene therapy.

Plasmid DNA is neither replicated nor integrated into the

host cell genome, but remains in its episomal form

(Nichols et al, 1995), and expression is generally short

term in tissues other than muscle. DNA injection does not

result in the production of anti-DNA antibodies (Jiao et al,

1992; Robertson, 1994), which allows for multiple

treatments. Since the efficiency of plasmid delivery is

lower than that of viral delivery, several methods have

been introduced to increase delivery in vivo, including

liposomes, microparticle bombardment and

electroporation.

During in vivo electroporation, electric pulses are

applied directly to the tissue to enhance uptake of

extracellular molecules (reviewed in Jaroszeski et al,

2000). The delivery of chemotherapeutic agents to tumors

by this method was first demonstrated in subcutaneously

injected hepatocellular carcinomas (Okino et al, 1987).

This technique, termed electrochemotherapy, results in

substantial, but localized tumor necrosis.

Electrochemotherapy is a highly effective anti-tumor

regimen and has been demonstrated preclinically in variety

of cutaneous and internal tumors, including rat (Jaroszeski

et al, 1997a) and rabbit (Ramirez et al, 1998)

hepatocellular carcinomas, Lewis lung carcinoma

(Kanesada, 1990), sarcomas and melanomas (Mir et al,

1991), mammary tumors (Belehradek et al, 1991), gliomas

(Salford et al, 1993), fibrosarcomas (Sersa et al, 1995), and

melanomas (Heller et al, 1995). This work was extended to

clinical trials in head and neck squamous cell carcinoma,

melanoma, basal cell carcinoma, Kaposi’s sarcoma, and

adenocarcinoma (Jaroszeski et al, 1997b; Heller et al,

1999). In these clinical trials, objective responses varied

from 72 to 100%.


Heller and Lucas: Delivery of plasmid DNA by in vivo electroporation

Table 1. Luciferase expression after muscle electroporation of plasmid DNA

Mir et al, 1999 Mathiesen et al, Vicat et al, 2000 Lucas et al,

1999

submitted

Pulse description Square LVLP Bipolar trains Square HVSP EEP

Electrode Caliper Wire pair Caliper Needle

µg plasmid 15 25 10 100

Muscle treated mouse tibial cranial

(transcutaneous)

Fold increase in

expression

Peak measured

expression

Observed length of

expression

rat soleus (surgically

exposed)

56

mouse tibialis anterior

(exposed)

mouse

gastrocnemius

(transcutaneous)

100 16 500 1000

Day 7 Day 3 Day 1 Day 2

9 months 3 days 6 months 7 days

LVLP, low voltage long pulses; HVSP, high voltage short pulses; EEP, exponentially enhanced pulses

II. Reporter gene delivery with in vivo

electroporation

Delivery of DNA to cells by the application of

electric pulses was first reported in 1982 (Wong and

Neumann, 1982). In vivo electroporation to enhance

plasmid delivery was later demonstrated in the skin cells of

newborn mice using exponential pulses with a clip

electrode (Titomirov et al, 1991). Optimal transformation

of skin cells was noted after two pulses in opposing

polarities of a field strength of 300-400 V/cm and a pulse

length of 0.1-0.3 ms. In experiments using three much

longer, higher amplitude exponential pulses and the

addition of pressure with a caliper electrode (1200V/cm,

10-20 ms), !-galactosidase expression was detected to a

depth of 370 µm in the skin of hairless mice after topical

administration of plasmid DNA (Zhang et al, 1996). More

recently, delivery to skin with square wave pulses after

intradermal injection has been demonstrated (Heller et al,

submitted). While plasmid delivery with caliper electrodes

and eight short (0.1 ms), high field strength (1500 V/cm)

pulses increased luciferase reporter expression10 fold in the

treated skin 24 hours after treatment, no increase was seen

using eight long (20 ms) lower field strength (100 V/cm)

pulses. Skin delivery of a plasmid encoding IL12 induced a

systemic response as well, in the form of increased serum

levels of interferon " . In these experiments, plasmid

delivery with a custom designed electrode induced higher

expression than delivery with simple calipers. These

results emphasize the importance of optimizing both

pulsing protocols and electrode configurations with respect

to tissue type to avoid damage and maximize protein

expression.

Electroporation enhances plasmid expression in other

tissues. Delivery to normal rat liver was first characterized

using six 0.1 ms pulses at a variety of field strengths

using a 6 needle array (Heller et al, 1996). Maximum

luciferase expression occurred at 1000 to 1500 V/cm,

electrical conditions very similar to those used for drug

delivery in clinical trials. 48 hours after treatment,

approximately 30% of cells in the electroporated area

expressed !-galactosidase and this expression was still

detectable 21 days later. The expression was dose

dependent, with peak expression occurring with delivery of

25 µg plasmid. GFP expression in normal liver from

delivery of plasmid DNA was examined using pulses that

were less intense but 500 fold longer (eight 50 ms at a

variety of field strengths) and a disk electrode (Suzuki et al,

1998). The highest expression was noted at 250 V/cm,

while the extreme damage noted at 500 V/cm probably

contributed to the lack of expression. This expression was

3.5 fold higher than with 50 ms pulses than with 25 or 99

ms pulses at this field strength. These investigators found

a DNA dose dependent expression to 80 µg plasmid DNA,

which may be due to the different electroporation

parameters used. Clearly, different pulsing parameters may

be used to effectively enhance plasmid DNA deliver to the

same tissue.

Delivery by lipid conjugation, microparticle

bombardment and electroporation using equal quantities of

plasmid DNA was directly compared in chicken embryos

(Muramatsu et al, 1997a). For liposome-mediated delivery,

DNA was complexed per manufacturer’s instructions

(Lipofectamine, Gibco BRL, USA) and injected. In

microparticle bombardment, DNA was bound to 0.9 mg

tungsten beads and delivered by nitrogen gas at 20 kfg/cm 2

at a distance of 6 cm. For electroporation delivery, tissue

received three 50 ms pulses at 31.25 V/cm with a plate

electrode after plasmid injection. No significant difference

was noted in embryo viability or number of embryos

positive for !-galactosidase staining 48 hours after

delivery, although electroporation appeared to transfect a

larger area with a greater intensity of expression.

Using CAT as a reporter, this group also compared

several electroporation conditions for optimization of DNA

delivery in surgically exposed mouse testes (Muramatsu et


Gene Therapy and Molecular Biology Vol 5, page 57

Table 2. Serum protein expression after muscle electroporation of plasmid DNA

Aihara et al, Rizzuto et al, 1999 Maruyama et al, Widera et al,

1998

2000

2000

Pulse description Square LVLP Bipolar trains Square LVLP Square LVLP

Electrode Needle pair Parallel wires Needle pair Needle pair

Muscle treated Mouse tibialis

anterior

(transcutaneous)

Fold increase in

expression

Peak measured

expression

Observed length of

expression

Mouse quadriceps

(exposed)

57

Rat thigh (exposed) Mouse tibialis

anterior

(transcutaneous)

119 >100 7 400

Day 5-7 Day 7 Day 7 Day 5

< 42 days 84 days >21 days 20 days

Reporter Interleukin-5 Erythropoietin Erythropoietin HBsAg

LVLP, low voltage long pulses; mice received 50 µg plasmid; rats received 400 µg plasmid

al, 1997b). In each case, eight low voltage, long pulses

were delivered. Tissue damage due to heat generation was

observed after severe conditions such as 100V for 50 ms,

so only conditions that caused minimal damage were

presented. The optimal conditions elucidated in this

experiment were 50 V for 10ms or 25 V for 50 ms.

Furthermore, CAT activity was examined after liposome,

microparticle bombardment, and electroporation deliveries

to mouse testes (Muramatsu et al, 1998). Microparticle

bombardment and electroporation both significantly

increased expression, while liposome delivery had no

significant effect on expression.

Much of the focus of in vivo electroporation has

been on muscle delivery. Mouse skeletal muscle injected

with plasmid DNA alone expressed reporter genes in both

dividing and non-dividing cells (Wolff et al, 1990). This

expression can be significantly augmented by the addition

of electric pulses (Aihara et al, 1998). In addition,

individual variability in transgene expression is reduced

after electroporation (Mir et al, 1999). The data from

several groups that have examined luciferase reporter

expression after muscle electroporation is summarized in

Table 1. Mir et al (1999) found maximum luciferase

expression after delivery of eight 20 ms pulses at 200

V/cm with a caliper electrode in mouse tibial cranial

muscles. This expression lasted 9 months. Electroporation

also enhanced luciferase plasmid DNA expression in

various mouse, rat, and rabbit muscles, and expression of

FGF1 was detected in muscle after plasmid electroporation

of mouse and monkey muscle. Similar long, low voltage

pulses (ten 40 ms) were compared to trains of bipolar

pulses (ten 1000 Hz trains of on thousand 0.4 ms pulses

each), both at a field strength of 100-180 V/cm

(Mathiesen, 1999). The pulsing protocols, which resulted

in the same total pulse time, induced similar levels of

expression. Although more muscle damage was observed

after the long pulses, the muscle cells had regenerated by 2

weeks after treatment.

While one group found a 50 fold decrease in

luciferase expression after plasmid delivery with eight 0.1

ms 1200 V/cm pulses (Mir et al, 1999), another found

increasing expression with increasing field strength with

0.1 ms pulses to an expression maximum at 1800 V/cm

(Vicat et al, 2000). This expression increased linearly

with DNA concentration up to 50 µg. This group also

reported more stable expression (>6 months) with shorter

pulses. This difference may be due to the more intense

immune infiltrate observed after longer pulses. A 20 fold

increase in expression after short, high voltage

electroporation delivery was confirmed using 0.1 ms

pulses at 1500 V/cm (Lucas et al, submitted), although in

these experiments, long, low voltage pulses or

exponentially enhanced pulses induced an even larger (1000

fold) increase in luciferase expression. Since these groups

compared similar pulsing parameters, other variables such

as the electrode used may be affecting the level of delivery.

Clearly, though, plasmid delivery to muscle is enhanced

by several different pulse types.

In vivo muscle electroporation also enhances

systemic expression of plasmid DNA. A comparison of

electroporation enhanced serum protein expression after in

vivo electroporation is summarized in Table 2. Since

several different serum reporter plasmids were used, the

increase in expression noted may not be directly

comparable. Electroporation significantly increased serum

expression of interleukin-5 after muscle injection (Aihara

et al, 1998). In this case, three 50 ms 100 V/cm square

wave pulses were delivered with a needle pair. Serum

expression of mouse erythropoietin was demonstrated after

electroporation of as little as 1 µg plasmid DNA into

muscle with ten 1 second pulse trains of on thousand

square bipolar pulses (0.2 ms each, 90 V/cm, Rizzuto et

al, 1999). Erythropoietin levels peaked at day 7 and were

elevated for at least 84 days. No muscle damage from

electroporation was detected histologically 24 hours after

treatment. At one week, areas of muscle fibers with central

nuclei and small necrotic areas with lympocyte


Heller and Lucas: Delivery of plasmid DNA by in vivo electroporation

infiltrations were observed, constituting up to 10-20% of

the total electroporated muscle. At one month after

treatment, muscles appeared normal. A plasmid encoding

rat erythropoietin was also delivered to rat muscle at 4

sites with 100 µg plasmid each with eight 50 ms 200

V/cm pulses delivered with a needle pair (Maruyama et al,

2000). In this case, serum erythropoietin expression

peaked at day 7 and continued for 32 days, with a

corresponding increase in hematocrit continuing at least to

day 32. Systemic expression of a hepatitis B surface

antigen was also observed after in vivo electroporation of

50 µg plasmid (Widera et al, 2000). After delivery of six

50 ms 100 V/cm pulses with a needle pair, antigen

expression peaked 5 days after treatment and continued at

least 20 days post injection in nude mice, while this

expression began to wane at day 13 in immunocompetent

mice, possibly due to a cellular immune response against

transfected muscle cells or clearance of antigen in antigenantibody

complexes. Electrically enhanced gene transfer to

muscle, which can result in both local and systemic

transgene expression, may prove an effective tool for

treating a variety of diseases.

A wide range of electrical conditions has also been

used to enhance plasmid delivery directly to several tumor

types. Increased plasmid encoded !-galactosidase

expression was first demonstrated after intra-arterial

injection of plasmid DNA, followed by eight 0.099 ms

600 V/cm electric pulses applied directly to rat brain

tumors (Nishi et al, 1996). Plasmid delivery by

hemagglutinating virus of Japan (HSV) liposomes,

microparticle bombardment and electroporation was

compared in rat bladder cancers (Harimoto et al, 1998). In

this experiment, eight 50 ms pulses at a field strength of

143-1000 V/cm were applied with a needle type electrode.

These researchers found that all three delivery methods may

be suitable for therapy of localized bladder tumors.

Expression of !-galactosidase was also detected in B16

mouse melanomas after intratumor injection of 12 µg

plasmid and application of ten 5 ms pulses at 800-900

V/cm with caliper electrodes (Rols et al, 1999). No gene

transfer was detected in these experiments with short (µs)

pulses. In contrast, 15% of B16 melanoma cells were

positive for !-galactosidase expression after intratumor

delivery of 100 µg plasmid with fourteen 0.1 ms pulses at

a field strength of 1500 V/cm (Niu et al, 1999). In these

experiments, pulses were delivered segmentally so that

each tissue area received only two pulses. The significant

difference between these two results may be due to different

electrode configurations or to the use of different reporter

plasmids. Using 0.1 ms pulses, a plasmid encoding

luciferase was delivered to rat liver tumors (Heller et al,

2000). Expression at 48 hours was highest at 2000 V/cm,

although this extreme field strength burned the tissue

immediately around the electrodes. Therefore, 1500 V/cm,

which caused no evidence of tissue damage, was used for

subsequent experiments. The level of luciferase expression

in response to electroporation increased as the amount of

plasmid increased up to 2 µg/mm 3 initial tumor volume.

Histochemical staining for !-galactosidase expression was

also enhanced by electroporation (Figure 1). In an

58

interesting set of experiments in mouse breast tumors, in

vivo electroporation of lipid complexed and naked DNA

was compared (Wells et al, 2000). Using caliper electrodes

and six 1 ms pulses, luciferase expression was augmented

2 orders of magnitude maximally 48 hours after delivery at

1100 V/cm. No statistical difference in expression was

noted between electroporation of naked plasmid or

lipoplexes.

III. Preclinical gene therapy with in

vivo electroporation

Electroporation enhanced plasmid delivery to muscle

may induce a clinically relevant response. After delivery of

a plasmid encoding erythropoietin, the responding

hematocrit is significantly increased in mice up to 6

months (Rizzuto et al, 1999) after delivery of as little as 1

µg plasmid or in rats up to 32 days after delivery of 400

µg plasmid (Maruyama et al, 2000). An immune response

to viral proteins can also be induced after muscle

electroporation of plasmid DNA. In mice, strong antibody

responses to HbsAg and to HIV gag protein were detected

two weeks after delivery of 3 µg or 10 µg plasmid

respectively (Widera et al, 2000). In addition, four weeks

after plasmid delivery, an anti-gag T cell response was

observed after challenge with a vaccinia virus expressing

HIV gag. Although there is no consensus as to the “best”

muscle electroporation conditions, the experiments

described here demonstrate that electroporation of plasmid

DNA into muscle has potential as a gene therapy in a

clinical setting, enhancing both intramuscular and

systemic transgene expression.

Electroporation enhanced plasmid delivery directly to

tumors may also induce a clinically relevant response.

After delivery of a plasmid encoding human monocyte

chemoattractant protein-1 into rat brain tumors, large

numbers of macrophages and lymphocytes were observed

in the tumor tissues (Nishi et al, 1996). Electroporation

enhanced delivery of a plasmid encoding a dominant

negative Stat3 variant into B16 mouse melanomas

inhibited tumor growth and caused tumor regression

mediated by apoptosis (Niu et al, 1999). The combination

of electrochemotherapy and cytokine plasmid delivery by

electroporation into B16 melanomas prevents tumor

recurrence and induces long term antitumor immunity in

mice (Heller et al, submitted). After electroporation of

plasmids encoding diphtheria toxin or herpes simplex

thymidine kinase followed by gancyclovir administration,

the growth of subcutaneously inoculated colon

adenocarcinomas was significantly inhibited (Goto et al,

2000).

IV. Conclusions and clinical

considerations

While electroporation is highly effective at

enhancing tissue expression of transgenes, it will be

necessary to optimize two variables for plasmid DNA

delivery, quite possibly for each tissue or tumor type: the

pulsing conditions and the electrodes used for pulse

delivery.


Gene Therapy and Molecular Biology Vol 5, page 59

Figure 1. Histochemical staining for !-galactosidase expression in rat hepatocellular carcinomas (Heller et al, 2000). 15 µm

frozen sections were stained for expression 48 hours after delivery. (a) Injection only of 50 µg pSV-!gal (Promega, Madison, WI,

USA); (b) Injection of 50 µg pSV-!gal followed by two 100 µs pulses at 1250 V/cm per tissue area.

Several pulse shapes will enhance DNA delivery in

vivo, including exponential, square wave, and bipolar

trains. In each case, the total number, length, and field

strength of each pulse can be varied considerably. Effective

pulse lengths range from 0.1 to 50 ms, while effective

field strengths range from 200 to 1800 V/cm. The

potential for damage resulting from delivery, which may

result from irreversible electroporation or from heat

damage after long or high intensity pulses, must be

balanced against the increased DNA expression. This

damage may be much more important in plasmid delivery

to healthy tissues such as muscle than delivery directly to

tumor tissue.

The potential damage from electric pulses to rabbit

liver, pancreas, kidney or spleen after delivery of eight 0.1

ms pulses at a field strength of 850 V/cm was assessed

histologically (Ramirez et al, 1998). Immediately after

pulse application, the primary effect was edema formation.

At days 2 and 7 after pulse delivery, localized necrosis and

fibrosis was limited to the electrode contact sites. In depth

studies of the effect of six 0.1 ms 1300V/cm pulses on

skin, muscle, nerves, and blood vessels in the hind limbs

of rats were also performed (Richard Heller, personal

communication). Histological analysis of the tissues after

one to three treatments showed short term (3 days),

localized necrosis to skin and muscle that started to resolve

within 14 days and completely resolved by 56 days after

treatment. In addition, all animals regained full limb

function within 6 minutes after the therapy was

administered. For clinical consideration, detailed

experiments elucidating damage from long, low voltage

pulses will also be necessary.

Electrode design must also be optimized (Gilbert et

al, 1997). Caliper or plate electrodes are easily available

and simple to use, although the treated tissue must be

accessible and in each case the voltage must be calculated

based on the thickness of tissue “gripped”. In this case,

electric pulses may be delivered over a large surface area,

59

but this delivery also requires higher voltages to maintain

the field strength. Needle electrodes increase the depth of

delivery and allow treatment of tissue without altering

tissue shape. The treated area can be better defined as well,

and the specific voltage used can be standardized from

sample to sample. Particularly for clinical use, it will be

necessary to design pulsing protocols and electrodes

specifically for each application of gene therapy.

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Gene Therapy and Molecular Biology Vol 5, page 63

Potential roles of p53 in recombination

Review Article

Gene Ther Mol Biol Vol 5, 63-79, 2000

Nuray Akyüz, Gisa S. Boehden, Christine Janz, Silke Süße, and Lisa Wiesmüller*

Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität Hamburg

_________________________________________________________________________________________________

*Correspondence: Lisa Wiesmüller, Ph.D., Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität

Hamburg, Martinistrasse 52, 20251 Hamburg, Germany; Tel.: +49-40-48051-235; Fax: +49-40-48051-117; Email: wiesmuel@uke.unihamburg.de

Key Words: double strand breaks / homologous recombination / nonhomologous end-joining / strand exchange / tumor suppressor

Received: 27 August 2000; accepted: 7 October 2000

Summary

Functional inactivation of p53 by mutation or by interactions with viral tumor antigens is associated with a deficit

to maintain the genomic stability. Until recently, p53 was believed to exclusively avoid the manifestation of DNA

damage by transcriptionally signaling transient cell cycle arrest or apoptotic cell death in response to cellular stress

situations. New ideas on a direct involvement of p53 in DNA repair originated from the discoveries of interactions

with replication and repair proteins and of repair-related enzymatic activities, such as the 3!-5! exonuclease activity.

Moreover, physical and genetic links were established between p53 and factors involved in homologous DNA

recombination processes, namely the initial strand transferase RAD51 and the RAD51 complex partner BRCA1

and BRCA2. Importantly, several groups reported that p53 suppresses spontaneous as well as radiation-induced

inter- and intrachromosomal homologous recombination events by at least one to two orders of magnitude. The

identification of cancer-related separation of function mutations demonstrated that p53 regulates homologous

recombination processes independently of its activities in transcription and growth control, and suggested that

functions in recombinative repair contribute to tumor suppression. Mechanistic studies indicated that the p53dependent

regulation of DNA-exchange events is tied to the generation of DNA strand exchange intermediates by

RAD51. The recognition of certain mismatched bases within these heteroduplex joints by p53 has lead to a model

suggesting that p53 monitors the fidelity of homologous recombination events in a manner complementary to the

mismatch repair factor MSH2. The possibility of an involvement of p53 in replication-associated recombination

processes is discussed.

I. Introduction

Clues to a role of p53 in DNA repair

Soon after having established p53 as the most

frequently altered gene in human tumors in the 1980s

(Caron de Fromentel and Soussi, 1992; Hollstein et al,

1994; 1996), p53 was understood as a major component of

the DNA damage response pathway (Lane, 1992; Ko and

Prives, 1996; Levine, 1997). After the introduction of

DNA injuries the level of posttranslationally modified p53

protein rises, which in turn induces a transient cell cycle

arrest or apoptotic cell death. The p53 response is

triggered most rapidly by DNA strand breaks, which can

be introduced either directly, e.g. via ionizing irradiation,

or indirectly after the conversion of DNA adducts or

single-stranded breaks by DNA repair or replication

(Nelson and Kastan, 1994). DNA damage activates p53

through posttranslational modifications by specific

kinases, such as the strand break sensor ATM, by

acetylases, and by the poly(ADP-ribose)polymerase,

which prevent proteolysis via the ARF-MDM2 pathway or

enhance binding of p53 to consensus sequences within the

genome (Lane, 1998; Wang et al, 1998; Lakin and

Jackson, 1999).

After the generation of mice nullizygous for p53, it

became clear that p53 not only responds to DNA damage,

but in fact represents a central player in genome

stabilization, thereby counteracting the multistep process


of tumorigenesis (Livingstone et al, 1992; Harvey et al,

1993; Ishizaki et al, 1994; Cleaver et al, 1999; Schwartz

and Russell, 1999). Loss of p53 function was shown to be

associated with genetic instabilities,which become

manifested as aneuploidies, allelic losses, as well as

increases in sister chromatid exchanges and in gene

amplification rates. Until the 1990s genome stabilizing

functions of p53 were explained by its ability to

transcriptionally signal cell cycle arrest in the G1 phase,

thereby giving time for DNA repair prior to the entry into

S-phase (Lane, 1992). Among the products of the p53

target genes, the cyclin-dependent kinase inhibitor

p21 WAF1/CIP1 is essential for the execution of the cell cycle

arrest at the G1/S transition and to sustain a G2 arrest

under certain circumstances (Levine, 1997; Bunz et al,

1998). However, different from p53 knockout mice, which

have a predisposition to form various tumors within

months (Donehower et al, 1992), mice nullizygous for

p21 WAF1/CIP1 did not display increased cancersusceptibilities

(Deng et al, 1995). As an alternative

pathway to avoid the manifestation of DNA damage, p53

induces apoptotic cell death in certain cell types and in

response to irrepairable DNA damage (Gottlieb and Oren,

1996; Levine, 1997). Apoptotic signaling involves

transcription-independent pathways and the activation of

target genes, such as BAX and IGF-BP3, encoding

antagonists of the survival factor BCL-2 and of the

insulin-like growth factor-1, respectively. However, even

the concept that p53-mediated apoptosis in addition to the

transcriptional upregulation of cell-cycle regulatory genes

prevents spontaneous tumorigenesis turned out to be

incomplete. This was indicated by the absence of tumor

formation after treatment of mice with "-ray at doses of 6-

8 Gy and a compound, which inactivates transcriptional

and apoptotic functions of p53 (Komarov et al, 1999). Due

to this observation it is also unlikely that the p53reponsive

gene GADD45 plays a central role in p53dependent

tumor suppression. GADD45 was proposed to

promote nucleotide excision repair (NER) downstream of

p53 (Smith et al, 1994). However, according to more

recent reports alternative functions of GADD45 in

chromatin remodelling and of GADD45-p21 WAF1/CIP1

complexes in cell cycle regulation seem to be more likely

(Kazantsev et al, 1995; Kearsey et al, 1995a, b; Carrier et

al, 1999).

Investigations on a direct participation of p53 in

DNA repair were stimulated by a number of biochemical

observations, which revealed activities of p53 in the

recognition of DNA damage, DNA reannealing, strand

transfer, and exonucleolytic DNA degradation

(Albrechtsen et al, 1999). The C-terminal 30 amino acids

of p53 (see Figure 1) recognize several DNA damagerelated

structures, such as dsDNA and ssDNA ends, DNA

gaps, and insertion/deletion mismatches (Bakalkin et al,

1994; Jayaraman and Prives, 1995; Reed et al, 1995; Lee

et al, 1997; Protopova et al, 2000). They are also required

for the RNA and DNA reannealing and strand transfer

Akyüz et al: p53 in recombination

activities (Oberosler et al, 1993; Brain and Jenkins, 1994;

Wu et al, 1995). Noticeably, the same central region

encompassing amino acids 83 to 323 within human p53,

where tumorigenic mutations are clustered (Hollstein et al,

1991), recognizes DNA sequence-specifically and is

necessary and sufficient for the 3´-5´ exonuclease activity

on DNA (Mummenbrauer et al, 1996; Janus et al, 1999).

Since its discovery the exonucleolytic activity intrinsic to

p53 has been confirmed by several groups (Jean et al,

1997; Huang, 1998; Shakked, 2000). This finding raised

the question, whether p53 is involved in fidelity control of

DNA replication, in the removal of unpaired regions

during the course of the post-replicative mismatch repair,

or in DNA end processing for conservative or

nonconservative pathways of double-strand break (DSB)

repair. In agreement with a possible role of p53 in DNA

replication, p53 was found in replicative foci of virusinfected

cells (Wilcock and Lane, 1991; Fortunato and

Spector, 1998). Furthermore, p53 physically interacts with

the single-stranded DNA binding protein RPA and with

polymerase # (Wold, 1997; Kühn et al, 1999). In

agreement with a possible role of p53 in DNA repair, p53

binds to a plethora of repair-related proteins, namely via

residues 300 to 393 to the two helicase components, XPB

and XPD, of the dual transcription initiation/repair factor

TFIIH, to CSB, a helicase involved in coupling

transcription to NER, to topoisomerase I (TOPO I) and II

(TOPO II), involved in transcription, replication, repair,

and recombination, and to the Werner's Syndrome Protein

(WRN), a helicase and exonuclease related to genomic

stabilization and with putative functions in replication

initiation (Xiao et al, 1994; Wang et al, 1994; Wang et al,

1995; Leveillard et al, 1996; Albor et al, 1998; Blander et

al, 1999; Gobert et al, 1999; Cowell et al, 2000). The

meaning of most of these interactions is not yet clear.

Thus, uncertainties exist on a direct participation of p53 in

NER by modulating TFIIH activities: Some groups

reported on defective NER in cells with reduced levels of

wild-type p53 (Smith et al, 1995; Wang et al, 1995;

Mirzayans et al, 1996; Ford and Hanawalt, 1997).

However, others saw an increase in sister chromatid

exchanges (SCEs) after UV irradiation in p53-deficient

cells rather than differences in repair (Ishizaki et al, 1994;

Cleaver et al, 1999).

II. Links to homologous recombination

A close functional relationship between p53 and the

human RecA counterpart, HsRAD51, must be deduced

from accumulating biochemical and genetic data (Lim and

Hasty, 1996; Stürzbecher et al, 1996; Tsuzuki et al, 1996;

Buchhop et al, 1997; Süße et al, 2000). In E. coli RecA is

sufficient to execute ATP-dependent homologous pairing

and strand exchange over a distance of 6 kb (Roca and

Cox, 1990; Kowalczykowski, 1991; Radding, 1991; West,

1992). Like RecA also the human RAD51 protein

(HsRAD51) binds to single- and double-stranded DNAs


(ssDNA and dsDNA), assembles cooperatively into helical

nucleoprotein filaments, synapses ssDNA with

homologous dsDNA, and catalyzes strand exchange (Sung

and Robberson, 1995; Baumann et al, 1996; Gupta et al,

1997, 1999; Baumann and West, 1999). However,

HsRAD51 hydrolyzes ATP and promotes strand exchange

at least 10 fold less efficiently than RecA, so that the

length of heteroduplex DNA formed is limited to 1.3 kb.

Therefore, homologous pairing and strand exchange by

RAD51 in vivo are facilitated by the DNA-end binding

protein RAD52, the DNA-dependent ATPase RAD54, and

the single-stranded DNA binding protein RPA (reviewed

in Baumann and West, 1998). Direct physical interactions

between wild-type p53 and HsRAD51 (Stürzbecher et al,

1996) were indicated from the results of

immunoprecipitation experiments with mammalian cell

extracts and Ni 2+ -NTA- or GST-pull-down assays with

recombinant proteins. The p53 point mutants p53(135tyr),

Gene Therapy and Molecular Biology Vol 5, page 65

p53(249ser), and p53(273his) showed weaker interactions

(Buchhop et al, 1997). According to the mapping analysis

by the same authors, two segments of the central domain

of p53, namely between amino acids 94 and 160 and

between 264 and 315, are important for HsRAD51

binding. The highly conserved amino acids 125 to 220 of

HsRAD51, comprising the nucleotide binding motif and,

in analogy to RecA, the putative homooligomerization

domain, were implicated in binding to p53. p53 has also

been shown to interact with the products of the two major

hereditary breast cancer susceptibility genes, BRCA1 and

BRCA2, which form stable complexes with RAD51 in the

nuclei of mitotic and meiotic cells (Scully et al, 1997;

Chen et al, 1998; Marmorstein et al, 1998; Ouchi et al,

1998; Zhang et al, 1998). Functions in DNA repair, in

histone acetylation, and in checkpoint signaling at the

G2/M transition have been ascribed to BRCA2 (Patel et al,

Figure 1. The tumor suppressor p53 displays molecular interactions and biochemical activities related to transcription and to DNA

repair. The amino acids, which are most frequently mutated during cancerogenesis, are indicated by vertical bars at the relative positions

within the p53 molecule. The height of each bar reflects the occurrence in cancer patients. Well-established phosphorylation (P) and

acetylation sites (A) and the modifying enzymes are indicated. The regions of p53, to which certain biochemical functions were

ascribed, are indicated according to the mapping studies cited in the text. The domains interacting with transcription or repair factors,

with the large tumor antigen of the Simian virus 40 (SV40Tag), and with the tyrosine kinase c-ABL are marked below the p53 scheme.


1998; Siddique et al, 1998; Chen et al, 1999a). BRCA1

was linked to transcriptional regulation, and

transcriptional transactivation of target genes, such as

p21 WAF1/CIP1 and GADD45, seems to underly cell cycle

control and the induction of apoptosis (Chen et al, 1999b;

Venkitaram, 1999). Furthermore, a role in recombination

was recently demonstrated, since BRCA1 directs DSB

repair from nonhomologous end-joining (NHEJ) into the

nonmutagenic pathway of homologous recombination

(Moynahan et al, 1999). Consistently, after irradiation

BRCA1 displays a mutually exclusive association with

either RAD51, the initial strand transferase of homologous

recombination, or with RAD50-hMRE11-p95 complexes,

which participate in DSB repair via NHEJ and via

homologous recombination pathways (Scully et al, 1997;

Haber, 1998; Zhong et al, 1999). Indicating functions

upstream of HsRAD51, BRCA1 and BRCA2 are required

for the assembly of ionizing-radiation-induced RAD51

complexes (Yuan et al, 1999; Bhattacharyya et al, 2000).

Indicating functions downstream of RAD51, the

disruption of BRCA2-RAD51 complexes leads to a loss of

the G2/M checkpoint control (Chen et al, 1999a). The

importance of these recombination proteins during

proliferation was convincingly demonstrated by the

embryonic lethal phenotypes of knockout mice with

deficiencies in either RAD50, RAD51, BRCA1, or

BRCA2 (Lim and Hasty, 1996; Hakem et al, 1997;

Ludwig et al, 1997; Luo et al, 1999). Strikingly, the

concomitant knockout of p53 partially suppressed the

arrest of embryo development in mice nullizygous for

RAD51, BRCA1, and BRCA2. Inactivation of the p53

target gene p21 WAF1/CIP1 had the same effect in BRCA1 and

BRCA2 knockouts. The p53-dependent G1/S checkpoint

response is significantly reduced in cell lines derived from

Nijmegen breakage syndrome patients, which are devoid

of p95, the subunit of the RAD50-MRE11-p95 complex

which is required for the DNA damage induced

phosphorylation of MRE11 (Jongmans et al, 1997; Carney

et al, 1998; Dong et al, 1999). Moreover, adding to the

complexity of this intricate regulatory network, the DSB

sensing kinase ATM directly regulates the activities of

p95, BRCA1, and p53 and indirectly promotes the

assembly of recombinative repair complexes via activation

of c-ABL-mediated RAD51 phosphorylation (Banin et al,

1998; Canman et al, 1998; Cortez et al, 1999; Chen et al,

1999c; Venkitaram, 1999; Lim et al, 2000; Morrison et al,

2000). Mice deficient of ATM are viable, but display a

defect early in prophase I during male gametogenesis,

resulting in apoptotis, whereas ATM/p53 or

ATM/p21 WAF1/CIP1 double mutant mice proceed to

pachytene (Barlow et al, 1997). p53 is highly expressed

during meiosis in spermatogenesis from preleptotene to

early pachytene (Rotter et al, 1993; Sjöblom and Lähdetie,

1996). This raises the question, whether interactions of

p53 with the recombination machinery, modulate the

transmission of signals by p53. Indeed, an early

hypothesis on the possible function of BRCA1 was to play

Akyüz et al: p53 in recombination

an auxiliary role in p53-dependent transcriptional

transactivation (Ouchi et al, 1998), although later studies

demonstrated additional p53-independent functions of

BRCA1 in transcriptionally upregulating GADD45, in

controlling the checkpoint during the G2/M-phase, and in

inducing apoptosis (Venkitaram, 1999). Therefore,

BRCA1 may link DSB repair and DNA damage signaling

via p53-dependent and -independent pathways.

III. p53 Inhibits homologous

recombination independent of its

functions in transcriptional

transactivation and in cell cycle control

DSBs arise spontaneously due to errors in

replication, recombination, or mitosis and can be induced

experimentally by ionizing radiation. DSBs trigger both

repair-associated and targeted recombination processes,

which in turn made the DSB responsive molecule p53 a

good candidate for being a regulatory factor of DNA

exchange processes. In 1994 Xia and colleagues (Xia et al,

1994) demonstrated that after X-ray treatment loss of

heterozygosity (LOH), due to inter-allelic homologous

recombination or gene conversion, was observed more

frequently in human lymphoblastoid cell lines with the

mutant p53(237ile), as compared to isogenic cells with

wild-type p53 (Figure 2). Unrestrained LOH in the same

mutant p53 cells was later also observed after treatment

with one of the chemical mutagens EMS, MMS, or

mitomycin C (Honma et al, 1997). With respect to

spontaneous recombination rates, several groups observed

5 to >100 fold rate elevations, when wild-type p53 was

inactive (Meyn et al, 1994; Wiesmüller et al, 1996;

Bertrand et al, 1997; Mekeel et al, 1997). In these studies,

systems for probing recombination were either based on

repeat sequences integrated into the cellular chromosomes

or on mutated variants of the Simian virus 40 (SV40)

genome, thereby taking advantage of the small, chromatin

packaged viral genome, which is amplified episomally.

Isogenic cell types of human and rodent origin, either

differing in the endogenous p53 status or ectopically

expressing wild-type p53 in a p53 null background or the

dominant negative mutants p53 (143ala) and p53 (175his)

in a wild-type p53 background, equally supported the

notion of an inhibitory role of p53 in homologous

recombination processes. Furthermore, a correlation could

be drawn between p53 neutralization by viral proteins and

the elevation of recombination rates. Overexpression of

the human papilloma virus 16 (HPV16) E6 protein, which

promotes p53 degradation by the ubiquitin-pathway,

reproducibly caused an increase in DNA exchange

frequencies by one to two orders of magnitude (Havre et

al, 1995; Mekeel et al, 1997). A possible influence of the

large tumorantigen of SV40 (Tag) in this process was

investigated by designing viral test genomes with a

mutation, leading to the single amino


Gene Therapy and Molecular Biology Vol 5, page 67

Figure 2. Separation of p53 functions in transcriptional transactivation, cell cycle control, and the inhibition of homologous

recombination. Several groups have provided evidence for a role of wild-type p53 in suppressing homologous recombination (HR)

independently of its transcriptional and checkpoint functions. In these studies transcriptional transactivation (TA) with respect to specific

target genes, G1 arrest induction after p53 upregulation, and the exchange between homologous sequences on viral or cellular

chromosomes (inhibition of HR) were monitored with respect to the endogenous p53 status, after expression of a mutated p53 transgene,

or after expression of a cellular (HDM2) or a viral (SV40Tag, HPV-E6) protein antagonizing wild-type (wt) p53 functions. p53 was

originating from mouse (mu), monkey (mo), or man (hu). Beyond the transforming p53 variants with wild-type p53 conformation

[p53(248gln), p53(273his)] or with mutant p53 conformation [p53(237ile), p53$237-239, p53(175his), p53(273pro)], the temperature

sensitive mutants p53(135val) and p53(143ala), the alternatively spliced (AS) form from mice, and variants with shortened C-terminus

were analysed [p53(1-363), p53 (1-333)]. The references were: a, Meyn et al, 1994; b, Xia et al, 1994; c, Wiesmüller et al, 1996; d,

Bertrand et al, 1997; e, Mekeel et al, 1997; f, Dudenhöffer et al, 1998; g, Saintigny et al, 1999; h, Dudenhöffer et al, 1999; i, Willers et

al, 2000.

acid exchange 402asp->his in Tag, thereby specifically

blocking p53-Tag interactions (Wiesmüller et al, 1996).

The corresponding analysis revealed that the suppression

of homologous recombination events by wild-type p53 can

be alleviated by complex formation with SV40Tag. Tag

was known to represent the causative agent of SV40 for

the elevation of recombination frequencies with cellular

and viral DNAs as well as for the stimulation of the

closely related gene amplification events (Perry et al,

1992; Ishizaka et al, 1995; Cheng et al, 1997). Now, p53

appeared to be the missing link between the viral protein

and the genomic instabilities conferred by SV40, and

possibly by other tumor viruses. Moreover, connections

between p53 and RAD51 became apparent, when cell

immortalization after stable transformation of human

fibroblasts with Tag was demonstrated to increase

chromosomal recombination in a RAD51-dependent

manner (Xia et al, 1997). Thus, protecting RAD51mediated

recombination from the interference by p53 via

Tag complex formation allows unrestrained strand

exchange by RAD51. Recently, Schiestl and coworkers

(Aubrecht et al, 1999) provided in vivo evidence for an

antagonistic role of p53 in homologous recombination

processes by use of the pink eyed unstable (p un ) mouse


model. Intrachromosomal homologous recombination,

resulting in deletions at the p un locus, was scored by black

spots on the gray fur of the offspring. Increased

frequencies were noticed with p53+/- and p53-/- mice as

compared to p53+/+ mice. This was true for spontaneous

events as well as for the exchange frequencies enhanced

by the administration of benzo[a]pyrene during

embryogenesis, whereas different results were obtained

after the corresponding treatment with X-rays (see chapter

V). Even further, chromosomal instabilities were noticed

in Ataxia telangiectasia (AT) patients, and ATM-/- mice

displayed significantly elevated spontaneous

recombination frequencies, suggesting that deregulated

DNA exchange events are due to the reduced p53 response

to DSBs in the absence of the upstream kinase ATM

(Bishop et al, 2000).

At this point the critical question was, whether the

regulation of spontaneous homologous recombination

processes is dependent on p53´s transcriptional activities

or on its growth regulatory functions, i.e. whether DNA

exchange processes are modulated indirectly by the

products of target genes or by secondary effects of cell

cycle control like the prevention of DNA synthesis.

However, already in 1995 p53-dependent G1 arrest

signaling via Rb was dissociated from functions in repair,

when it was observed that E6 from different HPVs, but not

HPV-E7 caused an increase in spontaneous mutagenesis

(Havre et al, 1995). Later on, by use of the SV40-based

recombination test system, it was shown that, after the

inactivation of p53, rate changes of viral DNA synthesis

did not correlate with the corresponding changes of

recombination frequencies (Wiesmüller et al, 1996).

Finally, p53 mutations were identified, which served to

demonstrate that p53 regulates homologous recombination

processes independently of its activities in transcription

and growth control (Dudenhöffer et al, 1999; Saintigny et

al, 1999; Willers et al, 2000). Striking examples were

cancer-related p53 mutants with an alteration at amino

acid position 273, which unveiled defects in the inhibition

of homologous recombination processes, while retaining

the ability to induce a G1 arrest (Figure 2). This finding

suggested that functions in recombinative repair contribute

to tumor suppression. Vice versa, a defectiveness in

transcriptional and cell cycle control functions without the

concomitant loss of the capacity to inhibit genetic

exchanges was noticed with the temperature sensitive

mutant p53(135val) and with wild-type p53 expressed

together with the p53-antagonist HDM2. Furthermore,

from the quantitative analysis of cell clones inducibly

expressing different amounts of exogenous wild-type p53,

it was noticed that low protein levels are sufficient for the

inhibition of recombination processes, whereas growthrelated

functions are exerted in a dose-dependent manner.

It can be envisioned that p53 guarantees the maintenance

of genomic stability in mitotically growing cells, because

1000 to 10000 p53 molecules are permanently available in

order to monitor DNA exchange processes. During

Akyüz et al: p53 in recombination

cellular stress situations modified p53 accumulates and

gains functions directed towards the regulation of growth.

Therefore, p53 might play a dual role as a tumor

suppressor in its latent and in its activated state, a model

which is compatible with the opposite regulation of the

exonuclease activity versus sequence-specific DNA

binding after phosphorylation (Janus et al, 1999).

IV. Possible mechanisms underlying

the regulation of homologous

recombination by p53

Given that wild-type p53 controls DNA

rearrangements independently of its transcriptional

functions, further investigations were aiming at clues to

the mechanism underlying the inhibition of recombination.

To identify candidate pathways of homologous

recombination, which are affected by p53, Lopez and

colleagues designed recombination substrates with

inverted or direct repeat sequences (Saintigny et al, 1999).

Inverted repeat recombination substrates allowed to focus

on mechanisms initiated by strand invasion, namely gene

conversion or crossing-over events, the latter of which can

lead to intrachromatid or unequal sister chromatid

exchanges. In yeast these pathways require the RAD52

epistasis group members RAD51, RAD52, RAD54, and the

RAD51 homologues RAD55, and RAD57, corresponding

to HsRAD51, HsRAD52, HsRAD54, and some of the

HsRAD51 homologues XRCC2, XRCC3, RAD51B,

RAD51H3, RAD51C, and RAD51D in human cells

(Lambert et al, 1999). In comparison, direct repeat

sequences allow nonconservative mechanisms of singlestranded

annealing (SSA) and replication slippage in

addition to strand invasion pathways. SSA in yeast

requires the NER endonuclease RAD1/RAD10, the

mismatch repair factors MSH2 and MSH3, and the

helicase SRS2, corresponding to XP-F/ERCC1, HsMSH2,

HsMSH3, and possibly an unknown SRS2 homologue in

human cells. Interestingly, wild-type p53 was shown to

affect inter- and intramolecular homologous

recombination processes with both types of substrates

without altering the ratio of gene conversion versus

crossing-over events (80 % versus 20 %). This indicated

an interference of p53 with recombination involving

RAD51-depedent strand invasion (Table 1).

Further clues to the involvement of p53 in

homologous recombination came from investigations on

its DNA binding activites. Applying gel retardation assays

and electron microscopy, wild-type p53 was demonstrated

to interact with Holliday junctions and with 3-stranded

recombination intermediates independent of the DNA

sequence content (Lee et al, 1997; Dudenhöffer et al,

1998). Interactions of p53 homotetramers with

recombination intermediates were characterized by a high

specificity and binding affinity (K D = 0.1 nM), which was

100 fold higher than for the binding of sequence-specific


Gene Therapy and Molecular Biology Vol 5, page 69

Reference Method Result

Lim and Hasty, 1996

Stürzbecher et al, 1996

Wiesmüller et al 1996

Xia et al 1997

Saintigny et al, 1999

Süße et al, 2000

genetic crossing

immunoprecipitation,

pull down

Table 1. Links between RAD51 and p53.

SV40 chromosome and

plasmid recombination

intrachromosomal recombination

between repeat sequences

gel retardation, strand exchange,

and exonuclease assay

transcriptional response elements (Dudenhöffer et al,

1998; Süße et al, 2000). Compatible with this finding, it

was noticed that supercoiled DNA efficiently competes

with p53-consensus-DNA in DNA binding assays with

p53 (Palecek et al, 1997). Others observed that even sitespecific

recognition of p53-response elements requires

DNA bending or a conformation distinct from B-DNA

(Kim et al, 1997; Nagaich et al, 1997). Furthermore,

strong binding of p53 to DNA junctions, which are

generated during homologous DNA exchange processes,

was in agreement with the observation that low p53

protein levels, such as in the absence of a DNA damage

stimulus, are sufficient for the suppression of homologous

recombination processes (see chapter III). Therefore,

strong junction DNA binding would provide the basis for

a specific and immediate response to RAD51-dependent

DNA exchange processes despite the multifunctionality of

p53. The idea of a direct interaction between p53 and the

recombination intermediate as a prerequisite for the

suppression of homologous recombination was supported

by DNA binding studies with differently mutated and

truncated forms of p53. For each p53 mutant analysed, a

perfect correlation was observed between the intensity of

binding to 3-stranded junction DNAs and the ability to

suppress homologous recombination (Dudenhöffer et al,

1999; Süße et al, 2000). These DNA binding studies were

also compatible with the notion that the integrity of p53

within its central DNA binding and 3´-5´ exonuclease

domain and within its C-terminally neighbouring

oligomerization domain are essential for recombination

inhibition. Substantiating the hypothesis of a direct

interaction with newly generated recombination

intermediates further, DNA-complex formation by p53

was found to be synergistically stimulated, when RAD51-

p53 knockout alleviates the embryonic lethal phenotype of RAD51

mutant mice.

physical interactions in vivo and in vitro

Complex formation between p53 and SV40Tag alleviates the p53dependent

suppression of homologous recombination.

Tag stimulates recombination in a RAD51-dependent manner.

p53 regulates spontaneous and irradiation-induced recombination

processes involving strand invasion.

formation of ternary RAD51-p53-DNA complexes on strand

exchange intermediates; stimulation of the exonuclease activity by

RAD51-mediated generation of heteroduplex joints

nucleoproteins were allowed to assemble on 3-stranded

DNA junctions before the association of p53 (Süße et al,

2000).

According to a novel model for the mechanism of

recombination control, it was suggested that p53 monitors

the fidelity of strand exchange events in close contact to

the recombinase RAD51 (Dudenhöffer et al, 1998). This

hypothesis was based on the discovery that high-affinity

binding of heteroduplex joints was enhanced 10 fold,

when certain types of mismatches were located within the

heteroduplex part. Strikingly, when SV40-virus based

recombination assays were designed to provoke the

generation of mispairings within the heteroduplex after

strand transfer, the same mismatches identified by in vitro

binding assays caused maximal inhibition of homologous

DNA exchange. The analysis of truncated p53 molecules

showed that the extreme C-terminus, which is responsible

for unspecific DNA binding, reannealing, and strand

transfer (Oberosler et al, 1993; Bakalkin et al, 1994; Brain

and Jenkins, 1994), is dispensable for recombination

suppression and 3-stranded junction DNA binding by p53,

but mediates the mismatch-dependent stimulation of

junction DNA binding by p53 (Dudenhöffer et al, 1999).

Therefore, this region negatively controls not only

sequence-specific DNA binding (Hupp and Lane, 1994)

and the exonuclease activity of the core (Janus et al,

1999), but also the inhibition of recombination. DNA

damage binding, truncation, the interaction with a

cofactor, acetylation by p300/CBP, and phosphorylation

by the kinases CKII and PKC can neutralize the negative

effect on sequence-specific DNA binding, and, by

analogy, might also modulate regulatory activities in

recombination (Bayle et al, 1995; Jayaraman and Prives,

1995; Lee et al, 1995; Reed et al, 1995; Selivanova et al,


1996; Steegenga et al, 1996; Gu and Roeder, 1997; Meek

et al, 1997).

Uncontrolled homologous recombination can be a

source of detrimental genome rearrangements, when

imperfectly homologous regions are involved. Erroneous

exchange events give rise to deletions, duplications,

contractions, and expansions of tandem repeat sequences

(reviewed in Belmaaza and Chartrand, 1994), which

accelerate the multistep process of tumor progression. The

activity of the mismatch repair system has been shown to

inhibit recombination between diverged sequences

(Kolodner, 1995; Modrich and Lahue, 1996). In MSH2-/mice,

deficient of the mammalian counterpart of MutS,

genomic instabilities are associated with mismatch repair

deficiencies and a hyperrecombinative phenotype (De

Wind et al, 1995). Parallels of p53 to the functions of this

mismatch repair factor in recombination immediately

arise, as MSH2 is well-known to strongly (K D = 0.5 nM)

bind Holliday junction DNAs (Alani et al, 1997;

Marsischky et al, 1999). Strikingly, p53 shows maximal

binding affinities for 3-stranded recombination

intermediates with A-G and C-T mispairings, whereas

MutS homodimers and MSH2/MSH6 heterodimers

recognize G-T and A-C mismatches best (De Wind et al,

1995; Dudenhöffer et al, 1998). This observation

suggested that p53 monitors the fidelity of strand

exchange events in a manner similar to the mismatch

repair factor MSH2. Indeed, synergistically increased

cancer susceptibilities of the double knockouts indicate

complementary anti-carcinogenic activities for these

multi-functional proteins (Cranston et al, 1997).

The question that remains is, if and how the

exchange process is attenuated when p53 encounters

mismatches in the strands aligned within heteroduplexes.

MutS binds Holliday junctions and inhibits RecA-exerted

strand exchange and branch migration upon encountering

mismatches in the heteroduplex (Worth et al, 1994). In

analogy, it is conceivable that p53´s interaction with

RAD51 serves to inhibit or interrupt strand exchange by

disturbing ATP-hydrolysis of RAD51 or the

oligomerization of monomers (Stürzbecher et al, 1996).

The association of p53 might be triggered or enhanced by

the recognition of certain heterologies within nascent

recombination intermediates (see Figure 3). Alternatively,

p53 could be envisioned to actively dissolve intermediates

comprising mispairings. Indeed, strand transfer and

exonuclease experiments indicated that p53 protein

performs exonuclease activity in a 3´-5´ orientation on the

double-stranded termini of 3-stranded junction DNAs

(Süße et al, 2000). As was predicted from the DNA

binding affinities of p53, degradation was faster, when

p53 was encountering an A-G mismatch within these

specifically recognized structures. Interestingly, for DSB

repair in yeast, Haber and coworkers (Paques and Haber,

1997; Sugawara et al, 1997) have shown that after strand

invasion extended nonhomologous 3´ ends are removed

by the RAD1/RAD10 endonuclease at the junction of the

Akyüz et al: p53 in recombination

duplex DNA. This NER endonuclease is recruited by the

mismatch repair proteins MSH2 and MSH3, which

recognize the branched DNA structure. Therefore, both

p53 and MSH2 have the potential to directly or indirectly

remove heterologies within recombination intermediates.

In summary, p53 and hRAD51 seem to execute their

recombinational repair functions in close neighbourhood

to each other, and interact during or shortly after strand

transfer possibly to facilitate the recognition of

heteroduplexes by p53. According to a new model for the

mechanism leading to fidelity control of recombination

processes by p53, recognition of recombinative DNA

structures by p53 would be followed by the nucleolytic

destruction of heteroduplexes, when encompassing

mispairings (Süße et al, 2000).

V. Possible roles in DNA end-joining,

meiosis, and replication-associated

recombination

Recombination represents the final and irreplaceable

repair mechanism under circumstances when DNA

double-strand breaks or gaps appear. Different from yeast,

where homologous recombination is the predominating

pathway of DSB repair, the relative contribution of

homology-directed repair of chromosomal DSBs in

mammalian cells seems to account for up to 50 %, as

studied by use of the rare-cutting, site-specific I-SceI

nuclease (Sargent et al, 1997; Taghian and Nickoloff,

1997; Liang et al, 1998; Lambert et al, 1999; Lin et al,

1999; Moynahan et al, 1999; Dronkert et al, 2000). To

check possible effects of p53 on the second major DSB

repair pathway, NHEJ, Yang and colleagues examined a

thyroid cell line with the temperature sensitive (ts) mutant

p53(138val) in transfection assays with a linearized

luciferase plasmid (Yang et al, 1997). At the permissive

temperature p53 was shown to enhance DNA end-joining

3-4 fold, and at the nonpermissive temperature 2-3 fold,

indicating a stimulatory influence of tsp53 in the wild-type

and in the mutant conformation. Interestingly, the

corresponding murine tsp53(135val) was shown to inhibit

homologous recombination processes again at both

temperatures (Willers et al, 2000). Therefore, one possible

explanation might be that the upregulation of NHEJ

simply reflects redirected repair after the downregulation

of homologous recombination. However, the stimulatory

effect on NHEJ was postulated to be dependent on the Cterminal

reannealing activity of p53, and this was

interpreted such that p53 joins broken DNA ends via short

homologies (Tang et al, 1999). Compatible with an active

participation of p53 in NHEJ, Schiestl and coworkers

(Aubrecht et al, 1999) concluded from their experiments

with p53+/+, p53+/-, and p53-/- mice that p53 is required

for efficient recombination after X-ray treatment.

Therefore, after irradiation RAD51-independent DSB

repair pathways, such as SSA and NHEJ, could be

enhanced by p53, whereas gene conversion and crossing


Gene Therapy and Molecular Biology Vol 5, page 71

Figure 3. Model for p53-dependent fidelity control of homologous recombination. During the generation of nascent heteroduplexes by

RAD51, RPA, and auxiliary factors from the RAD52 epistasis group, mismatches can arise as a result of DNA exchange between

imperfectly homologous sequences. MSH2, within heterodimers with MSH6 or MSH3, and p53 homotetramers independently surveil

the fidelity of the initial strand exchange by transient interactions with the heteroduplex joint. Depending on the type of mismatch

created, either the interactions by MSH2 or by p53 (shown here) are stabilized, thereby abrogating further strand exchange by RAD51

and/or allowing nucleolytic correction of the misaligned region.

over events would be suppressed in parallel. However, this

interpretation might hold true only for certain

recombination substrates or cell types, since other groups

observed totally different results, which indicated that p53

suppresses NHEJ after irradiation (Mallya and Sikpi,

1999), and reduces the integration of linearized plasmid

into the host chromosome (Lee et al, 1999). Furthermore,

when monitored by the neutral Comet assay, DSB

rejoining during and after the exposure to ionizing

radiation was shown to increase with mutant versus wildtype

p53 (Bristow et al, 1998). Therefore, the role of p53

in NHEJ must be considered far from being understood.

There is some evidence for an indirect involvement

of p53 in V(D)J joining. In pre-B cells, the accumulation

of wild-type p53 induces cell differentiation, which is

manifested by immunoglobulin % light-chain gene

expression after successful V(D)J recombination (Aloni-

Grinstein et al, 1995; Bogue et al, 1996). However, the

upregulation of p53 might simply be explained by V(D)J

recombination triggering a p53-dependent DNA damage

checkpoint (Guidos et al, 1996). In scid mice the knockout

of p53 can rescue rearrangement at multiple TCR loci,

which most likely involves a homology-dependent bypass

pathway (Guidos et al, 1996). Thus, the proposed indirect

involvement of p53 in V(D)J recombination can be

explained by its regulatory role in homologous

recombination processes.


Homologous recombination processes are important

for eukaryotic organisms not only during the mitotic life

cycle in order to repair DNA damage, but also to create

diversity and to ensure proper segregation of

chromosomes during meiosis (Kucherlapati and Smith,

1988). From the fact that p53 expression in testes is

stronger than in other tissues, p53 was proposed to be

connected to meiotic recombination (Rotter et al, 1993;

Sjöblom and Lähdetie, 1996). More specifically, its

expression in mice and rats is most prominent in zygotene

- early pachytene spermatocytes, at the meiotic stage when

homologous chromosomes synapse for genetic exchange.

Still, p53 expression can further be increased by radiation

treatment. However, Gersten and Kemp (1997) did neither

observe elevated rates for the targeted types of DNA

exchange during meiosis nor during antigen receptor

rearrangements in p53 knockout mice. Therefore, it is

conceivable that during meiosis p53 only serves to

eliminate defective meiotic spermatocytes by irradiationinduced

apoptosis (Odorisio et al, 1998). Homologous

DNA exchange processes in mitotically growing cells are

suppressed by a factor of 1000 as compared to rates during

meiotic recombination, so that complex control

mechanisms must be involved to allow recombination

between homologous chromosomes during meiosis and to

avoid detrimental genome rearrangements during growth

(Haber, 1997). Considering the experimental data

describing the regulatory role of wild-type p53 in RAD51dependent

DNA exchange processes (Table 1), p53mediated

surveillance of homologous recombination

seems to contribute to the control mechanisms, which

suppress spontaneous DNA exchange events specifically

in mitotically growing cells.

In mitotically growing cells recombination processes

are frequently associated with DNA replication in order to

allow the bypass of unrepaired lesions, and to repair DSBs

generated at replication forks passing a single-strand break

(Cox, 1997; Haber, 1999; Cox et al, 2000; Flores-Rozas

and Kolodner, 2000). Due to this vital function of

recombination processes during the normal life cycle of

cells, the lack of the central enzyme function causes high

mortality rates in E. coli devoid of RecA (Roca and Cox,

1990), an early embryonic lethal phenotype of mice

nullizygous for RAD51 (Lim and Hasty, 1996; Tsuzuki et

al, 1996), and results in an extreme sensitivity towards

ionizing radiation and methyl methanesulfonate (MMS) of

S. cerevisiae cells carrying mutations in RAD51 (Game

and Mortimer, 1974; Shinohara et al, 1992). Consistent

with the sister chromatid being the preferred homologous

template for the repair of damaged DNA, homologous

recombination represents the predominant DSB repair

pathway in replicating cells, as opposed to

nonhomologous end-joining (reviewed in Haber, 1999;

Lambert et al, 1999; Cox et al, 2000; Flores-Rozas and

Kolodner, 2000). RAD51 levels rise at the beginning of Sphase

and cause the formation of S-phase specific nuclear

foci (Flygare et al, 1996; Tashiro et al, 1996; Chen et al,

Akyüz et al: p53 in recombination

1997; Scully et al, 1997). Most recently, Tashiro and

colleagues (Tashiro et al, 2000) concluded from their

combined pulse labeling and microirradiation studies that

even within 15 min RAD51 is recruited to sites of DNA

damage in regions of replicative DNA synthesis.

Interestingly, p53 binds RPA, which participates in

replication and recombination, and forms functional and

extremely stable complexes with DNA polymerase #

(Wold, 1997; Kühn et al, 1999), the DNA polymerase for

lagging-strand synthesis, which in the yeast system has

been shown to be involved in DSB repair (Holmes and

Haber, 1999). p53 was also found to excise mispaired

nucleotides from DNA in a polymerase # based in vitro

replication assay (Huang, 1998). Therefore, p53 might

function on heteroduplexes after strand invasion or/and

during strand synthesis, namely by controlling the

replicative extension of the 3´ invading end, especially

during RAD51-mediated replication-associated

recombination processes.

VI. Conclusions

DNA in eukaryotic cells undergoes continuous

damage and resynthesis. Therefore, multiple repair,

fidelity control, and cell cycle checkpoint systems exist to

avoid the accumulation of mutations, and to create barriers

against the multistep process of cancerogenesis (Loeb and

Loeb, 2000). During the last years numerous genetic

studies have revealed overlapping functions in DNAmetabolizing

processes and checkpoint control for several

proteins, which are as divergent as 3´-5´ exonucleases

(Lydall and Weinert, 1996; Onel et al, 1996), DNA

polymerase & (Navas et al, 1995), and the mismatch repair

factors hMSH2 and hMLH1 (reviewed in Kunkel, 1995).

Until the 1990s, tumor suppression by p53 was solely

explained by its cell cycle control functions in response to

DNA damage (Lane, 1992). As soon as DNA repairrelated

activities of p53 were discovered, new models of a

dual role of p53 in checkpoint control and in repair were

arising (Albrechtsen et al, 1999).

So far, evidence for a role of p53 as a proofreader of

DNA polymerases was mainly based on in vitro

experiments (Mummenbrauer et al, 1996; Jean et al, 1997;

Huang, 1998; Janus et al, 1999; Kühn et al, 1999; Shakked

et al, 2000), although recently, p53 was found to reduce

the frequency of chemically induced point mutations in

vivo (Courtemanche and Anderson, 1999). More

convincingly, in vitro and in vivo data from several groups

substantiated that p53 suppresses spontaneous inter- and

intrachromosomal homologous recombination by at least

one to two orders of magnitude (Meyn et al, 1994; Xia et

al, 1994; Wiesmüller et al, 1996; Bertrand et al, 1997;

Mekeel et al, 1997; Dudenhöffer et al, 1999; Saintigny et

al, 1999; Willers et al, 2000; Süße et al, 2000). Accurate

recombination is especially important in actively dividing

cells, when DSBs arise spontaneously during the process

of DNA replication (Haber, 1999; Cox et al, 2000).


Therefore, a hierarchy of regulatory factors coordinates

recombinative repair. Thus, BRCA1 is engaged in

channeling DSB repair from DNA end-joining into the

precise pathway of homologous recombination in

mammalian cells (Moynahan et al, 1999). In S. cerevisiae,

with predominantly coding sequences and hardly any

repeats, DNA end-joining is rare, and this might explain

why a BRCA1 homologue and possibly other fidelity

systems, such as p53, did not develop. The recognition of

mismatches seems to enable MSH2 to prevent strand

exchange (Alani et al, 1997), thereby creating a barrier to

the recombination between repeated sequences that are

diverged (Kolodner, 1995; Modrich and Lahue, 1996).

Similar to the proposed role of MSH2, p53 might monitor

the correct alignment of homologous sequences during

recombinational repair. As would be expected for the

unrestrained exchange of mismatched sequences,

spontaneous and UV-induced sister chromatid exchanges

rise in p53-deficient cells (Ishizaki et al, 1994; Cleaver et

al, 1999; Schwartz and Russell, 1999). Consistently, a role

of p53 in creating a threshold for recombination between

short versus long homologies was proposed from studies

on the stability of repetitive sequences (Gebow et al,

2000). Moreover, a role in surveillance of the fidelity of

RAD51-dependent strand transfer processes by

exonucleolytic correction of errors was proposed for p53

(Dudenhöffer et al, 1998; Süße et al, 2000). In agreement

with this hypothesis, a correlation was recently reported to

exist between p53 mutations in non-small cell lung cancer

and the appearance of microsatellite instabilities at certain

tetranucleotide repeats (Ahrendt et al, 2000).

Importantly, some cancer-related p53 mutants are

defective in modulating recombination, but still arrest

cells in G1. This suggests that, after mutation of p53,

deregulated recombination contributes to accelerated

tumor formation. Therefore, characterizing both functions

for different p53 mutants seems critical to the

understanding of resistance phenotypes during cancer

therapeutic treatments. Indeed, p53 has profound effects

on the responses to genotoxic treatments, and these effects

vary dramatically depending on the treatment (McGilland

and Fisher, 1999) and depending on the p53 mutation

(Benchimol, 1999). Thus, p53-deficient colon carcinoma

cells are resistant towards an inhibitor of DNA synthesis,

5-Fluorouracil, whereas they respond extremely sensitive

towards adriamycin or irradiation. Taken together, it

seems that as a consequence of the individual p53

mutation and of the specific anti-cancer treatment applied

either the impairment of DNA repair or the inability to

execute apoptosis produce different cellular responses.

The final goal will be to raise the curing rate by individual

cancer therapies, developed according to the functional

status of p53 in the respective tumor.

Gene Therapy and Molecular Biology Vol 5, page 73

Acknowledgements

Our work was supported by the Deutsche

Forschungsgemeinschaft, grants Wi 1376/1-4 and -5 and

grant 10-1417-De4 by the Deutsche Krebshilfe, the Dr.

Mildred Scheel Stiftung. S.S. was supported by the

FAZIT-Stiftung, Frankfurt a.M. The Heinrich-Pette-

Institut is supported by the Freie und Hansestadt Hamburg

and by the Bundesministerium für Gesundheit.

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Gene Therapy and Molecular Biology Vol 5, page 79


Akyüz et al: p53 in recombination


Gene Therapy and Molecular Biology Vol 5, page 81

81

Gene Ther Mol Biol Vol 5, 81-86, 2000

Characterisation of the p53 gene in the rat CC531

colon carcinoma

Research Article

Sacha B Geutskens 1 , Diana JM van den Wollenberg 1 , Marjolijin M van der Eb 1,2 ,

Hans van Ormondt 1 , Aart G Jochemsen 1 , Rob C Hoeben 1*

Departments of 1 Molecular Cell Biology and 2 Surgery, Leiden University Medical Center, Wassenaarseweg 72, 2333 AL

Leiden, The Netherlands

_________________________________________________________________________________________________

* Correspondence: Rob C Hoeben, PhD, Department of Molecular Cell Biology, Leiden University Medical Center, Wassenaarseweg

72, 2333 AL Leiden, The Netherlands; Phone: 00 31 71 5276119; Fax: 00 31 71 5276284; E-mail: r.c.hoeben@lumc.nl

Key words: colorectal cancer, mutated p53

Received: 6 November 2000; accepted: 7 November 2000

Summary

The p53 gene was characterised in CC531 colon carcinoma of the Wag/Rij rat, a model frequently used for the

evaluation of anti-cancer treatments. The gene incurred a large in-frame deletion, with junctions in exons 4 and 8,

and encodes a protein of approximately 32 kDa, lacking the entire DNA-binding domain. No wild-type p53 allele is

retained. Functional analysis shows that the mutated protein can repress the function of wtp53 protein.

I. Introduction

Colorectal cancer is one of the most common

malignant tumours and a major cause of cancer death in

developed countries. Over 700, 000 men and women are

found to have colorectal cancer globally each year (Soussi

et al, 1996).

The liver is the first vascular bed in which

disseminating colorectal cancer cells are trapped. Liver

metastases are detected in 20% of all patients undergoing

resection of their primary colorectal tumour (Wanebo et

al, 1978). Ultimately, about 75% of all colorectal cancer

patients develop liver metastases. At present, the only

chance of long-term survival is complete resection of these

metastases, an operation that is exclusively performed on

patients with no signs of irresectable extra-hepatic disease.

Animal models are indispensable in the search of

new approaches for the treatment of colorectal tumour

metastases. One of the few well-characterised animal

models for hepatic colorectal cancer makes use of the rat

CC531 cell line. The CC531 colon carcinoma cell line is

derived from a 1,2-dimethyl-hydrazine-induced tumour

and syngeneic with the Wag/Rij rat (Thomas et al, 1993).

It is a representative model for secondary liver metastases

and frequently used for studying effects of various anticancer

treatments (Marinelli et al, 1991; Oldenburg et al,

1994; Veenhuizen et al, 1996; Griffini et al, 1997).

Two-thirds of all colorectal tumours contain

mutations in the p53 gene, illustrating the essential role of

p53 in the aetiology of colorectal cancer (Soussi et al,

1996). Loss of p53 function, as frequently occurs in cancer

cells, causes loss of growth control and abrogation of

programmed cell death (Bellamy, 1997). Reintroduction of

wild-type p53 (wtp53) arrests cell growth and may induce

apoptosis, as has been described for various tumour cell

lines (Xu et al, 1997; Anderson et al, 1998). A relationship

has also been established between the presence of a

mutation in the p53 gene and the clinical prognosis

(Hamelin et al, 1994).

In view of the prominent role of the p53 gene for

colorectal cancer, we assessed the integrity of the p53

gene in the CC531 cancer cell line, and discovered a

homozygous in-frame deletion within the coding region of

the p53 mRNA, resulting in removal of the entire DNAbinding

domain. The mutated protein is capable of

inhibiting transcription activation by wtp53. Our data not

only reveal the p53 status of this model, but also provide a

genetic marker that allows development of a sensitive


Geutskens et al: Characterisation of p53 in the rat CC531 cell line

PCR-based assay for the detection of cancer cells amid

normal cells.

II. Results

A. PCR amplification and sequence

analysis of the p53 mRNA and chromosomal

DNA in CC531 cells

To study whether the p53 gene of the CC531 cell

line contains a mutation, RT-PCR was performed to

amplify the entire coding region of the p53 gene. Instead

of the expected 1200-bp fragment, only a fragment of ca.

82

600 bp was amplified, under various conditions (Figure

1A). Moreover, when internal primers (annealing to exons

4 and 8) were used no amplified product was detected

(Figure 1A), suggesting a deletion within the coding

region. Indeed, sequence analysis and comparison with the

published sequence of the rat p53 gene (Hulla and

Schneider, 1993), revealed that a large part of the coding

region was absent. This in-frame deletion encompasses

part of exon 4, exons 5-7 and part of exon 8 and removes

amino acids 105 to 326.

Figure 1.

A: Lane 1 shows the 600-bp p53-cDNA fragment obtained by PCR-amplification on RNA isolated from CC531 cells. In lanes 2, 3 and

4, a PCR fragment is absent if primer combinations are used spanning exon 1/exon 4, exon 8/exon 10 and exon 4/exon 8 respectively.

Fragment sizes have been estimated by comparison to a 100 bp DNA marker (first lane).

B: Schematic representation the genomic configuration of the coding region of p53 gene in CC531 cells. The exons are indicated. Below

the graph the sequence of the fused exons 4 and 8 is given. The filled triangle, below the sequence marks the junction.

C: PCR amplification of a 200-bp DNA fragment encoding the fused exons 4 and 8 (primers F4/R8) of the p53 gene from CC531derived

chromosomal DNA (lane 2). No fragment is detected if primers F7/R8 are used (lane 1). A 300-bp fragment containing the 5’

exon 7/ 5’ exon 8 (F7/R8 primers) region is amplified if chromosomal DNA from Wag/Rij rat hepatocytes is used (lane 3). No fragment

is detected if this DNA is amplified with F4/R8 primers (lane 4). Lane 5 and 6 depict H 2O controls for primer pairs F7/R8 and F4/R8

respectively. Fragment sizes have been estimated by comparison to a 100-bp DNA marker (first lane).


Thus, the resulting p53 protein lacks the entire

specific DNA-binding domain, but still contains the

domain responsible for tetramerization with other p53

proteins (Figure 1B).

To verify that the deletion is not the result of aberrant

RNA splicing, chromosomal DNA from CC531 cells was

analysed. Therefore, forward primers were developed

binding in the remaining part of exon 4 (F4) and in exon 7

(F7) and one reverse primer downstream of the junction in

exon 8 (R8). If a wild-type p53 allele were present, it

would be expected to yield a PCR-product of about 300-bp

when F7 is combined with R8. If, on the other hand, the

deletion occurs on the chromosomal DNA, a !200-bp

product is expected if F4 is combined with R8. If the

mutated allele is the only one present, no product should be

seen with F7 and R8.

Amplification of chromosomal DNA derived from

CC531 cells yielded a 200-bp product with the F4/R8

primers and no product with the F7/R8 primers. In contrast,

PCR amplification of chromosomal DNA isolated from

Wag/Rij rat hepatocytes yielded no product with F4/R8

primers, but a fragment of ca.300-bp when F7/R8 primers

were used (Figure 1C). These data demonstrate that the

deletion is present on the chromosomal DNA level.

Moreover, the CC531 cell line is homozygous for the

mutated p53 allele.

B. Analysis and function of the mutated

p53 protein

Because the deletion does retain the p53 open reading

frame, a p53 protein lacking the central domain be

synthesised. To investigate this possibility, the CC531 p53encoding

cDNA was cloned in an expression vector and

transfected into the human Hep3B (no p53) and HepG2

(wtp53) cell lines. After 48 h, these cells and, as controls,

Gene Therapy and Molecular Biology Vol 5, page 83

83

rat CC531 and BxC22 cells, were analysed for presence of

human and rat p53. Indeed, a smaller p53-protein of ca. 32

kDa was detected in CC531 cells, which co-migrates with a

band seen in the Hep3B and HepG2 cells transfected with

pCMVCC53 (Figure 2). Since the CC531-derived p53

protein lacks the DNA-binding domain, but still contains

the tetramerization part, it was hypothesized that

Figure 2

Immunoblot analysis on cell lysates of BxC22 (lane A; rat

wtp53), CC531 (lane B, rat mutp53), HepG2 (lane C, human

wtp53) and Hep3B (lane F, human, no p53). HepG2 and Hep3B

cells transfected with 10 µg (lane D and G) or 20 µg (lane E and

H) of pCMVCC53.

Blots have been incubated with PAb122, recognising the C

terminus of rat and human wtp53 (blot A) or with DO-1,

recognising human wtp53 (blot B). Both BxC22 and HepG2

express normal-size (ca. 53 kDa) wtp53. CC531-derived cell

lysates and those transfected with the CC53-construct express a

shorter p53 protein of ca. 32 kDa. Protein sizes have been

estimated by comparison to a broad-range protein marker.

Figure 3

Luciferase activity in HepG2 cell

lysates. Lysates were made 48 h

after transfection of HepG2 cells

with the various constructs. Cells

(1x10 5 /well) were transfected with

the calcium-phosphate precipitation

method, with 1.5 µg pXAluc or

pOLXALuc, 20 ng of either

pCMVneo, pCMVwtp53 or

pCMVCC53, and 20 ng pCMVlacZ

as indicated. Precipitates were

made in duplicate and the

experiment was performed three

times. Luciferase activities depicted

are corrected for differences in

transfection efficiencies determined

with the ß-galactosidase assay.

.


Geutskens et al: Characterisation of p53 in the rat CC531 cell line

the protein might act as a dominant-negative mutant. To

investigate whether the 32-kDa p53 protein can affect

transcription activation of wtp53, the pCMVCC53 vector

was transfected into HepG2 cells, together with a p53responsive

luciferase-reporter plasmid. Transfection of

pOLXALuc, which contains a p53-responsive element

(Steegenga et al, 1995) yields a higher luciferase activity

than transfection with pXALuc, which lacks a p53reponsive

element (Peltenburg and Schrier, 1994) (Figure

3). Co-transfection of a plasmid encoding human wtp53

further increased expression of pOLXALuc, but not that of

pXALuc, indicating the validity of the approach. The

activity of pOLXALuc was clearly reduced upon cotransfection

with pCMVCC53, while pXALuc expression

was not affected. This suggests that the 32-kDa p53

protein acts as a dominant inhibitor of wtp53 function.

Expression of a non-p53-regulated reporter, lacZ, which is

driven by the CMV promoter, did not show manifest

differences between various co-transfections.

III. Discussion

Genetic alteration of the tumour-suppressor gene p53

is frequently found in cancer, especially in the DNAbinding

domain that spans exons 5 through 8 (Tullo et al,

1999; Veldhoen et al, 1999). In many studies, only this

region is screened for the presence of mutations in clinical

samples. The rat colon carcinoma studied here, showed a

large deletion, removing amino acids 105 to 326. A smaller

p53-protein of ca.32 kDa is translated which is recognized

by a human and rat-p53 specific antibody, PAb 122, that is

known to bind to the C-terminus of the wild-type protein.

Although the DNA-binding domain is deleted, the 32-kDa

p53 protein can impair transcriptional activation by

regulation of wtp53. Such a dominant-negative effect has

been described earlier (Chen, 1998; Roemer, 1999) and

probably results from disruption of DNA binding of wtp53

by forming hetero-tetramers with the mutant p53 (Deb et

al, 1999). This type of mutants is also thought to exhibit a

gain of function by generating genomic instability,

increasing oncogenic transformation (Gualberto et al,

1998).

CC531 is a valuable model for secondary liver

metastases in the rat and often used for studying the

therapeutic effect of various anti-neoplastic agents

(Marinelli et al, 1991; Oldenburg et al, 1994; Veenhuizen

et al, 1996). The p53 protein is essential in the induction of

apoptosis by several anti-cancer therapeutics (Tishler and

Lamppu, 1996; Hagopian et al, 1997; Anderson et al,

1998). Mutations in the p53 gene are associated with drug

resistance, so the p53 status of this model is highly

important. The p53 deletion characterised here might

explain the resistance of CC531 to cisplatinum treatment

(Gheuens et al, 1993) that activates p53-dependent

apoptotic pathways. Despite the dominant negative activity

of the CC531 p53 protein, transfer of wtp53 rendered the

CC531 colon carcinoma susceptible for apoptosis (Van der

84

Eb et al, manuscript in preparation). Combining

chemotherapeutics with the transfer of wtp53 might give a

higher efficacy of anti-tumour treatment than with the antineoplastic

agent alone.

In addition, the characterisation of the junction of this

deletion has allowed the differentiation of CC531 tumour

and non-tumour cells via PCR. This technique permits

detection and quantification of minute amounts of tumour

cells in extra-hepatic tissues, such as lymph nodes and

lungs and it will make the CC531 model even more

valuable for exploration of new avenues for treatment of

colorectal cancer.

IV. Materials and Methods

A. Tissue culture and cell lines

The CC531 cell line is a moderately differentiated

adenocarcinoma of the colon, syngeneic with Wag/Rij rats

(Thomas et al, 1993). BxC22 is a wtp53-expressing Ad5 E1transformed

Wag/Rij baby rat kidney cell line. HepG2 and

Hep3B are human hepatoma cell lines expressing wtp53 (Hosono

et al, 1991) or lacking p53 expression (Farshid and Tabor, 1992)

respectively. All cell lines were maintained in high-glucose

Dulbecco’s modified Eagle’s medium (DMEM) supplemented

with 10% foetal-calf serum (GIBCO Laboratories, Grand Island,

NY, USA), at 37 °C/5% CO 2.

B. Polymerase chain reaction (PCR)

For reverse transcription-PCR (RT-PCR), RNA was

isolated directly from tissue culture with RNAzolB according to

the manufacturers protocol (Campro Scientific, Veenendaal, The

Netherlands). RNA was treated with DNase I (Roche

Diagnostics, Almere, The Netherlands) to degrade contaminating

DNA. First-strand-complementary DNA (cDNA) of 1 µg of

RNA was synthesized with SuperscriptII RNase H** reverse

transcriptase (Gibco/Life Technologies, Breda, The Netherlands)

and an Oligo-dT primer. The cDNA (1 µg) was amplified with

primers complementary to the 5’ and 3’ ends of the coding

region of wild-type rat p53 (forward primer: 5’ GTG GAT CCT

GAA GAC TGG ATA ACT GTC 3’; reverse primer: 5’ AGT

CGA CAG GAT GCA GAG GCT G 3’) (Van den Heuvel et al,

1990), with Pfu polymerase (Stratagene, Amsterdam, The

Netherlands) in buffer supplied by the manufacturer. Primers

complementary to internal sequences of the p53 coding region

(forward primer: 5’ TAC CAC TAT CCA CTA CAA GTA CAT

G 3’; reverse primer: 5’ TTT CTT CCT CTG TCC GAC GGT

CTC 3’) were used as a control. The amplified 600-bp fragment

was sequenced by BaseClear (Leiden, The Netherlands).

Chromosomal DNA was isolated with the NP40 protocol

(Maniatis et al, 1989). DNase was heat-inactivated and RNase

(Merck, Darmstadt, Germany) was added to degrade RNA. One

microgram of DNA was amplified with primers annealing to

exon 8 (R8): 5’ AAT CCA ATA ATA ACC TTG GTA CCT T

3’, exon 7 (F7): 5’ TGT GCC TCC TCT TGT CCC 3’ or exon 4

(F4): 5’ CGA CAG GGT CAC CTA ATT CC 3’ of wild-type rat

p53 chromosomal DNA with Taq polymerase (Roche

Diagnostics, Almere, The Netherlands).


C. Western immunoblotting

Cells (total of 3x10 6 ) were lysed in 750 µl NP40/SDS

(2%/0.2%) buffer (25 mM Tris pH 7.4, 50 mM NaCl, 0.5%

deoxycholate). Protein lysates (40 µl) were size-fractionated by

gel electrophoresis in 10% SDS-polyacrylamide. Proteins were

transferred to Immobilon-P (0.45 µm, Millipore Corporation,

Bedford, USA) and incubated with an antibody specifically

recognizing human wtp53, DO-1, or with PAb 122, recognizing

both human and rat p53 (Schmieg and Simmons,1984). As a

second antibody horse-radish-peroxidase (HRP)-conjugated

antibody, G"M-IgG (Jackson Immunoresearch Laboratories,

Westgrave, USA) was used. The blots were visualized by

exposion to Kodak XAR-films. The broad-range protein marker

was used as a standard (BioRad laboratories, Veenendaal, The

Netherlands).

D. Luciferase reporter assay

For the expression of the mutant p53 cDNA, a vector

containing the p53-coding region of CC531 under the regulation

of the CMV promoter (pCMVCC53) was made by digesting the

purified RT-PCR fragment with BamHI and SalI and subsequent

ligation into pcDNA3.1+ (Invitrogen, Leek, The Netherlands)

digested with BamHI and XhoI. Plasmid constructs expressing

the neomycin resistance gene (pCMVneo), the E.coli ßgalactosidase

gene (pCMVlacZ), or the human wtp53 cDNA

(pCMVwtp53) were used and have been described earlier

(Steegenga et al, 1995). The luciferase reporter constructs,

pXALuc and pOLXALuc (containing the p53-consensus binding

sequence) have also been described before (Peltenburg and

Schrier, 1994; Steegenga et al, 1995).

Cells (1x10 5 /well) were transfected with the calciumphosphate

precipitation method (van der Eb and Graham,1980),

with 1.5 µg pXAluc or pOLXALuc, 20 ng of either pCMVneo,

pCMVwtp53 or pCMVCC53, and 20 ng pCMVlacZ. Precipitates

were made in duplicate and the experiment was performed three

times.

After 48 h, cells were lysed in 250 µl of cell-culture lysis

reagent (Promega, Madison, WI, USA) and luciferase activity of

100 µl of lysate was determined as described before (Steegenga

et al, 1995). The #-galactosidase activity resulting from cotransfection

of the control plasmid pCMVlacZ was determined in

a ß-galactosidase assay (Maniatis et al, 1989) and served as an

internal control to correct for variations in transfection

efficiency. In none of the experiments, ß-gal activity varied more

than 20%.

Acknowledgements

The sequence of the CC531 p53 cDNA is deposited

in Genbank (accession number AY009504).

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Huang W, Wills KN, Gregory RJ, Sutjipto S, Fen Wen S,

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Oosterom A, De Bruijn E (1993) Multidrug resistance in rat

colon carcinoma cell lines CC531, CC531mdr+ and

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Griffini P, Smorenburg SM, Verbeek FJ, Van Noorden CJ (1997)

Three dimensional reconstruction of colon carcinoma

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Hosono S, Lee CS, Chou MJ, Yang CS, Shih CH (1991)

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Steegenga WT, Van Laar T, Terleth C, Van der Eb AJ,

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transcription and cell-cycle regulation by the large (54 kDA)

and small (21 kDA) adenovirus E1B proteins. Virology 212,

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.


Gene Therapy and Molecular Biology Vol 5, page 87

Gene Ther Mol Biol Vol 5, 87-100, 2000

Recombinant adenoviruses as expression vectors

and as probes for DNA repair in human cells

Review Article

Andrew J. Rainbow 1 , Bruce C. McKay 2 and Murray A. Francis

1Department of Biology, McMaster University, Hamilton, Ontario L8S 4K1, Canada

2Canada Centre for Cancer Therapeutics, Ottawa Regional Cancer Centre, 501 Smyth Rd, Ottawa, Ontatio K1H 8L6,

Canada.

_________________________________________________________________________________________________

*Correspondence: Dr. Andrew J. Rainbow, Department of Biology, McMaster University, Hamilton, Ontario L8S 4K1, Tel: (905) 525-

9140 Ext. 23544 Fax: (905)-522-6066; E-mail: rainbow@mcmaster.ca

Key Words: recombinant adenovirus, host cell reactivation, nucleotide excision repair, enhanced reporter gene expression,

inducible DNA repair, p53 tumour suppressor, ultraviolet light

Received: 25 August 2000; accepted: 10 September 2000

Summary

There is widespread interest in the use of recombinant adenovirus (Ad) vectors for gene therapy of cancer and as

tools in molecular biology research. There are also potential benefits to be gained by combining strategies for Adbased

gene therapy of cancer with radiotherapy and chemotherapy. However, there is limited information available

on the effects of cytotoxic agents on transgene expression which would allow a rational approach to combining

these modalities. We have used recombinant nonrepilcating Ad expressing the lacZ gene under the control of the

cytomegalovirus (CMV) immediate early promoter to assess the effects of cytotoxic agents on the expression of a

reporter gene in human cells. Using this approach we are able to examine both constitutive and inducible

expression of the reporter gene. Pretreatment of normal human cells with low UV fluence results in an enhanced

expression of the reporter gene. The enhanced expression occurs at lower doses of the DNA damaging agent in cells

deficient in the transcription coupled repair (TCR) pathway of nucleotide excision repair (NER) suggesting that the

enhancement in human cells is triggered by persistent damage in actively transcribed genes. The enhancement is

also reduced or absent in SV4O-transformed cells and cells expressing the human papilloma virus ('WV) E7 gene,

but not in Li-Fraumeni syndrome (LFS) cells or cells expressing the 'WV E6 gene. Since SV4O-transformation and

HPV E7 expression both abrogate the retinoblastoma (pRb) family of proteins, whereas 'WV E6 abrogates p53 and

LFS cells express mutant p53, these results indicate that the enhanced expression depends on one or more of the

pRb family of proteins, but not on p53. We have also used recombinant Ad expressing a lacZ reporter gene as a

probe for DNA repair in human cells. Using this approach we have examined constitutive as well as inducible DNA

repair of a UV-damaged reporter gene in human cells. We detected enhanced host cell reactivation (HCR) of a UVdamaged

reporter gene in pre-heat-shock treated or pre-UV treated TCR proficient but not in pretreated TCR

deficient human fibroblasts or LFS cells. These results suggest the existence of an inducible repair response for UVdamaged

DNA in human cells which is dependent on the TCR pathway of NER and the wild type p53 tumour

suppressor. These results have important implications for the use of recombinant Ad-based expression vectors

under the control of the CMV promoter in gene therapy for cancer when used in combination with DNA damaging

agents.

I. Introduction

Adenovirus (Ad) vectors are a very efficient method

for delivering foreign genes into mammalian cells both in

vitro and in vivo (Hitt et al, 1997) and show great promise

for gene therapy of cancer (Boulikas, 1998; Stewart et al,

1999). Ad infects both dividing and non-dividing cells in a

wide variety of tissues and cell types of many different

species.


Rainbow et al: Recombinant adenoviruses as expression vectors

The Ad genome is relatively easy to manipulate using

standard molecular biology techniques such that both

replication proficient and replication deficient vectors can

be easily produced and putified on a large scale (Graham

and Prevec 1991; Hitt et al, 1995). Replication proficient

Ad vectors with only the early region 3 (E3) deleted can

accommodate up to about 4 kb of foreign DNA, whereas

replication deficient Ad vectors deleted in both E3 and El

can accommodate up to about 8 kb.

Several reports have proposed the use of Ad

transgene delivery in combination with radiation therapy

and chemotherapy (for a recent review see Stackhouse and

Buchsbaum 2000). To combine gene therapy and radiation

therapy or chemotherapy into an effective combination of

modalities for the treatment of cancer it is essential to

understand the effects of radiation treatment and

chemotherapy treatment of cells and transgenes on

transgene expression. We have used a recombinant

nonreplicating human Ad, either Ad5HCMVsp1lacZ

(Morsy et al, 1993) or AdCA35 (Addison et al, 1997),

expressing the lacZ gene under the control of the

cytomegalovirus (CMV) immediate early promoter to

assess the effects of cytotoxic agents on the expression of

a reporter gene in mammalian cells. Using this approach

we are able to examine both constitutive and inducible

expression of the reporter gene in cells treated with DNA

damaging agents (Figure 1 and 2).

In addition we have used the same non-replicating

Ad expressing the lacZ reporter gene as a probe for DNA

Figure 1. Enhanced expression of a

recombinant adenovirus based reporter gene

following pretreatment of human cells with

UV. Cells were seeded in 96 well microtitre

plates at a density of 2 x 10 4 cells/well, 18-24

h to treatment. For UV treatment of cells, the

growth medium was aspirated and replaced

with 40 ml phosphate buffered saline (PBS)

and the cell monolayers were either left

untreated or UV-irradiated with a range (A to

D) of fluences. UV irradiation of cells was

performed using a germicidal lamp (General

Electric model G8T5) emitting predominantly

254 nm at a fluence rate of I J/m 2 /s. After UV

treatment, cells were either infected or at

mock infected for 90 mm at 37 ºC with

untreated Ad5HCMVSPlIaCZ in a total

volume of 40 ml and the infected cells were

incubated for a period of time (usually 12 - 48

h) before harvesting and scoring for !galactosidase

activity as reported previously

(Francis and Rainbow 2000). Lysates from

mock infected wells served as a measure of

background levels for !-galactosidase activity.

repair in mammalian cells. Using this approach were are

able to examine both constitutive and inducible DNA

repair of a UV-damaged reporter gene in several different

mammalian cell types including normal human fibroblasts,

repair deficient human fibroblasts and several different

human tumour cells (Figures 3 and 5).

II. Recombinant adenovirus expression

vectors for gene transfer into mammalian

cells

A. Adenovirus based transgene expression

levels in mammalian cells

The level of expression in cells infected by Ad

vectors is greatly influenced by the promoter controlling

expression of the transgene. Xu et al, (1995) have

demonstrated that the human CMV immediate early

promoter directs the highest level of expression in the

widest variety of mammalian cell types in vitro when

compared to that directed by the human b-actin, Ad major

late, and SV4O early and late promoters. In addition,

Addison et al, 1997 showed that the murine CMV

immediate early promoter (Dorsch-Hasler et al, 1985) is

also an extremely strong promoter and rivals the human

CMV promoter for in vitro expression in both murine and

human cells. In vivo, the highest level of expression

reported to date occurred following intravenous delivery


to the mouse of an Ad vector under the control of the

human CMV promoter (Kolls et al, 1994).

B. Enhanced expression of a lacZ reporter

gene driven by the CMV immediate early

promoter following pretreatment of human

cells with DNA damaging agents

The CMV immediate early (IE) promoter is one of

the most commonly used promoters in eukaryotic

expression vectors, due primarily to its ability to yield

high expression levels in many different mammalian cell

types. Both stress-activated MAP protein kinases

(Bruening et al, 1998) and ionising radiation (Tang et al,

1997) can each up regulate expression of transgenes

driven by the CMV promoter. Our protocol for assessing

transgene expression in cells treated with DNA damaging

agents is outlined in Figure 1. We report that up

regulation of transgene expression from the CMV

promoter in human cells also results from pretreatment of

cells with UV light (Francis and Rainbow 1997, 2000,

Figure 2) or cisplatin (Francis 2000). UV radiation results

in damage to DNA and the activation of cell surface

receptors and their downstream signalling pathways.

Although UV-induced DNA damage acts to directly block

transcription, and cellular RNA levels immediately

decrease following UV exposure (Mayne and Lehmann

1982), the expression of several cellular and viral genes

are enhanced following UV exposure (Herrlich et al, 1994;

Bender et al, 1997). These UV-inducible genes can be

divided into those which respond immediately after UV

exposure and those which have a response which is

delayed and not observed until several hours after UV

exposure (Herrlich et al, 1994; Bender et al, 1997). While

the extent of the immediate response appears to depend on

the magnitude of the initial UV insult, the strength and

duration of the delayed response appears to be affected by

the cells ability to repair UVinduced DNA damage,

particularly to its DNA (reviewed in Bender et al, 1997).

Evidence for this comes from studies using cell lines

which are deficient in repair of UV-damaged DNA

(Miskin and Ben-Ishai 1981; Blattner et al, 1998).

UV-induced up regulation of the CMV driven

transgene in human cells appears to be a delayed response

and expression from CMV-driven constructs is enhanced

following lower UV exposures to TCR deficient compared

to TCR proficient human fibroblasts (Francis and

Rainbow 1997, 2000; Figure 2). In addition, pre-infection

of human fibroblasts with a UV-damaged Ad construct

containing an actively transcribing gene can induce

expression from a CMV driven transgene, while preinfection

with a UVdamaged Ad control vector which

does not contain an actively transcribing gene (which

presumably has minimal transcription activity) does not

(Francis and Rainbow 2000). Taken together, these data

strongly suggest that persistent and unrepaired damage in

active genes plays a direct role in eliciting enhanced

Gene Therapy and Molecular Biology Vol 5, page 89

expression from CMV promoters. It is possible that it is

the persistent stalling of RNA pol II at sites of unrepaired

damage which acts as a trigger for this response following

UV exposure as has been suggested for other UV-induced

cellular responses (Yamaizumi and Sugano 1994;

Ljungman and Zhang 1996; Ljungman et al, 1999).

The p53 protein has been implicated in numerous

cellular responses to UV, including DNA repair, and can

regulate the expression of a large number of cellular genes

(reviewed in McKay et al, 1999, Lakin and Jackson 1999,

elDeiry 1998). We have examined UV-induced expression

of a CMV-driven reporter construct in HeLa cells, 5V40transformed

fibroblasts, Li-Fraumeni syndrome (LFS)

fibroblasts, and spontaneously immortalised LFS sublines

(Francis and Rainbow 2000).

These cells have impaired p53 function as a result of

human papilloma virus (HPV) E6-expression (Seedoif

1987; Scheffner et al 1990; Mietz et al, 1992), SV4O large

Figure 2. Enhanced expression of tbe CMV-driven bgalactosidase

transgene in Ad5HCMVSp1lacZ following pretreatment

of human fibroblasts with UV light. !-galactosidase

reporter activity was quantitated 24 h following UV irradiation

and subsequent infection at 10-20 plaque forming units per cell

of normal (GM 38 (!)), XP-A (GM XP12BE (")), and CS-B

(CS lAN (#)) fibroblasts with a highly purilied preparation of

AdlHCMVsp1IacZ. Values were normalised to unirradiated

controls. Each point is the average of 2 independent experiments

(+1- SEM), each performed in 6 replicates. Adapted from

Rainbow and Francis 2000


Rainbow et al: Recombinant adenoviruses as expression vectors

T antigen (SV40LT) expression (Segawa et al, 1993),

geamime transmission of a mutant p53 allele (Malkin et

al, 1990), and spontaneous loss of the wild type p53 allele

from LFS fibroblasts (Yin et al, 1992), respectively.

Although no UV induced expression of the CMV-driven

lacZ gene from Ad5HCMVsp1IacZ was observed in any

SV404ransformed line examined, a significant UVenhancement

of reporter expression was observed in both

ReLa and all LFS cell strains (Francis and Rainbow,

2000). These data suggest that p53 does not play an

essential role in the UV-induced expression from CMV

promoters, whereas some protein or pathway altered by

SV4O-transformation does play an essential role in this

response. Candidate proteins altered by SV40transfonnation

include members of the retinoblastoma

(Rb) family, which are known to play important roles in

stress signalling, repair, and transcription (along with

several other pathways). Since the pRb family of proteins

are also altered in HeLa cells due to expression of the

HPV E7 gene, the results of UV-enhanced expression of

the reporter in HeLa cells n'ight suggest that pRb does not

play an essential role. However, it has been reported that,

although HPV E7 binds pRb and its family members p107

and p130, only pRb is targeted for degradation, while the

levels of the two other proteins are not significantly

altered by E7 expression (Berezutskaya et al, 1997)

Furthermore, even pRb remains at significant levels and

accumulates still higher in ReLa cells following UV

exposure (Pedley et al, 1996). Thus it is possible that

sufficient levels of one or more of the pRb family of

proteins remain in HeLa cells to induce expression of

reporter activity. In addition, more recent experiments

using pRb-null or p53-null mouse embryo fibroblasts

(Francis and Rainbow 1999a; Francis 2000) and human

fibroblasts transformed with the HPV E7 or HPV E6 gene

(Francis and Rainbow, unpublished data) support our

earlier data indicating that p53 does not play an essential

role and suggest a role for the pRb protein(s) in UVenhanced

expression from a CMV-driven reporter. Since

the pRb proteins and other pathways involving stressactivated

MAP protein kinases (Bruening et al, 1998) are

frequenfly altered in human tumour cells, the outcome of

protocols combining gene therapy and radiotherapy or

chemotherapy using CMV-driven transgenes may be

tumour cell4ype specific.

Other reports have suggested the use of ionizing

radiation-activated gene therapy vectors for combined

gene therapy and radiotherapy (Joki et al, 1995; Seung et

al, 1995; Mauceri et al, 1996; Takahashi et al, 1997; Tang

et al, 1997; Marples et al, 2000; Scott et al, 2000;

Stackhouse and Buchsbaum 2000). Gamma-ray enhanced

expression from a reporter gene is both cell-type specific

and promoter specific (Tang et al, 1997; Marples et al,

2000) and gamma-ray enhanced expression from the CMV

promoter was only detected when cells were irradiated and

the transgenes were not. No amplification of the transgene

was detected when both host cells and transgene were

subjected to irradiation (Tang et al, 1997). In contrast,

gamma-ray induced expression of a plasmid born reporter

gene under the control of a synthetic radio-responsive

transcriptional enhancer could be repeated by additional

radiation treatments in human tumour cells (Marples et al,

2000). These results suggest that depending on the

promoter of the transgene, the timing sequence of genetherapy

and radiotherapy or chemotherapy may be an

important determinant of clinical outcome. It thus appears

likely that with additional information on the various

parameters controlling the up regulation of transgene

expression, adenovirus-mediated gene therapy and

radiotherapy or chemotherapy can potentially be

formulated into synergistic protocols for the treatment of

cancer.

III. Recombinant adenovirus as a

probe for DNA repair in mammalian cells

A. Nucleotide excision repair of damaged

DNA The integrity of the human genome is constantly

being compromised by alterations induced by a wide

variety of exogenous physical and chemical agents, as

well as by products of cellular metabolism. Several highly

conserved repair pathways have evolved to remove

damage from cellular DNA and disiuption of each of these

DNA repair pathways is associated with carcinogenesis

(as reviewed in Friedberg et al, 1995). The nucleotide

excision repair (NBR) pathway repairs a wide range of

bulky DNA adducts induced by numerous carcinogenic

and antineoplastic compounds, including ultraviolet (UV)

irradiation from the sun. NER can be divided into two

interrelated subpathways: (1) transcription coupled repair

(TCR) which preferentially removes DNA damage at a

faster rate from the transcribed strand of actively

transcribed genes, and (2) global genomic repair (GGR)

which removes damage from throughout the entire

genome and from the non-transcribed strand of active

genes (Mellon et al, 1986, 1987). Individuals with the

genetic diseases xeroderma pigmentosum (XP) have some

deficiency in NER and show an increased incidence of a

variety of skin cancers (Wei et al, 1993, Kraemer et al,

1994). Several additional links between NER and

carcinogenesis have been reported. Mutations in the p53

tumour suppressor gene, the most commonly altered gene

in malignancy (Hollstein et al, 1991), have also been

shown to result in reduced NER (Ford and Hanawalt 1995;

Smith et al, 1995; Wang et al, 1995; McKay et al, 1997;

McKay et al, 1999; Therrien et al, 1999). Mismatch repair

proteins have also been implicated in NER (Mellon et al,

1996) and mutations within genes encoding these proteins

are associated with hereditary non-polyposis colorectal

cancer (Fishel et al, 1993). Decreased NER has also been

observed in a variety of tumour and transformed cell lines

(Squires et al, 1982; Rainbow 1989). These reports

suggest that DNA repair mechanisms are disrupted


in tumour cells and that DNA repair contributes to

resistance to neoplasia. XP is composed of a minimum of

seven complementation groups (XP-A-G), each displaying

a general deficiency in NER which compromises at least

the GR subpathway and usually TCR as well. The

exception is XP-C which retains viable TCR in spite of a

severe deficiency in GGR (Venema et al, 1990; Venema et

al, 1991). The genetic disease Cockayne syndrome (CS) is

also associated with a deficiency in NER, although unlike

XP patients, CS patients do not display an increased

risk of skin cancer. Two complementation groups of CS

(CS-A and CS-B) have been identified, each of which

exhibits a deficiency in TCR, while the GGR pathway

appears to function normally (Venema et al, 1990a; van

Hoffen et al, 1993). The XPB and XPD proteins are

components of the transcription factor THIH (Schaeffer et

al, 1993, Schaeffer et al, 1994) which plays a role in both

NER and transcription by RNA polymerase II (RNAPII)

(Drapkin et al, 1994). The CSA and CSB proteins coimmunoprecipitate

(Henning et al, 1995) and are required

for TCR. CSA interacts with THIH directly (Henning et

al, 1995) whereas CSB does so via XPG (Iyer et al, 1996).

TFIIH, XPG and the RPAIXPA/XPFIERCC1 complex

(Park and Sancar 1994; Matsuda 1995) are required for

both subpathways of NER, whereas the XPC/HHR23B

complex (Masutani et al, 1994) appears to be required

only for GGR (Venema et al, 1990a; Venema et al, 1991;

Evans et al, 1993).

Gene Therapy and Molecular Biology Vol 5, page 91

Figure 3. Host cell reactivation of a UVdamaged

recombinant adenovirus based

reporter gene in human cells. Cells were

seeded in 96 well microtitre plates at a

density of 2 x 10 4 cells/well and 18-24 h

later infected for 90 mm at 37 °C with

unirradiated or UV-irradjated AdS

HCMVSp1lacZ in a total volume of 40

ml. AdSHCMVsp1lacZ was UV-irradiated

with range of Iluences (A to D) using a

germicidal lamp (General Electric model

G8T5) emitting predominantly at 254 nm

at an incident fluence rate of 2 J/m 2 /s. 12-

48 h later, infected cells were harvested

and scored for !-galactosidase activity as

reported previously (Francis and Rainbow

1999). Lysates from wells infected with

heavily irradiated AdSHCMVspIIacZ

(10,000 J/m 2 ), X, served as a measure of

background levels for !-galactosidsse

activity

.

B. Host cell reactivation of DNA-damaged

reporter genes in mammalian cells

Host cell reactivation (HCR) of reporter gene activity

has been assessed in mammalian cells using either

recombinant Ad or plasmid constructs by a number of

different laboratories. For plasmid constructs, this

approach has typically involved the transfection of a

DNA-damaged plasmid carrying a reporter gene into the

cells of interest (Protic et al, 1988; Ganesan and Hanawalt

1994; Smith et al, 1995, Stevsner et al, 1995; Ganesan et

al, 1999). Since primary human fibroblasts take up

exogenous DNA 10-100 times less efficiently than either

rodent or human cell lines derived from tumour tissue or

transformed by viral antigens (Murname et al, 1985;

Canaani et al, 1986; Hoejmakers et al, 1987), most

experiments examining the reactivation of plasmid born

UV-damaged reporters has been peiformed in tumour and

transformed cell lines rather than primary human

fibroblasts. However, many tumour and transformed cells

have been reported to have a reduced DNA repair capacity

for UV-damaged DNA (Squires et al, 1982; Rainbow

1989; McKay and Rainbow 1996; McKay 1997).

Furthermore, the cellular response to DNA damage is

stimulated by at least some transfection procedures

(Renzing and Lane 1995; Seiget et al, 1995), leading to

cell cycle arrest (Renzing and Lane 1995), suggesting that

the transfection procedure itself may affect the outcome of

DNA repair experiments. In contrast, recombinant nonreplicating

Ad reporter constructs have the ability to infect

and express high levels of recombinant gene products in

most cell types including primary human fibroblasts.

Furthermore, non-replicating Ad reporter constructs do not

appear to elicit the DNA damage response, or inhibit host


Rainbow et al: Recombinant adenoviruses as expression vectors

DNA synthesis following infection (Blagoskionnay and

el-Deity 1996).

Recombinant non-replicating Ad constructs have been

used to introduce UV-damaged (Valerie and Singhal 1995;

McKay and Rainbow 1996; McKay et al, 1997; Francis

and Rainbow 1999) or cisplatin damaged (Moorehead et

al, 1996) reporter genes into non-treated human and rodent

cells in order to assess the repair of damaged DNA in the

absence of cellular stress using bost cell reactivation

(HCR) of reporter gene activity as an endpoint. UVinduced

lesions in the template strand of active genes

inhibit progression of RNA polymerase II (Donahue et al,

1994) and a single UVinduced cyclobutane pyrimidine

dimer (CPD) is thought to be sufficient to inhibit reporter

gene expression (Protic-Sabaji and Kraemer 1985; Francis

and Rainbow 1999). UVinduced DNA lesions are

removed from plasmid born (Ganesan and Hanawalt 1994,

Ganesan et al, 1999) and recombinant adenovirus born

(Boszko 2000, Boszko and Rainbow 2000) reporter genes

when introduced into repair proficient human cells and the

removal is reduced when the same reporter genes are

introduced into NER deficient

Figure 4. Nucleotide excision repair deficent cell strains show

reduced HCR of UV-irradiated reporter activity. Lines represent

repair proficient normal GM 3440 ("); and repair deficient XP-C

(XP3BE (!)), XP-G (XP2BJ ($)) and CS-B (CS lAN ("))

primary human fibroblasts. Each point is the average of 4

replicates, error bars represent one standard error. Untreated cells

were infected with unirradiated or UV-irradiated virus at 10-20

plaque forming units per cell and scored for !-galactosidase

activity 40-44 h later. Adapted from Francis and Rainbow 1999.

ceUs. Thus HCR of reporter gene activity is thought to

require the repair of transcription blocking DNA lesions

and reflectrepair of DNA lesions in the transcribed strand.

A typical protocol to examine HCR of reporter gene

activity for UV-irradiated recombinant Ad expressing the

lacZ gene is shown in Figure 3. Using this approach we

have shown that HCR of reporter gene activity for UV

damaged DNA is reduced in several different nucleotide

excision repair (NER) deficient cells of both human and

rodent origin, including skin fibroblasts from patients with

xeroderma pigmentosum from complementation group C

(XPC) which showed HCR levels ranging from 25-75%

that obtained in NER proficient normal primary human

fibroblasts (Figure 4, McKay and Rainbow 1996; Francis

and Rainbow 1999). The result for XP-C is surprising

since XP-C cells are reported to be proficient in

transcription coupled repair (TCR) and thus would be

expected to reactivate UV induced lesions in the

transcribed strand of the reporter gene. Blockage of RNA

polymerase II by UV induced DNA lesions does not

appear to be sufficient to promote the preferential repair of

these transcription blocking lesions in non-UV-treated

XP-C cells (McKay and Rainbow 1996; Francis and

Rainbow 1999). Also of interest was the finding that CS

fibroblast strains retained a considerable ability to repair

the UV-damaged reporter gene in non-treated cells (57-

90% of normal levels) in spite of their being characterized

as deficient in repair of the transcribed strand of active

genes following UV irradiation of the cell (Francis and

Rainbow 1999). It is therefore apparent that in the absence

of UV exposure to the cell, damage in the transcribed

strand of the recombinant Ad-based reporter gene is

repaired to a large extent by the global genomic repair

(GGR) pathway of NER in primary human fibroblasts,

although significant TCR must also occur since HCR is

also reduced in the CS strains. Since the Ad genome is

approximately 5 orders of magnitude smaller than the

human genome, the number of lesions introduced into

cells with a UV-irradiated virus in our experiments is

minimal compared to the number introduced into the host

cell genome following UV treatments used to examine

repair in cellular DNA.

Several studies using plasmid born reporter genes

have reported decreased HCR in NER deficient XP cells

(Lehmann and Ooman 1985; Protic-Sabljic and Kraemer

1985; Barrett et al, 1991) and CS cells (Barrett et al 1991,

Klocker et al, 1985) compared to repair proficient human

cells. These studies have generally used 5V40-transformed

repair deficient XP and CS cell lines and repair proficient

"normal" cell lines derived from human tumours rather

than primary fibroblast strains. SV4O transformed cells

and many human tumours have alterations in the p53

tumour suppressor, the pRb tumour suppressor and other

stress activated pathways which have been shown to affect

the GGR and/or TCR pathway of NER (Ford and

Hanawalt 1995, 1997; ; McKay et al, 1997; 1999; Ford et

al, 1998; Therrien et al; 1999).


Therefore, a direct comparison of the relative contribution

of TCR and GGR to repair in the transcribed strand of

plasmid born reponter genes using 5V40-transformed cells

and tumour cells with that obtained using an Ad based

reponter in primary human fibroblasts may not be

appropnate.

B. Enhanced host cell reactivation of a

UV-damaged reporter gene following

pretreatment of mammalian cells with DNA

damaging agents

1. Evidence for inducible DNA repair.

Examination of DNA repair in mammalian cells generally

requires that the cells are treated with a DNA damaging

agent in some manner, which makes it difficult to

determine if the repair pathways are constitutively active

or induced by the DNA damaging agent. In contrast, the

use of viral probes for DNA repair allows the virus and

cell to he treated with a DNA damaging agent

independently and thus allows an examination of both

constitutive and inducible pathways affecting survival of

the virus or expression of the reponter gene. There are

manyreponts showing that pretreatment of a variety of

different mammalian cells with chemical or physical DNA

damaging agents results in an increased survival (or

enhanced reactivation) for several nuclear replicating

Gene Therapy and Molecular Biology Vol 5, page 93

Fignre 5. UV-enhanced host cell reactivation of

a UV-damaged recombinant adenovirus based

reporter gene in hmnan cells. Cells were seeded

in 96 well microtitre plates at a density of 2 x

10 4 cells/well, 18-24 h prior to UV-treatrnent of

cells. The growth medium was then aspirated,

replaced with 40 ml phosphate buffered saline

(PBS) and sets of cell monolayers were either

UV-irradiated with a fluence F or were mockirradiated

and received no UV. After treatment,

both UV-irradiated and non4rradiated sets of

cells were infected for 90 min at 37 ºC in a total

volume of 40 ml with unirradiated

Ad5HCMVsp1lacZ or AdiHCMVsp1lacZ

which had been UV-irradiated with arange of

fluences (A to D). Infected cells were incubated

for a period of time (usually 12 - 48 h) before

harvesting and scoring for b-galactosidase

activity as reported previously (Francis and

Rainbow 1999). Lysates from wells infected

with heavily irradiated Ad5HCMVspllacZ

(10,000 J/m 2 ), X, served as a measure of

background levels for !-galactosidase activity.

UV irradiation of cells and virus was as for

Figures 1 and 3 and as described previously

(Francis and Rainbow 1999). Enhanced HCR in

UV-treated compared to non-treated cells

suggests inducible repair of the UV-damaged

reporter gene

.

double stranded DNA viruses damaged by UV or ionising

radiation (for a review see Rainbow 1981, Defais et al,

1983). It has been suggested that the enhanced reactivation

of DNA-damaged viruses results, in part at least, from an

induced DNA repair pathway (Jeeves and Rainbow 1983,

1983a, 1983h; Bennett and Rainbow 1988; Brown and

Cenrutti 1989).

We and others have examined HCR of a UV-damaged

reporter gene in pre-treated compared to non-treated cells

(McKay et al, 1997; Li and Ho 1998; Francis and

Rainbow 1999; Boszko and Rainbow 2000). A typical

protocol for these enhanced HCR experiments is shown in

Figure 5 Using this approach we show that pre-treatment

of normal human fibroblasts with low UV fluences

(McKay et al, 1997; Francis and Rainbow 1999) as well as

heat shock (McKay and Rainbow 1996; McKay et al,

1997) results in enhanced HCR of the UV-damaged

reporter suggesting the presence of inducible DNA repair

in human cells. Prior exposure of cells to low UV fluences

or heat shock resulted in enhanced HCR for expression of

the UV-damaged reporter gene in normal and XP-C

fibroblast strains, but not in TCR deficient XP and CS

strains (Figure 6). These results suggest that UV or heat

shock treatment results in an induced repair of UVdamaged

DNA in the transcribed strand of the reporter

gene in


Rainbow et al: Recombinant adenoviruses as expression vectors

Figure 6. Pre-UV irradiation of cells results in enhanced HCR of

UV-irradiated reporter activity in normal and XP-Cbut not in

other TCR deficient cells. Results of typicalexperiments

representing unirradiated (") and UV-irradiated (#) primary

human fibroblasts. UV exposures to cells are indicated on the

figure and cell strains presented are GM 3440 (normal), XP3BE

(XP-C) CS1BE (CS-B), and XP2BI (XP-G). Immediately

following UV exposure cells were infected with unirradiated

orUV-irradiated virus at 10-20 plaque forming units per cell and

scored for !-galactosidase activity 40-44 h later. Each point is

the average of 4-6 replicates, error bars represent one standard

error. Adapted ftom Francis and Rainbow 1999.

Figure 7. Pre-UV irradiation of cells results in enhanced HCR of UV-irradiated reporter activity in normal but not in Li-Fraumeni

syndrome cells. Results of typical experiments representing unirradiated (") and UV-irradiated with 15 J/m 2 (#) normal human

fibroblasts and Li- Fraumeni syndrome (LFS) cells. Cell strains and cell lines presented are GM 9503 (normal), LFS: 087 mutlwt

(heterozygous for a mutation in p53), LFS 087 mut (expressing only mutant p53). Immediately following UV exposure cells were

infected with unirradiated or UV-irradiated virus at 10-20 plaque forming units per cell and scored for !-galactosidase activity 40-44 h

later. Each point is the average of 3-6 replicates, error bars represent one standard error. Adapted from McKay et al, 1997 and Francis

2000.


normal and XP-C cells through an enhancement of TCR

or through a mechanism which involves the TCR

pathway. More recently we have used a novel quantitative

polymerase chain reaction (PCR) technique to examine

direct removal of UV-induced photoproducts from lacZ

reporter gene in AdHCMVsp1lacZ following infection of

human fibroblasts. Using this technique we show a

significant removal of UV photoproducts after infection of

normal human fibroblasts, hut a reduced removal of

lesions, compared to normal fibroblasts, after infection of

NER deficient XP and CS fibroblasts. In addition, we

show that pre-UY exposure of normal human fibroblasts

results in an enhanced rate of removal of photoproducts

from the reporter gene, giving evidence that UV-enhanced

HCR for expression of the UVdamaged reporter gene

results from enhanced removal of UV-induced lesions

from DNA (Boszko 2000, Boszko and Rainbow 2000).

Other evidence for damage-induced DNA repair pathways

in mammalian cells comes from a number of studies

including the enhanced DNA repair capacity of

mammalian cells following carcinogen treatment (Protic et

al, 1988), the p53-mediated enhancement of NER by the

DNA damaged induced GADD45 gene (Smith et al, 1996,

Smith et al, 1994) and the identification of a novel DNA

repair response which is induced by irradiation of cells at

the GuS border (Leadon et al, 1996). Pretreatment of

normal human lung fibroblasts with the drug emodin

enhances NER of UV and cisplatin damaged DNA (Chang

et al, 1999) and pretreatment of cells with dinucleotides

prior to UV irradiation increased the repair of UV-induced

DNA damage as assessed by unscheduled DNA synthesis

(Eller et al, 1997). Pre-treatment of human cells with

quinacrine mustard resulted in an enhanced removal of

UV-induced CPD from both the transcribed and the nontranscribed

strand of the p53 gene (Ye et al, 1999), also

giving evidence for an inducible NER response in human

cells. Most of these studies provide evidence for an

induction of the GGR rather than the TCR pathway of

NER (as reviewed in McKay et al, 1999). Some

mammalian cells exhibit a hypersensitivity to low doses of

x-rays or cisplatin, but increased resistance following

higher doses of these agents (Skov et al, 1994, Joiner et al,

1996, Caney et al, 2000). The increased radioresistance at

higher x-ray doses is absent in some DNA repair deficient

cell lines (Skov et al, 1994), and hypersensitivity at low

doses of x-rays or cisplatin can he removed by

pretreatment of cells with "priming doses" of a DNA

damaging agent (Joiner et al, 1996, Caney et al, 2000),

suggesting an inducible DNA repair response in

mammalian cells. Pre- exposure of human cells with low

"priming" doses of ionising radiation leads also to an

enhanced removal of thymine glycols after higher doses

(Le et al, 1998) providing evidence for an inducible repair

of base damage in human cells.

Gene Therapy and Molecular Biology Vol 5, page 95

2. Evidence for the involvement of p53 in

NER

Over the past few years it has become clear that p53

and/or p53 regulated gene products contribute to NER of

UV-induced DNA damage in mammalian cells (Smith et

al, 1994, 1995, 1996; Ford and Hanawalt 1995, 1997;

Wang et al, 1995; McKay et al, 1997, 1997a; Ford et al,

1998; Li and Ho 1998). We have reported that UV and

heat shock enhanced HCR for expression of the UVdamaged

reporter gene was absent in Li-Fraumeni cells

expressing mutant p53 (Figure 7, McKay et al, 1997,

1999) indicating a role for p53 in the induced DNA repair

response. A similar p53 dependent enhancement in HCR

for a CMV driven plasmid based and UV-damaged

reporter gene has been reported in UVB-treated murine

fibroblasts (Li and Ho 1998). In addition, thymine

dinucleotides have been shown to induce the reactivation

of a UV-damaged reporter gene under control of the

SV4O early promoter, by a process which may also

involve p53 (Eller et al, 1997). Furthermore, Huang et al

1998 report that transcription from a p53 driven promoter

in the presence of wild-type p53 results in up regulation of

both transcription and repair of a UV-damaged reporter

gene, and that the enhanced DNA repair of the reporter

gene is a separate and distinct activity of p53, but is

dependent on p53 driven transcription. As discussed

above, UV and heatshock enhanced HCR of the

recombinant Ad-based and UV-damaged reporter gene are

thought to reflect an induction of TCR or a repair process

dependent on TCR. The absence of UV and heat-shock

enhanced HCR of the UV-damaged reporter in LFS cells

suggests further that either TCR or a repair process

dependent on TCR requires functional p53.

Hwang et al. 1999 have reported that transcription

from the p48 gene, which is mutated in GGR-deficient,

damage-specific DNA binding (DDB) protein deficient,

XP-E cells (Hwang et al, 1998), is up regulated (in a p53dependent

manner) in response to UV treatment in human

cells. This provides a model for a UV-inducible GGR

response in human cells which is dependent on p&8

transcription. A UV-induced increase in p48transcription

would require removal of UV-induced lesions from the

p48 gene and therefore be dependent on TCR as has been

reported for other p53 responsive genes (McKay et al,

1999, McKay and Ljungman, 1999). Thus the UV-induced

up regulation of p48 leading to enhanced GGR would be

expected to be dependent on both wild-type p53 and TCR.

It is thus possible that the p53 and TCR dependent UVenhanced

repair of the UV-damaged reponter gene results

from an up regulation of GGR in the transcribed strand of

the reporter gene mediated by a UVinduced up regulation

of the p48 gene product. Previous reports have also

suggested that the DDB protein is responsible for the

enhanced repair of UV-damaged expression vectors

(Protic et al, 1989). However, some recent reports suggest

that TCR also may be up regulated by a p53 dependent

mechanism. Pre-treatment of human cells with low doses


Rainbow et al: Recombinant adenoviruses as expression vectors

of quinacrine mustard resulted in an enhanced rate of

removal of CPD by NER (Ye et al, 1999). Although the

enhanced rate of removal was greater for non-transcribed

strand, an enhanced rate of removal also occurred for the

transcribed strand of an exon 9 portion of the p53 gene,

such that both GGR and TCR may be up regulated by pretreatment.

In addition, Therrien et al, 1999 showed that the

rate of repair of UV-induced CPD was reduced along both

the transcribed and the non4ranscribed strands of the p53

and/or c-jun loci in Li-Fraumeni syndrome (LFS) cells

expressing mutant p53 and human fibroblasts expressing

the human papilloma virus (HPV) E6 oncoprotein that

functionally inactivates p53. The reduction in the rate of

CPD repair for the LFS cells compared to normal cells

was considerably greater in the transcribed (6 fold)

compared to the non-transcribed strand (3 fold) providing

evidence that both TCR and GGR are dependent on wildtype

p53 in UV-irradiated human cells. Our results for

UV-enhanced HCR of a UV-damaged reporter gene are

therefore also consistent with a model in which pretreatment

of cells with UV results in an up regulation of

TCR through a p53 dependent mechanism. It is possible

that a p53 dependent up regulation of both GGR and TCR

can contribute to UV-enhanced HCR of a UV-damaged

reporter gene.

3. Gene therapy using Ad vectors

expressing p53 and p53 responsive genes

The p53 tumor supressor and several p53 responsive

genes also play a role in arresting the cell cycle at the GI

checkpoint in response to DNA damage and in inducing

apoptosis in cells that have received extensive radiation

damage (for a review see Hartwell and Kastan, 1994;

Hinds and Weinberg, 1994). The p53 gene and other

tumor suppressor genes have been found to be mutated in

a variety of tumours and many of these mutations are

thought to be responsible for the proliferative capacity and

resistance of these cells to radiotherapy and

chemotherapy. On this basis, both p53 and the p53

responsive gene p21 waf1 have been proposed as gene

therapy vectors to prevent replication of tumor cells.

p21 waf1 is a member of the family of cyclin-dependent

kinase (CDK) inhibitors and plays a role in the

maintenance of the cell cycle checkpoints and cell

progression (Harper et al, 1993). Following infection of

cells with Ad expressing a p53 transgene in vitro, the

biological effects of p53 are readily detected, including the

upregulation of p21wafl, an overall growth suppression,

and an increased number of cells undergoing apoptosis for

a variety of tumour cell lines carrying p53 mutations

(Bacerietti and Graham, 1993; Liu et al, 1994; Yang et al,

1995). Furthermore, administration of p53 expressing Ad

vectors has been found to be efficacious in several tumor

models (Fujiwara et al, 1994; Lui et al, 1994; Yang et al,

1995). In vitro infection of a variety of tumour cells with

p21 waf1 recombinant Ad vectors induces a growth arrest at

the G 0/G1 checkpoint without inducing apoptosis (Eastam

et al, 1995; Katayose et al, 1995; Yang et al 1995) and

p21waf1 expressing Ad vectors have been reported to

suppress tumour growth in vivo (Eastam et al, 1995; Yang

et al, 1995).

Ad constructs expressing p53 have been suggested as

a means of sensitizing tumor cells to conventional

radiotherapy and chemotherapy (Fugiwara et al, 1994).

However, this approach may be detrimental in some

situations. Down regulation of the p53 responsive

GADD4S gene decreased DNA repair and sensitized cells

to UVirradiation and cisplatin (Smith et al, 1996) whereas

upregulation of the p53 responsive p21waf1 gene by Admediated

transgene expression results in an increased

resistance of cells to UV and cisplatin (McKay et al, 1998,

Bulmer and Rainbow, unpublished data). Recently it has

been reported that p53 expression protects against or

confers sensitivity to UV-induced apoptosis depending on

the timing of p53 expression relative to the genotoxic

stress (McKay et al, 2000). Thus it is possible that

upregulation of p53 and p53 responsive genes such as

p21waf1 and GADD45 through the use of gene therapy

vectors may result in the upregulation of p53 protective

functions, including DNA repair, resulting in an enhanced

resistance of tumor cells to radiation and chemotherapy.

Furthermore, we have found that expression of p53

regulated gene products is both positively and negatively

regulated by DNA damage depending on the cell type and

the extent of such damage (McKay et al, 1998). Therefore,

it may be difficult to predict the net effect of protective

and cytotoxic functions of p53 in combined therapies.

Acknowledgements

We thank Todd Bulmer, Cathy Hill, Jim

Stavropoulos, Katharine Sodek, Ihor Boszko and Colleen

Caney for their contributions to this work. This work was

supported by the National Cancer Institute of Canada with

funds from the Canadian Cancer Society.

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Gene Therapy and Molecular Biology Vol 5, page 101

Gene Ther Mol Biol Vol 5, 101-110, 2000

Chromatin remodeling and developmental gene

regulation by thyroid hormone receptor

Review Article

Laurent M. Sachs 1 , Peter L. Jones 2 , Victor Shaochung Hsia 2 , and Yun-Bo Shi 2,3

1 Laboratoire de Physiologie, MNHN, UMR CNRS 8572, PARIS cedex 05, FRANCE, and

2Unit on Molecular Morphogenesis, Laboratory of Molecular Embryology, National Institute of Child Health and Human

Development, NIH, Bethesda, MD USA

_________________________________________________________________________________________________

* Correspondence: Yun-Bo Shi, Building 18T, Rm. 106, NICHD, NIH, Bethesda MD, 20892; Tel: (301)-402-1004; Fax: (301)-402-

1323; E-mail: Shi@helix.nih.gov

Key Words: Xenopus laevis, Amphibian metamorphosis, histone acetylation, chromatin remodeling, thyroid hormone receptor

Received: 15 November 2000; accepted: 21 November 2000

Summary

Thyroid horruone (TH) receptors (TRs) are dual function transcription factors. They activate or repress

transcription in the presence or absence of TH, respectively. Using the Xenopus laevis oocyte as an in vivo system to

assemble TH target promoters into chromatin under conditions mimicking somatic cells, we have shown that

transcriptional repression by unilganded TR involves histone deacetylase while transcriptional activation by THbound

TR leads to chromatin disruption. Using Xenopus laevis development as a developmental model, we have

demonstrated that TR is constitutively bound to its target genes in chromatin. Transcriptional activation induced

by TH is accompanied by the release of at least one histone deacetylase and increase in local histone acetylation.

These studies together with the developmental expression profiles of TR genes suggest that TH-induced changes in

chromatin remodeling play an important role in the dual functions of TR in frog development: gene repression in

premetamorphic tadpoles when TH is absent and gene activadon during metamorphosis, a process induced by the

endogenously synthesized TH.

I. Introduction

Thyroid hormone (TH) plays important roles during

development (Shi, 1999). In humans, TH tectable in the

embryonic plasma by 6 months rises to high levels around

birth (Tata, 1997). is postembryonic period, extensive

tissue and organogenesis take place. TH deficiency during

human development leads to developmental, such as

mental retardation, short stature, and in the e form,

cretinism (Hetzel, 1989; Shi, l999).Likewise, TH is critical

for amphibian development. In fact, anurans depend upon

TH to develop into adult, frogs (Dodd and Dodd, 1976;

Shi, 1999). endogenous synthesis of TH leads to the f

giant tadpoles that cannot metamorphose ion of exogenous

TH to premetamorphic tadpoles causes precocious

metamorphosis. Furthermore, most, if not all, organs are

genetically predetermined to undergo specific changes and

these changes are organ autonomous. Such properties have

made anuran metamorphosis one of the best-studied

postemhryonic developmental process at morphological,

cellular, and biochemical levels and paved way for current

molecular investigations of the underlying mechanisms.

Here we summarize some recent advances from studies in

Xenopus laevis.

II. Chromatin remodeling by TRs

The biological effects of TH are mostly, if not

entirely, mediated by thyroid hormone receptors (TRs).

TRs belong to the superfamily of nuclear hormone

receptors, with two subfamilies of TRs in vertebrates,

TR! and TR". TR can be divided roughly into 5 domains,

A/B, C, D, E, and F, respectively, from the amino- to

carboxyl-terminus (Krust et al, 1986). The DNA binding

domain (domain C) is located in the amino half of the


protein and is the most highly conserved domain among

different receptors of the supeifamily. The large ligand

binding domain (domain F) is in the carboxyl half of the

protein and is conserved among TRs in different species.

The other domains vary in sizes and sequences among

different nuclear receptors. The N-terminal A/B domain is

highly variable in sequence and length, the shortest being

the TRs in Xenopus laevis (Yaoita et al, 1990). At least in

some TRs, this domain contains a transactivation function

(AF), although its role in amphibian TRs is unclear.

Another transactivation function domain is the AF-2

domain, which is located at the very C-terminus (F

domain and part of the E domain).

TH can both up- and down-regulate gene expression

in target tissues or cells. The vast majority of the known

TH response genes are up-regulated by the hormone and

most studies of receptor function have been on these upregulated

genes. The discussions here focus only on the

mechanisms for this class of genes.

Transcriptional activation by TH requires the binding

of TRs, most likely as heterodimers with RXRs (9-cis

retinoic acid receptors), to TREs (TII response elements)

present in the regulatory regions of the TH-response

genes. The binding of TREs by TR/RXR heterodimers is,

however, independent of TH both in solution and in

chromatin (Perlman et al, 1982; Wong et al, 1995). In the

absence of TH, TR/RXR represses transcription of target

promoters, while in the presence of TH, TR/RXR

enhances transcription from these same promoters

(Fondell et al, 1993; Tsai and O'Malley, 1994; Wong et al,

1995).

A. Chromatin disruption by liganded

TR/RXR

Most of the functional studies of hormone receptors

have been carried out in vitro or by transient transfection

experiments in tissue culture cells. However, genomic

DNA in eukaiyotic cells is associated with histones and

other nuclear proteins and assembled into chromatin.

Thus, to understand the mechanism of TR action, it is

important to use properly chromatinized templates.

We have made use of the ability of Xenopus oocyte to

assemble exogenous DNA into chromatin (Almouzni et al,

1990) to investigate the mechanism of TR action. When

single-stranded plasmid DNA is injected into a frog

oocyte nucleus, it is quickly replicated (1-2 hr) and

assembled into chromatin in a replication-coupled

chromatin assembly pathway, mimicking the chromatin

assembly process in somatic cells. The resulting template

often produces low level of transcriptional activity. In

contrast, when double-stranded promoter-containing

plasmid DNA is injected into an oocyte nucleus, it is

chromatinized more slowly (5-6 hr) with less well defined

nucleosome arrays such that the transcnption from the

promoter is often at high levels, Thus, by using different

forms of promoter-containing plasmid DNA, it is possible

to study the transcriptional regulation under different

Sachs et al: Gene repression and activation by TRs

chromatin conditions. Xenopus oncytes have little

endogenous TR to affect the transcription of a TREcontaining

promoter (Wong and Shi, 1995). However,

when exogenous Xenopus TRs and RXRs are cointroduced

into the oocytes by injecting their mRNA into

the cytoplasm, they can repress the transcription from both

single-stranded and double-stranded DNA containing a

TRE (Figure 1) (Wong and Shi, 1995; Wong et al, 1995;

Hsia et al, 2000). On the other hand, maximal regulation

by TH occurs when the single-stranded DNA is used. This

is mainly due to more effective repression of the promoter

by unliganded TR/RXR during replication-coupled

chromatin assembly process (Wong et al, 1995). We have

used two independent assays to investigate the effects of

TR/RXR on chromatin structure (Wong et al, 1997a).

These are the plasmid DNA supercoiling assay for

measuring nucleosomal density andlor DNA wrapping

conformation in the plasmid minichromosome, and the

micrococcal nuclease digestion assay for determining the

nucleosomal array stmcture of the plasmid

minichromosome.

Both assays have shown that the binding of TR/RXR

alone deacetyla has little effect on the gross chromatin

structure. On the by unlig other hand, the addition of TH

to TR/RXR-containing (Figure 1), templates causes the

disruption of the ordered chromatin. Furthermore, this

chromatin disruption occurs even when transcription

elongation is blocked. Thus, TH-bound leads TR/RXR

heterodimers can disrupt chromatin structure through an

active process, although the nature of the disruption is yet

unclear.

Figure 1. Histone deacetylases is involved in transcnptional

repression by TR. A double-stranded plasmid (pHL10)

containing HIV- 1 promoter, which is regulated by TH (Hsia et

al, 2000), was microinjected into frog oocytes with or without

prior injection of TR"/RXR! mRNAs. The injected oncytes

were treated with or without 5 ng/ml TSA or 50 nM T3 as

indicated and the promoter activity was analyzed by primer

extension. Note that the addition of TSA activated the promoter

slightly. The presence of unliganded TR/RXR repressed the

promoter activity. The addition of either T3 or TSA reversed the

inhibition and further activated the promoter, supporting a role of

histone deacetylase in the repression by unliganded TR/RXR.

The plasmid pCMV-CAT containing a cytomegatovirus

promoter driving the expression of CAT reporter gene was used

as an internal control (Kass et al, 1997).


B. Regulation of histone acetylation levels

through histone acetyltransferases and

deacetylases

Both transcriptional repression by unliganded TRs

and activation by TH-bound TRs involve TR-interacting

cofactors (Chen and Li, 1998; McKenna et al, 1999; Xu et

al, 1999; Rachez and Freedman, 2000). Many such factors

have been isolated based on their ability to interact c with

TRs in the presence or absence of T 3

Gene Therapy and Molecular Biology Vol 5, page 103

or under both

conditions. The corepressors bind preferentially or

exclusively to unliganded TR while the coactivators

generally require TH for binding to TR.

Interestingly, the corepressors appear to form

multimeric complexes containing histone deacetylases

while many coactivators themselves are histone

acetyltransferases or acetylases (McKenna et al, 1999; Xu

et al, 1999; Burke and Baniabmad, 2000; Hu and Lazar,

2000; Urnov et al, 2000). Our studies have suggested the

existence of multiple corepressor complexes, both with

and without histone deacetylase activity, in the frog oocyte

(Jones et al, unpublished data). This raises the possibility

that histone acetylation status may play a role ii

transcriptional regulation by TR/RXR.

Histone acetylation has long been implicated

influence gene expression (Allfrey et al, 1964; Wolffe

1986; Struhl, 1998). Histone acetylation occurs at lysine

residues on the amino-terminal tails of the histor leading

to the neutralization of the positive charges histone tails

and reduced affinity toward DNA (Hon al., 1993).

Although we have failed to detect any gross changes in

chromatin structure under conditions exp to alter histone

acetylation levels of plasmid minichromosome (Wong et

al, 1998), alteratic histone acetylation levels will likely

chang nucleosomal conformation and chromatin access

thus influencing transcription.

Indeed, our studies in the oocyte have provided

evidence for a role of histone acetylation in promoter

activation (Figure 1) (Wong et al, 1998; Hsia et al, 2000)

First, addition of a specific inhibitor of deacetylase, TSA

(trichostatin A), can reverse the repression by unliganded

TR/RXR, mimicking the addition of TH (Figure 1),

indicating the involvement of histone deacetylase in the

repression by TR/RXR. Conversely, overexpression of the

catalytic subunit of a frog histone deacetylase complex

(Rpd3) leads to transcriptional repression of a THinducible

promoter. This deacetylase-induced repression

can be reversed by either TR/RXR in the presence of TH

or TSA (Wong et al, 1998).

C. A model for gene regulation by

TR/RXR

Although the studies so far are supportive of an

important role for histone acetylation in transcriptional

activation, other pathways are likely involved. First, we

have shown that transcriptional activation by liganded

TR/RXR leads to chromatin disruption but over-

expression or blocking the function of histone

deacetylases has no such effect despite dramatic

influences on transcription. In addition, many cofactors

can interact with the transcriptional machinery directly

(Burke and Baniahmad, 2000; Hu and Lazar, 2000;

Rachez and Freedman, 2000). Finally, at least one

coactivator complex, the DRIP/TRAP complex, has no

histone acetyltransferase activity but can activate

transcription from chromatin templates (Rachez and

Freedman, 2000). Thus, transcriptional regulation by

TR/RXR is likely to involve a complex, multi-step, multicomponent

process. A potential model for TR/RXR

function is outlined in Figure 2. Tn the absence of TH,

TR/RXR recruits a corepressor and its associated

deacetylase complex to the promoter, leading to histone

deacetylation and transcriptional repression. Upon TH

binding, the corepressor complex is dissociated and one or

more coactivator complexes are recruited to the promoter.

This recruitment may lead to increased histone acetylation

(Utley et al, 1998; Sachs and Shi, 2000), chromatin

disruption, and transcriptional activation.

III. Dual function of TRS in frog

development

Four TR genes, two TR! and two TR" genes, are

present in Xenopus laevis (Figure 3) (Yaoita et al, 1990).

The total dependence of anuran metamorphosis on TR

offers an opportunity to study TR/RXR function during

development. Expectedly, both TR! and TR" genes are

highly expressed luring metamorphosis in Xenopus

(Yaoita and Brown, 1990; Shi, 1999). In addition, RXR

genes are also expressed during metamorphosis (Wong

and Shi, 1995). More importantly, the expression of both

TR and RXR genes correlates temporally w

metamorphosis of individual organs. Thus, high levels of

both TR and RXR mRNAs are present in the limb during

early sages of metamorphosis (Stage 54-58) when limb

morphogenesis takes place. Subsequently as limb

undergoes growth with little morphological changes, both

TR and RXR genes are down regulated. On the other

hand, both TR and RXR genes are upregulated toward the

end of metamorphosis (after stage 60), which corresponds

to the period of tail resorption. Such correlation argues

that TR/RXR heterodimers are indeed the mediators of the

controlling effects of TII on metamorphosis in all organs

(Shi et al, 1996).

Interestingly, TR! and TR" genes are differentially

regulated dunng development (Figure 3) (Yaoita and

Brown, 1990). The TR! genes have little expression prior

to metamorphosis and are themselves direct TH-response

genes (Ranjan et al, 1994; Machuca et al, 1995). Their

expression is upregulated by the rising concentration of

endogenous TH during metamorphosis (Figure 3). In

contrast, the TR! genes are activated shortly after the

completion of embryogenesis and their mRNAs reach high

levels by stage 45 when tadpole feeding begins (Figure 3)


Sachs et al: Gene repression and activation by TRs

Figure 2. A model for transcriptional regulation by TRs. TR functions as a heterodimer with RXR. In the absence of TH, the

heterodimer represses gene transcription through the recruitment of a corepressor complex containing the corepressor such as N-CoR,

Sin3A and histone deacetylase such as Rpd3. This leads to histone deacetylation and transcriptional repression. When TH is present, the

corepressor complex is released and a coactivator complex containing coactivators such as SRC-l, CBP/p300, and P/CAF, and/or the

DRIP/TRAP coactivator complex is recruited. The DRIP/TRAP complex may contact RNA polymerase directly to activate gene

transcription. On the other hand, the SRC-l, CBP/p300, and P/CAF complexes may function through chromatin modification as they

possess histone acetylase activity. In addition, transcriptional activation is associated with chromatin disruption, which may be due to

the recruitment of chromatin remodeling machinery by TR/RXR.


Gene Therapy and Molecular Biology Vol 5, page 105

Figure 3. Developmental expression of TR genes suggests dual functions for TR in frog development. The TH-inducible gene

stromelysin-3 (ST3) is expressed during late embryogenesis when little TR mRNA is present. As the TRa genes are activated, ST3 is

repressed. When endogenous TH levels rise after stage 54 both ST3 and TR" genes are activated. The TR and RXR mRNA levels are

based on (Yaoita and Brown, 1990; Wong and Shi, 1995). The ST3 mRNA levels are based on (Patterton et al, 1995). Thyroid hormone

T 4 levels are from (Leloup and Buscaglia, 1977).

The expression profiles together with the ability of

TR to both repress and activate TH-inducible genes in the

absence and presence of TH, respectively, suggest dual

functionsfor TRs during development. In premetamorphic

tadpoles, TRs, mainly TR!, act to repress TH-response

important also for the gene regulation by TR. Thus,

TR/RXR heterodimers function as transcriptional

repressors of TH-inducible genes in premetamorphic

tadpoles when TR is absent, and as transcriptional

activators during metamorphosis when TH is available.

IV. Constitutive DNA-binding and

involvement of histone acetylation in

developmental gene regulation by TRS

The studies in the frog oocyte and other model

systems have provided strong evidence that TR/RXR may

regulate gene transcription at least in part by recruiting

histone deacetylase or acetylase (acetyltransferase)

complexes, depending upon the absence or presence of

TH, respectively. To investigate the possible involvement

of histone acetylation in gene regulation by TR in vivo, we

have treated tadpoles with TH or TSA, a specific drug for

blocking histone deacetylases, and analyzed the effect on

the expression of TH response genes (Sachs and Shi,

2000). Surprisingly, no detectable upregulation of TH

response genes by TSA can be detected in whole animals,

although T 3 induces the expression of TH response genes

as expected (Figure 4B) Since TR-treatment leads to a

large array of very different changes in the

premetamorphic tadpoles, it is possible that the regulation

of TH response genes may be tissue/organ-specific,

depending upon the changes in the tissues/organs. Thus,

we have chosen the intestine and the tail to investigate the

role of histone acetylation further. These two organs are

among the few well-characterized organs that undergo

extensive remodeling and are known to have the most

dramatic upregulation of TH-response genes during

metamorphosis. Premetamorphic tadpole intestine consists

predominantly of a single tissue, the larval epithelium,

which undergoes apoptosis and is replaced by the adult

epithelium (Yoshizato, 1989; Shi, 1996). The tail, on the

other hand, completely absorbs through an apoptotic

pathway (Dodd and Dodd, 1976; Yoshizato, 1989; Shi,

1999). Thus, these two organs offer relatively

homogeneous tissues for study tissue specific changes in

gene expression and chromatin remodeling. Indeed, our

studies on these two organs indicate that TSA induces

precocious expression of most TH response genes

analyzed, including the only two genes that have been

shown to contain TREs (Ranjan et al, 1994; Machuca et

al, 1995; Furlow and Brown, 1999), the TRb and TH/bZIP

genes Figure. 4A) (Sachs and Shi, 2000). On the other

hand, TSA had little effect on the expression of TR!

genes, which are not direct TH response genes. Thus,

these data support the involvement of histone deacetylase

in the repression of TH response genes by unliganded

TR/RXR.


It has long been known that TR is chromatinassociated

in somatic cells (Penman et al, 1982).

Furthermore, in the frog oocyte system, we have shown

that TR/RXR can bind to TRE both prior to and

subsequent of replication-coupled chromatin assembly

(Wong and Shi, 1995; Wong et al, 1997a). However, a

direct demonstration of TR/RXR binding to the TREs of

its target genes is lacking in any developmental system. If

TR/RXR indeed functions to repress TH response genes in

premetamorphic tadpoles as suggested above, we would

expect that they are bound to TREs of endogenous TH

response genes independent of TH. To test this possibility,

we have made use of the sensitive chromatin

irnmunoprecipitation (ChIP) assay using antibodies

against TR or RXR (Sachs and Shi, 2000). PCR analysis

of the immunoprecipitates for the binding of TR or RXR

to the TRE regions of the Xenopus TRb and TH/bZip

genes, have demonstrated clearly that both TR and RXR

are bound to the TREs in the intestine and tail (Figure 4C)

(Sachs and Shi, 2000). Furthermore, tbe binding is

independent of TH or TSA treatment, in agreement with

studies in vitro and in the frog oocyte.

The ChIP assay also offers an opportunity to study

whether local histone acetylation levels change in

response to TH binding to TR/RXR. This has been done

by using an antibody against acetylated histone H4 on the

two TH response genes (TRb and THIbZip) in Xenopus

laevis intestine and tail. The results have shown that TH

treatment of premetamorphic tadpoles leads to an increase

of histone acetylation specifically at the TRE regions of

TH response genes (Figure 5A) (Sachs and Shi, 2000)

without affecting global histone acetylation or the

acetylation of chrornatin far away from the TRE (Figure

5B). On the other hand, TSA treatment of premetamorphic

tadpoles elevates global histone acetylation levels,

including the TRE regions of TH response genes.

Similarly, ChIP assay using an antibody against the

histone deacetylase Rpd3, the only characterized

deacetylase in Xenopts laevis, demonstrates that Rpd3 is

present at the TRE regions of TH response genes and its

binding is reduced upon TH treatment of premetamorpbic

tadpoles (analyzed in whole animals as Rpd3 was not

detectable in prernetamorphic intestine, Sachs and Shi,

2000). Thus, these data together suggest that TR/RXR is

bound to TREs assembled into chromatin in vivo. In the

absence of TH, TR/RXR recruits histone deacetylase

complexes to silence transcnption, at least in the intestine

and tail. In the presence of TH, histone deacetylase

complexes are released and histone acetylase complexes

are likely recruited by TR/RXR, resulting in increased

histone acetylation and gene activation.

Sachs et al: Gene repression and activation by TRs

V. Conclusion

TH regulates a wide range of biological processes

across most animal species by influencing gene

transcription through TR. The roles of TR! and TR" in

regulating anuran metamorphosis are supported by their

temporal and spatial expression profiles during

development. Furthermore, these receptors appear to have

dual functions depending upon the cell types and

developmental stages when they expressed. In

premetamorphic tadpoles, they are likely to function as

unliganded transcriptional repressors to block the

expression of TH response genes that are involved in

metamorphosis, thus ensuring a proper period of tadpole

growth. When TM becomes available during

metamorphosis, it binds to the receptors and converts them

into activators to upregulate the TH-inducible genes, thus

initiating metamorphosis.

Our studies involving over-expression of TRJRXR in

embryos have provided some in vivo evidence that

supports the involvement of TR/RXR heterodimers in

repressing TM-inducible genes in the absence of TM and

in activating them when TM is present. ChIP assays have

directly shown that TRs are bound to TREs assembled into

chromatin. Furthermore, our data support the model that in

the absence of TM, they recruit histone deacetylase

complexes to silence transcription at least in some

organs/tissues. The binding of TM to chromatin-bound TR

leads to local histone acetylation likely due to the release

of deacetylase complexes and possible recruitment of

acetylase complexes. These findings are also consistent

with those from in vitro studies and from analyses in the

frog oncyte system, where it has been shown that histone

acetylation plays an important role in gene regulation by

TR and that transcriptional activation by TM leads to

additional chromatin remodeling. Thus a model for TR

action based on a TM-dependent switch between

transcriptional repression and activation involving

chromatin remodeling provides one possible molecular

mechanism for the dual functions of TRs in development.

Acknowledgements

We would like to thank Ms. K. Pham for preparing