01. Gene therapy Boulikas.pdf - Gene therapy & Molecular Biology

Gene Therapy and Molecular Biology Vol 1, page 77
MeP-dR treatment. Representative animals from each of 4 groups at completion of the study (62 days) are shown: Group 1: nude mice
were injected with D54MG cells, vehicle treated. Group 2: nude mice were injected with D54MG cells, MeP-dR treated. Group 3: nude
mice were injected with D54-PNP cells, vehicle treated. Group 4: nude mice were injected with D54-PNP cells, MeP-dR treated. From
Parker WB, King SA, Allan PW, Bennett LLJr, Secrist JAIII, Montgomery JA, Gilbert KS, Waud WR, Wells AH, Gillespie GY, and
Sorscher EJ (1997) In vivo gene therapy of cancer with E. coli purine nucleoside phosphorylase. Hum Gene Ther 8, 1637-1644. With
the kind permission from the corresponding author (Eric Sorscher, University of Alabama at Birmingham) and Mary Ann Liebert, Inc.
the CEA gene; isolation of 14.5 kb of 5' flanking
sequences for this gene followed by subcloning into
luciferase pGL2 basic vectors and testing for luciferase
activity in transfected LoVo, SW1463, Hep3B, and HuH7
cell lines (the first two express CEA whereas the other two
do not) has identified the CEA promoter between bases -
90 and +69, and two enhancers one at -13.6 to -10.7 and
the other at -6.1 to -4.0 kb (Richards et al, 1995); these
sequences were able to sustain high levels of expression of
the CD gene into CEA-expressing cell lines.
Regulatory sequences from the CEA gene (-322 to
+111 bp) were also used to express the HSV thymidine
kinase gene in pancreatic and lung neoplasms (Dimaio et
al, 1994; Osaki et al, 1994).
XXIV. Transfer of drug resistance
genes
A. Principles and genes used
An attractive approach to circumvent chemotherapyinduced
myelosuppression is the use of gene-transfer
technology to introduce new genetic material into
hematopoietic cells. Protection of bone marrow progenitor
cells by introduction of a drug resistance gene allows
larger and curative doses of chemotherapy to be
administered to the patient as was shown in several preclinical
studies. Drug resistance genes under experimental
consideration are shown on Table 4. Clinical trials are
now under way to evaluate the potential use of two gene
sequences: MDR1 (protocols #43, 44, 59, 89, and 100)
and O 6 -methylguanine DNA methyltransferase (#101 see
Appendix 1) (see also Lee et al, 1998, this volume).
Dose-limiting hematopoietic toxicity produced by the
cytosine nucleoside analogue cytosine arabinoside (Ara-C)
is one of the major factors that limit its use in the
treatment of neoplastic diseases. Deamination of Ara-C by
cytidine deaminase results in a loss of its antineoplastic
activity. Transfer of human cytidine deaminase into
murine fibroblast and hematopoietic cells conferred drug
resistance to Ara-C protecting them from drug toxicity
(Momparler et al, 1996). It is worth mentioning that
apolipoprotein B mRNA editing involves the deamination
Table 4. Drug resistance gene designs
77
of cytidine by the cytidine deaminase catalytic subunit that
creates a new termination codon and produces a truncated
version of apo-B (apo-B48); the cytidine deaminase
catalytic subunit (apo-B mRNA-editing enzyme catalytic
polypeptide 1) of the multiprotein editing complex has
been identified (Yamanaka et al, 1995).
B. Mechanism of MDR1 resistance
A great deal of our knowledge of basic insights on
drug uptake and molecular mechanisms of drug action
were elucidated from the study of resistance of tumor cells
to chemotherapeutic agents. The P-glycoprotein or p170
encoded by the multidrug resistance MDR1 gene uses the
energy of ATP to extrude a variety of drugs apparently
unrelated; the only chemical similarity is that they contain
condensed aromatic rings and have a positive charge at
neutral pH; these drugs, most of which are effective
against a variety of human tumors, include molecules
found in nature such as colchicine, doxorubicin (also
called adriamycin, member of the anthracycline family),
actinomycin D, vinblastine, etoposide, taxol, vinca
alcaloids, and epipodophyllotoxins collectively called
MDR-type of drugs (reviewed by Gottesman and Pastan,
1988; see Lee et al, 1998 this volume).
Cell lines resistant to drugs accumulate far less
amounts of drug compared with parental cells because of
overexpression of the MDR1 gene; development of
multidrug resistance by tumor cells poses a major
impediment to successful cancer chemotherapy. A number
of cell lines with multidrug resistance have been derived
like KB and K562 cells (Marie et al, 1991; Fardel et al,
1995). The P-glycoprotein is a 1280 amino acid molecule
in human cells (Chen et al, 1986) or 1276 amino acid
molecule in mouse cells with 80% sequence similarity to
the human protein (Gros et al, 1986). P-glycoprotein has
12 hydrophobic domains grouped into pairs representing
transmembrane domains. The molecule has a 500 amino
acid duplication; each duplicated segment possesses an
ATP-binding site on the cytoplasmic side; it also has
several site of glycosylation near the N-terminus to the
exterior side. Its gene is amplified in multidrug resistant
Drug resistance gene Confers resistance to Reference
MDR1 (multidrug resistance) Daunomycin, doxorubicin, taxol Galski et al, 1989; Podda et al, 1992;
Sorrentino et al, 1992 (see below)
Mutant dihydrofolate reductase Methotrexate (MTX) Williams et al, 1987; Corey et al, 1990;