Recombination at chromosomal sequences ... - Carcinogenesis

Carcinogenesis vol.25 no.8 pp.1305--1313, 2004
doi:10.1093/carcin/bgh092
Recombination at chromosomal sequences involved in leukaemogenic
rearrangements is differentially regulated by p53
Gisa S.Boehden 1,2 , Anja Restle 1 , Rolf Marschalek 3 ,
Carol Stocking 2 and Lisa Wiesmuller 1,2,4
1 Universitatsfrauenklinik, Prittwitzstrasse 43, D-89075 Ulm, 2 Heinrich-Pette-
Institut fur Experimentelle Virologie und Immunologie an der Universitat
Hamburg, Martinistrasse 52, D-20251 Hamburg and 3 Institut fur
Pharmazeutische Biologie, Johann Wolfgang Goethe Universitat, Biozentrum,
N230, R303, Marie-Curie-Strasse 9, D-60439 Frankfurt/Main, Germany
4 To whom correspondence should be addressed
Email: lisa.wiesmueller@medizin.uni-ulm.de
Chromosomal translocations and retroviral integration
events at breakpoint cluster regions (bcrs) have been associated
with leukaemias. To directly compare the effect of
different cis-regulatory sequences on recombination, we
adapted our SV40 based model system to the analysis of
correspondingly selected bcrs from the TAL1, LMO2, retinoic
acid receptor a (RARa) and MLL genes. We show that
a 399 bp fragment from the MLL bcr is sufficient to cause a
3--4-fold stimulation of spontaneously occurring DNA
exchange and to respond to etoposide by up to 10-fold
further elevated frequencies, i.e. to mimic the fragility of
the 8.3 kb bcr during chemotherapy. To analyse
the regulatory role of p53 in recombination involving
leukaemia-related sequences, we stably expressed wtp53
and a transactivation negative mutant. Consistent with
the proposed role of p53 as a suppressor of error-prone
recombination, both p53 proteins down-regulated recombination
with most of the sequences tested, even with the
MLL bcr after etoposide treatment. Surprisingly, however,
p53 stimulated recombination, in constructs carrying the
RARa bcr fragment. This is the first study, which provides
evidence for a stimulatory role of p53 in homologous
recombination. Our data further indicate that inhibition
of topoisomerase I can mimic the effects of p53 on stimulating
recombination on the RARa bcr. Thus, these data
also firstly describe a biological role of the biochemical
interactions between p53 and topoisomerase I that may
have implications for a gain-of-function phenotype of
certain p53 mutants in genetic destabilization.
Introduction
Chromosomal rearrangements, such as translocations, deletions
and gene amplifications, initiate haematopoietic malignancies
(1). Among the biochemical events potentially
underlying these chromosomal instabilities, dysfunction of
double-strand break (DSB) repair has closely been linked to
tumorigenesis. The DSB repair pathway of non-homologous
end-joining (NHEJ) reseals DSBs in mitotically growing cells
Abbreviations: bcrs, breakpoint cluster regions; BLM, Bloom syndrome
protein; DSB, double-strand break; HR, homologous recombination; RARa,
retinoic acid receptor a; RGC, ribosomal gene cluster.
and assembles variable regions during V(D)J recombination in
developing lymphocytes. Homologous recombination (HR)
enables genetic mixing during meiosis and repairs DSBs and
other DNA lesions that remain unrepaired until being encountered
by a replication fork (2). HR by gene conversion
predominates among DSB-induced interchromosomal recombination
events (3).
Molecular studies of genes involved in chromosome aberrations
of leukaemia patients unveiled a striking clustering of
translocation breakpoints, raising questions about the causes of
local fragilities. In close to 30% of patients with T cell acute
lymphoblastic leukaemia (T-ALL), alterations in the TAL1
(T-cell acute leukaemia 1) gene (also called SCL and TCL5),
either as a consequence of a t(1;14) chromosome translocation
or a deletion (4,5), are detected. The t(11;14) translocation,
which involves the LMO2 (LIM domain only 2) gene (also
called Rhombotin 2, Rbtn2 and TTG-2), is found in 5--10% of
T-ALL cases (6). Acute promyelocytic leukaemia (APL) is, in
the majority of patients, associated with a reciprocal translocation
between chromosome 15 and chromosome 17, and the
breakpoint on chromosome 17 lies within the retinoic acid
receptor a (RARa) locus (7,8). The MLL (mixed lineage leukaemia)
gene (also called ALL-1, HRX and Htrx1) on chromosome
11 is rearranged in ALL and AML (9). So far, 430
hybrid genes of MLL with different fusion partners have been
identified. In addition, MLL is a hotspot for chromosomal
rearrangements after treatment with topoisomerase inhibitors
(10,11).
Support for a direct involvement of p53 in HR processes
came from the discovery of p53 mutants with separable functions
in transcriptional transactivation, growth control and
apoptosis induction versus HR (12--15). Moreover, by use of
an I-SceI meganuclease coupled DSB repair test system, an
indirect effect stemming from an involvement of p53 in other
DNA repair pathways was excluded (15). With respect to the
underlying mechanism it is interesting that p53 recognizes
mispaired recombination intermediates with high affinities
in vitro and suppresses inappropriate recombination between
mispaired DNA sequences and between sequences with short
homologies in living cells (15--17). The mismatch repair factor
MSH2, which similarly recognizes mispairings in heteroduplex
joints and restrains the exchange between divergent
sequences, stimulates p53 binding to Holliday junctions and
shows a common nuclear subcompartmentalization with p53 at
sites of recombinative repair complexes (18,19). This has led
to the proposal of complementary or even synergistic roles of
p53 and MSH2 in the fidelity control of HR (16,19). Additionally,
p53 was shown to interact with other surveillance factors
of DSB repair, namely with BRCA1, which channels DSB
repair into the non-mutagenic pathway of HR, with BRCA2,
which promotes DSB repair via the conservative HR pathway,
and with the anti-recombinogenic RecQ helicases, Bloom syndrome
protein (BLM) and Werner syndrome protein (WRN)
(20--25). p53 was further reported to counteract Holliday
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G.S.Boehden et al.
junction unwinding by BLM and WRN in vitro, suggesting
that p53 regulates recombination indirectly by modulating
other surveillance activities (24). However, cell based studies
revealed that p53 and BLM act on complementary recombination
regulatory pathways and indicated that p53--BLM interactions
may rather be required to recruit p53 to sites of
aberrant DNA exchange processes (25). Finally, p53 was also
demonstrated to bind HR proteins, namely Rad51, the initial
strand transferase, and Rad54, a member of the SWI2/SNF2
family of helicase-like proteins (25--28), and recombination
down-regulation by p53 was found to depend on the Rad51
pathway (12,15,28). Co-localization of p53 with Rad51 and
Rad54 was observed at putative processing sites of DNA
breaks and at stalled replication forks, consistent with the
observed involvement in the control of replication-associated
recombination processes (29,30). In conclusion, current
models on the anti-recombinogenic role of p53 involve physical
interactions with the DNA intermediates of recombination
and/or with Rad51 as the key events that are influenced by the
presence of other proteins. The functional relevance of the
association of p53 with recombination surveillance factors
other than BLM awaits further clarification.
It is still difficult and time-consuming to quantitatively evaluate
genetic alterations by conventional cytogenetics. Moreover,
deletions or smaller rearrangements escape detection by
this method. In order to elucidate cis-acting mechanisms for
genomic instability, we introduced a set of leukaemia-related
DNA fragments into our SV40 based assay (31) and assessed
the impact of recombination regulation by p53 on elementspecific
rearrangements.
Materials and methods
DNA-cloning procedures
Retroviral integration sites from growth factor-independent mutants after
retroviral infection of either the TF-1 or FDC-P1(M) cell lines (32,33) were
isolated by either inverse-PCR or by cloning in lambda vectors. TF-1 mut 33
and FDC-P1(M) mut 2GM20 contained a single provirus in either the TAL1 or
LMO2 gene locus at position 145233--145232, GenBank accession number
NT_032977, or 142546--142547, genome clone RP23-358C5.
The Cla linker was introduced into XbaI/BamHI digested, Tag intron negative
pUC-SV40-tsVP1(286S) (16) via PCR amplification between SV40 genome
positions 2338 and 2682, and XbaI/BglII cloning. To generate further
pUC-SV40-Cla vectors, we transferred the Cla linker after BamHI/ApaI or SfiI/
XcmI cleavage. Next, we introduced PCR amplified fragments into the ClaI
site. The DEGFP (15) fragment encompassed position 742--1058 in pEGFP-N1
(Clontech, Palo Alto, CA), the TAL1 bcr 145372--145111, accession number
NT_032977, the LMO2 bcr 25314640--25314212, accession number
NT_009237, the RARa bcr 91920--91640, accession number AC090426,
the MLL bcr 7036--6638, accession number X83604. Each vector set with a
pUC-SV40-Cla, a pUC-SV40-tsVP1(290T)-Cla and a pUC-SV40-tsVP1
(196Y)-Cla derivative was sequenced and carried the foreign sequence in the
same orientation.
Cell lines, virus, DNA synthesis and HR
CV1, COS1, LLC-MK2(p53her), LLC-MK2[p53(138V)her] and LLC-
MK2(neo) cells were clonally established, cultivated and activated by 1 mM
b-estradiol (Sigma, Taufkirchen, FRG) as described (13,16). Virus particles
were generated in COS1 cells and MOIs and PFUs determined as in ref. (31).
The deletion of the foreign DNA insert during viral amplification was ruled out
by restriction analysis of isolated viral DNAs. De novo synthesis of viral DNA
was quantified as described (30). Briefly, cells on 60-mm plates were labelled
with 30 mCi of [ 3 H]thymidine at various times post-infection (hpi) for 1 h, viral
genomes were isolated, and [ 3 H]thymidine incorporation determined. Rates of
viral DNA synthesis were expressed as counts per minute (c.p.m.) per 10 5 cells
and mean values including SEs calculated. HR assays were executed and
evaluated as before (16). The statistical significance of differences was determined
using Student's unpaired t test.
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Gel mobility shift assays
Fractions enriched for p53 were obtained after protein over-expression in
insect cells and sequential nuclear extraction as described (13,16). 32 P-
Labelled DNA fragments were synthesized as in Wiesmuller et al. (31). The
RARa bcr fragment was amplified as described for cloning. The ribosomal
gene cluster (RGC) repeat was amplified after SmaI/HincII subcloning from
pSK-45-13-2-PyCAT (34) into the Cla linker of pUC-SV40-Cla by use of the
primer pair 5 0 -AGGAGGAGATCTATCGATACCCGGGCAATTGT TGTTG-
TTAACTTGTTTATTG-3 0 /5 0 -GTTAACAACAACAATTGCCCC-3 0 .Forpurification
of the radiolabelled fragments we used the QIAquick PCR purification
kit (Qiagen, Hilden, FRG) according to the manufacturer's instructions, for
the determination of DNA concentrations DNA Dipsticks (Invitrogen,
Karlsruhe, FRG). Labelled DNA fragments (100 pM), competitor oligonucleotide
(1000 nM) and 0.7--2.9 nM p53 proteins were mixed, incubated on ice for
30 min, electrophoresed on a native 4% polyacrylamide gel, and autoradiographed
as in Dudenhoffer et al. (16).
Results
Assay to test cis-regulatory sequences in recombination
To study the influence of disease-related sequences on recombination,
we utilized our assay based on genetic exchange
between differently mutated SV40 minichromosomes (31). It
relies on ts SV40 variants (SV40-tsVP1), which enabled us to
produce virus particles at the permissive temperature of 32 C
and to assay recombinative reconstitution of SV40-wtVP1
after co-infection with two SV40-tsVP1 mutants at the nonpermissive
temperature of 39 C (Figure 1A). To obtain HR
frequencies, the ratios between the values from double infections
with SV40-tsVP1 mutants and from control infections
with the same infectious units of SV40-wtVP1 were determined.
This procedure also served to exclude rate deviations
caused by growth regulatory and cytotoxic effects, and alterations
in virus propagation.
In order to permit packaging of additional DNA pieces of up
to 0.7 kb, the SV40-tsVP1 genomes and the wtSV40 control
chromosome were made smaller. This was achieved by removing
269 bp from the large tumour antigen (Tag) intron
(Figure 1a). To allow the uptake of foreign DNA, we separated
the early and late polyadenylation signals by a PCR strategy,
which concomitantly inserted two new restriction sites (Cla
linker).
Into the Cla linker of the corresponding SV40-wtVP1,
SV40-tsVP1(196Y) and SV40-tsVP1(290T) genomes, we
chose to introduce fragments of 50.5 kb that were derived
from genomic bcrs for which frequent chromosomal breakage
was expected from a clustering of deletions or translocations
(Figure 1b). Thus, we PCR-amplified a 0.3 kb region within
the RARa bcr from APL patients, where two neighbouring
t(15;17) translocation sites were identified at the molecular
level (7,8). Additionally, we selected a 0.4 kb fragment from
the MLL bcr, within which we have identified previously eight
out of 24 and four out of 36 t(4;11) translocations in infants
and other ALL patients, respectively (35). Furthermore, from
the analysis of retroviral integration sites of factor-independent
mutants generated by retroviral insertional mutagenesis of two
myeloid progenitor cell lines (32), we found that two out of a
total of 42 integration sites mapped to bcrs in ALL: one at the
TAL1 locus associated with t(1;14) translocations (4,5) and one
within the LMO2 gene, where t(11;14) translocations have
been identified in patients (6) (Figure 1B). Since retroviral
integration represents another type of genetic alteration related
to chromosomal breakage, which is underlying the development
of leukaemia (36), we also amplified the 0.3 and 0.4 kb
regions encompassing the retroviral integration locus adjacent
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Cis-regulatory sequences in recombination
Table I. Recombination at distinct chromosomal sequences
DNA sequence Size a Recombination
frequency ( 10 4 ) b
wtSV40 n.a. 9.7 1.3 n.a.
Cla linker 13 10.4 3.1 1.1
DEGFP 315 12.5 2.5 1.3
TAL1 bcr 280 21.4 3.5 2.2
LMO2 bcr 448 21.2 3.2 2.2
RARa bcr 300 22.0 6.5 2.3
MLL bcr 418 33.6 5.3 3.5
Stimulation
a
Non-viral DNA sizes within SV40-Cla virus genomes include cloning sites
and cis-acting elements.
b
Recombination assays were performed with LLC-MK2(neo) cells using
SV40, SV40-tsVP1(290T) and SV40-tsVP1(196Y) virus (wtSV40), the
corresponding Cla linker viruses, and derivatives comprising the indicated
sequences. Recombination frequencies and SEs were calculated from mean
values for two clones and 2--14 independent measurements per clone
(independent measurements per clone with wtSV40, n ˆ 6--8; with Cla linker,
n ˆ 2--8; with TAL1 bcr, n ˆ 3--8; with LMO2 bcr, n ˆ 2--9; with RARa bcr,
n ˆ 4--11; with MLL bcr, n ˆ 5--14) and for one representative clone and three
independent measurements with DEGFP virus.
n.a., not applicable.
to the TAL1 and the LMO2 bcr, respectively. Finally, a 0.3 kb
fragment from a mutated EGFP gene (DEGFP) (16), which
has not been implicated in genome instability, served as a
control.
Recombination is stimulated by the MLL bcr sequence
First, we measured HR in LLC-MK 2(neo) cells for the wtSV40
virus set [SV40-tsVP1(290T), SV40-tsVP1(196Y) and
wtSV40] and the SV40-Cla virus set, which demonstrated
that the mutations created within the Cla linker genomes did
not interfere with HR (Table I). Control viruses with the
DEGFP sequence recombined at a frequency not significantly
different from Cla linker viruses, indicating that changes in the
total homology length (5439 versus 5137 bp) did not affect the
HR frequency. The frequencies determined for TAL1, LMO2
and RARa bcr viruses indicated a 2-fold stimulation in relation
to the wtSV40 virus set, although compared with DEGFP virus
the significance of the changes was limited (TAL1, P ˆ 0.042;
LMO2, P ˆ 0.041; RARa, P ˆ 0.175). The MLL bcr fragment
caused a 3--4-fold increase in the basal frequency (MLL,
P ˆ 0.002), i.e. maximal enhancement of spontaneous recombination
among the cis-regulatory sequences investigated in
this work.
Fig. 1. Test system for recombination at cis-regulatory DNA sequences. (a)
Test principle. SV40 genomes were designed for insertion of foreign DNA
by removal of the Tag intronic sequences and the introduction of a Cla linker
and tsVP1 mutations. Virus particles were produced at the permissive
temperature of 32 C and used for co-infection at the non-permissive
temperature of 39 C, to select for SV40-wtVP1-Cla reconstitution by HR.
SV40-wtVP1-Cla virus release was scored by plaque assays at 39 C. (b)
Genomic organization. Squares represent exons; black lines introns (TAL1,
GenBank accession number NT_032977, LMO2: NT_009237; RARa,
AC090426; MLL, Z69744-Z69780). Coding sequences are marked with grey
colour, untranslated sequences with grey stripes. tal d deletion and t(1;14)
translocation sites found in the TAL1 gene of T-ALL patients (4,5), bcrs in
the LMO2 gene of T-ALL patients (6) and the RARa gene of APL patients
(7,8) are indicated. The t(4;11) translocation sites, which show a clustering 5 0
to exon 12 of MLL in ALL patients below 1 year of age at diagnosis, are
marked by black bars (35). Relevant features within PCR amplified bcr
fragments are indicated (bottom).
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G.S.Boehden et al.
Table II. Effect of p53 on recombination at bcrs
DNA sequence Relative recombination frequency (%) a
wtp53 p53(138V)
wtSV40 26.5 5.9 29.3 2.3
Cla linker 33.8 4.5 37.0 0.3
TAL1 bcr 54.1 18.0 64.5 4.5
LMO2 bcr 45.6 2.6 58.9 17.3
RARa bcr 392 71 293 101
MLL bcr 40.4 12.1 31.7 6.8
a Virus strains without foreign sequence (wtSV40), containing Cla linker only
or, additionally, the indicated bcr sequences were subjected to recombination
measurements in wtp53her and p53(138V)her clones. Frequencies in LLC-
MK2(neo) cells without exogenous p53 were taken as 100% (see Table I) and
relative recombination frequencies including SEs calculated. Four LLC-
MK 2(p53her) and two LLC-MK 2[p53(138V)her] clones were analysed by
two to six independent measurements per clone [independent measurements
per wtp53 clone with wtSV40, n ˆ 2--6; with Cla linker, n ˆ 2--3;
with TAL1 bcr, n ˆ 2--3; with LMO2 bcr, n ˆ 2--4; with RARa bcr, n ˆ 2--4;
with MLL bcr, n ˆ 2--5; independent measurements per p53(138V) clone:
n ˆ 2--3].
Recombination regulation by p53 as a function of the DNA
sequence
To test whether the non-viral sequences, influence the potential
of wtp53 to regulate HR, we altered the p53-status in the
SV40-infectable monkey cell line LLC-MK 2, devoid of wtp53
(31). We newly established independent cell clones ectopically
expressing the wtp53 hybrid protein p53her (data not shown),
which is activated in a b-estradiol-dependent fashion (13,16).
To dissect functions in transcriptional transactivation and
growth control versus HR, we also stably expressed the transactivation
defective (14,15) mutant p53(138V)her in two independent
cell clones.
In accord with previous SV40 based studies, wtp53 downregulated
HR (16,31). On average, the frequency for the
wtSV40 virus set was reduced by 4-fold in four LLC-
MK2(p53her) clones (2.6 10 4 ) as compared with LLC-
MK2(neo) cells (Tables I and II). The frequency measured
with Cla linker viruses was not significantly different than
that found with the wtSV40 virus set. Similarly, the recombination
frequencies observed after TAL1 bcr, LMO2 bcr and
MLL bcr virus infections of LLC-MK 2(neo) cells, were
decreased by a factor of 2--3 in the LLC-MK2(p53her) clones
(Table II). These findings indicate that wtp53 has the capacity
to counteract DNA exchange events, even at MLL bcr
sequences that cause a 3--4-fold stimulation of spontaneous
recombination.
In contrast, with RARa bcr virus, the frequencies were
elevated 4-fold (P ˆ 0.009) in a wtp53-dependent manner
(Table II). Moreover, we found that in the two LLC-
MK2[p53(138V)her] lines, the recombination frequencies
were not significantly different from the frequencies in LLC-
MK2(p53her) cells for wtSV40, Cla linker, TAL1 bcr, LMO2
bcr, MLL bcr and RARa bcr virus infections. Clearly, both
recombination inhibitory as well as stimulatory activities were
exhibited by wtp53 and by mutant p53(138V). From this, both
activities must be considered to be independent of the transcriptional
transactivation functions of p53.
Role of cis-regulatory sequences in replication
HR is tightly linked to replication fork stalling and replicationassociated
HR processes were reported to be regulated by
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Fig. 2. De novo DNA synthesis. LLC-MK 2(neo) (black rhombus) and LLC-
MK 2(p53her) (grey squares) cells were infected with the indicated virus
strains and [ 3 H]thymidine incorporation into viral DNA determined at
various times post-infection (hpi). The maximum DNA synthesis rate for
wtSV40 in LLC-MK2(neo) cells 24 hpi was taken as 100% (26 10 2 c.p.m./
10 5 cells) and the relative rates calculated. Values are the means SEs from
two to four independent measurements each.
p53 (2,29,30). In order to study the impact of replication on
recombination with respect to the selected DNA sequences,
we measured [ 3 H]thymidine incorporation into the viral
genomes at different times after infection (hpi) (Figure 2).
wtSV40 replication was described previously to remain either
unaffected or to become slightly reduced by wtp53 (30,31).
In LLC-MK2(neo) cells, similar replication patterns were
seen with wtSV40, TAL1 bcr, LMO2 bcr and MLL bcr virus.
Compared with wtSV40, replication of the RARa bcr genome
was increased 2-fold, 12 hpi, 24 hpi and 36 hpi. However,
in LLC-MK 2(p53her) cells no significant differences were
found between the [ 3 H]thymidine incorporations into
wtSV40, TAL1 bcr, LMO2 bcr, RARa bcr and MLL bcr
virus genomes. Thus, the incorporation of the RARa bcr
sequence, which causes HR stimulation in a wtp53-dependent
manner, did not alter viral replication in cells carrying wtp53.
Analysis of RARa bcr DNA in complex formation with p53
In an attempt to understand the stimulatory effect of p53 on HR
between RARa bcr virus genomes, we tested whether the
RARa bcr element represents a substrate for specific p53interactions.
For this purpose, we compared p53--DNA complex
formation by electrophoretic mobility shift assays with
the 300 bp RARa bcr fragment and with a 301 bp p53-specific
binding sequence, namely the RGC repeat, which was characterized
previously by Prives and colleagues (34). First, the
RARa and the RGC sequences were radioactively labelled
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y PCR amplification. Then, the DNA substrates were incubated
together with protein fractions, highly enriched for baculovirally
expressed human wtp53, the DNA contact mutant
p53(248P), or the conformational mutant p53(273P) (13).
After native gel electrophoresis of these mixtures, we detected
p53--RGC DNA complexes exclusively with wtp53, as indicated
by the complete retardation of the labelled input DNA
within distinct bands (Figure 3a). This picture did not emerge
when we mixed the RARa bcr probe with wtp53 at the same
protein concentrations (Figure 3b). Thus, at 2.9 nM wtp53
caused the formation of p53--DNA aggregates in the loading
well, at lower concentrations (1.4--0.7 nM) the DNA was not
retarded to a discrete position. Moreover, a similar smearing
Fig. 3. Binding of p53 to RGC versus RARa bcr sequences. Baculovirally
produced p53 proteins were purified and applied to electrophoretic mobility
shift assays as described (16). wtp53 (lanes 2--4), p53(248P) (lanes 5--7) and
p53(273P) (lanes 8--10) were added at the indicated concentrations.
Radioactively labelled DNA fragments were prepared by PCR amplification
and included into the mixture at a final concentration of 100 pM in the
presence of excess amounts of competitor oligonucleotide. The positions in
the autoradiograph of substrate bands and the p53-specific band shifts are
indicated by arrows and schematic illustrations. (a) Band shift analysis with
the RGC fragment. (b) Band shift analysis with the RARa bcr fragment.
Cis-regulatory sequences in recombination
was observed with the p53 mutant proteins (Figure 3b, lanes
5--10). In summary, our data allowed us to draw the conclusion
that p53 does not specifically and stably bind the RARa bcr
sequence, which stimulated recombination in a p53-dependent
manner. This finding was consistent with the absence of a p53
consensus sequence within the RARa bcr.
Sequence-specific recombination induction by topoisomerase
inhibitors
p53 was reported to interact with topoisomerase IIa, IIb and
topoisomerase I that function in transcription, replication,
recombination and DNA damage recognition (37--41). To test
the hypothesis that topoisomerase mediated cleavage within
the RARa bcr caused the p53-dependent increase of HR, we
treated LLC-MK 2(neo) and LLC-MK2(p53her) cells with the
topoisomerase II inhibitor etoposide during recombination
assays with RARa bcr virus. For comparison, we analysed
MLL bcr virus, because it has been proposed that topoisomerase
II inhibition is involved in recombination associated with
therapy-induced AML and ALL (42).
Etoposide exposure caused a 5--6-fold stimulation of recombination
between MLL bcr virus chromosomes as compared
with the 2-fold elevated DMSO values, which resulted in an
overall increase by one order of magnitude (Figure 4a). Interestingly,
LLC-MK2(p53her) cells were largely resistant to
etoposide-induced recombination (0 mM, 7 10 4 ; 100 mM,
15 10 4 ), which indicated a 24-fold inhibition by wtp53. In
contrast, neither recombination between wtSV40 nor between
RARa bcr viruses was affected by etoposide application, which
excluded a major influence of topoisomerase II on the RARa
bcr sequence during recombination. When we administered
the topoisomerase I inhibitor camptothecin to LLC-
MK 2(neo) cells, 3--4-fold elevated recombination frequencies
with RARa bcr virus (73 10 4 versus 23 10 4 ) were
detectable (Figure 4b). After p53her expression, both DMSO
and camptothecin treated cells recombined at similarly elevated
frequencies (82 10 4 versus 64 10 4 ). Under the
same conditions, camptothecin did not significantly influence
recombination between wtSV40 or MLL bcr viruses. Taken
together, the data suggest that topoisomerase I is responsible
for p53 downstream events that might explain the striking
finding that p53 stimulates recombination at the RARa bcr
fragment.
Discussion
In this work, a set of short sequences that are involved in
leukaemogenic genome alterations was characterized with
respect to their potential to regulate recombination in cis and
the role of p53 in trans on this regulation. SV40 has been
exploited previously as a powerful tool to examine the role
of specific DNA structures (16). Here, we manipulated the
virus in a way to enable us to quantitatively evaluate spontaneous
and drug-induced recombination between chromatinpackaged
circular SV40 genomes containing the sequence of
interest.
Cis-acting mechanisms in leukaemogenic rearrangements
With respect to the individual sequences, we focused on loci
where recurrent, non-random chromosomal translocations
have been recognized in acute leukaemia patients. To narrow
down the precise region, we combined two strategies: first, we
took advantage of characterized breakpoint clusters available
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G.S.Boehden et al.
Fig. 4. Recombination stimulation by topoisomerase inhibitors. (a)
Induction of recombination by etoposide. LLC-MK 2(neo) ( wtp53) and
LLC-MK2(p53her) (‡wtp53) cells were subjected to recombination assays
with wtSV40, RARa bcr and MLL bcr virus strains. 12 hpi the medium was
supplemented with DMSO ( etoposide) or with 100 mM etoposide
(‡etoposide) for 1 h and the cultivation continued until virus harvest at 84
hpi. (b) Induction of recombination by camptothecin. Recombination was
measured with wtSV40, RARa bcr and MLL bcr virus strains in response to a
1 h treatment with DMSO ( camptothecin) or with 300 nM camptothecin
(‡camptothecin) 12 hpi.
from patient data, and, secondly, we chose regions in which
both chromosomal translocations and retroviral integrations
were found.
Neither the TAL1 nor the LMO2 bcr fragment, which also
corresponded to retroviral integration sites, caused a recognizable
destabilizing effect in our assay, in contrast to the other
two loci investigated. This may be due to the fact that
sequences that dictate susceptibility to chromosome breaks
were not included in the fragment examined or that the rhesus
monkey kidney cells, in which these assays were performed,
do not express the appropriate enzymatic machinery involved
in this break. In this context it is important to note that both
these bcrs are associated with T-cell ALLs and that the chromosomal
joining occurred at the TCR a/d chain locus (4--6).
Indeed, it has been demonstrated that heptamer/nonamer-like
sequences within these loci function as weak V(D)J recombination
signals (43,44).
1310
Is it possible that the V(D)J recombination machinery also
plays a role in the retroviral integrations characterized in this
work? Interestingly, both the LMO2 and TAL1 integrations are
found upstream (396 and 485 bp, respectively) of imperfect
heptamer/nonamer-like sequences. Conceivably, misrecognition
and DNA cleavage by the RAG complex and/or recruitment
of DSB repair enzymes by RAG binding may have
provided an entry site for the provirus, mediated by the viralencoded
integrase. Several recent reports have demonstrated
the interplay between retroviral integration, and DNA repair,
and thus lend support to such a hypothesis (45,46). At least one
of the two cell lines in which these retroviral sites were
identified also express RAG1 and RAG2 (M.Ziegler and
C.Stocking, unpublished data). Further, V(D)J recombination
was documented previously for a fraction of immortalized cell
lines of the B-, T- and myeloid lineages (47). Retroviral
integrations in the LMO2 locus have also been reported
recently in the neoplastic clonal expansion of T-cells in two
patients, following the successful gene therapeutic treatment
of 11 children with severe combined immunodeficiency
(SCID) (48). Although, integration into this locus has most
probably influenced the selective growth of these cells, it
cannot be presently excluded that preferential integration into
this loci due to the V(D)J machinery contributes to the high
incidence of this event. A better understanding of the potential
interaction of V(D)J machinery and retroviral integration is
thus required to assess the significance of these observations.
As our assay was designed to identify cis-acting mechanisms
in homology-directed recombinative repair rather than in
V(D)J recombination, we can only conclude that the TAL1
and LMO2 regions tested did not influence HR in the absence
of V(D)J recombinase.
In sharp contrast, we established recombination stimulation
by the MLL bcr sequence in the SV40 based assay. This is the
first report, which describes a cis-stimulatory role in recombination
for a MLL sequence from the 8.3-kb bcr as small as 399
bp. Within this bcr two hotspots of chromosomal fusion sites
were identified, one for infants below 1 year and one for
patients above this age (35). Strikingly, within the first hotspot,
which is covered by the MLL fragment chosen in this study,
specific DNA cleavage, in response to treatment with topoisomerase
II inhibitors, was described to occur (11,12). In
agreement with these reports, we noticed a pronounced recombination
induction after treatment with etoposide. Conversely,
the MLL bcr was resistant to topoisomerase I inhibition under
conditions, which caused a pronounced effect on recombination
at the RARa bcr. Surprisingly, the 399-bp MLL bcr fragment
does not carry a topoisomerase II consensus site,
although recognition sequences were identified at neighbouring
positions between exon 11 and 14 (42). Alternative explanations
for the MLL bcr fragility have come from observations
indicating that it maps to the centre of a high affinity matrix
attachment region (MAR), that MLL bcr subfragments still
function as MARs, and that a correlation exists between the
density of MLL translocation breakpoints and scaffold association
(49). Topoisomerase II is enriched at MAR sequences, so
that, after treatment with topoisomerase II inhibitors, the
enzyme might preferentially cleave within the MLL bcr.
Recombination inhibition by p53 is sequence-independent
p53 was shown to be capable of inhibiting HR and NHEJ, and
the recognition of aberrant exchange events may represent
the underlying mechanism (15--17). In agreement with these
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findings we saw down-regulation of ectopic recombination at
the TAL1 bcr, LMO2 bcr and the MLL bcr in cell lines stably
expressing wtp53 protein. Recombination frequencies were
kept low, even when a dramatic rise of the exchange events
at the MLL locus was provoked by etoposide treatment. Notably,
rearrangements within the MLL bcr underly therapyrelated
leukaemias (42), and were observed to be closely
related to mutations in the p53 gene (50). The consequence
of this is that mutation of p53 increases the leukaemia risk,
because of the failure to control DSB repair.
p53(138V) is defective in transcriptional activation and cell
cycle regulatory activities. However, p53(138V) expressing
cells were fully active in down-regulating ectopic recombination
on TAL1 bcr, LMO2 bcr and MLL bcr virus genomes.
These data further support the hypothesis that wtp53 directly
controls recombination (12--15). From our present knowledge
there are three mechanisms possibly underlying recombination
inhibition by p53 (15--17,24--28): first, by analogy to MSH2, it
can be envisioned that p53 blocks continued strand exchange
by the recombinase Rad51 after strongly binding to nascent
intermediates of HR. Secondly, exonucleolytic proofreading of
mispaired heteroduplexes may dissolve recombination junctions.
Thirdly, p53 may act anti-recombinogenic via modulation
of BLM activities, which disrupt recombinogenic
molecules that arise at sites of defective processing of DNA
replication intermediates.
Recombination stimulation by p53 is sequence-dependent
In sharp contrast to the data discussed so far, we saw a 4-fold
p53-dependent recombination stimulation rather than an inhibition
after introduction of the RARa bcr fragment into the
viral genome. It is well-established that replication fork pausing
leads to elevated recombination activities (2). However, in
LLC-MK 2(p53her) cells, the replication curve for the RARa
bcr virus was indistinguishable from other curves such as the
TAL1 bcr or LMO2 bcr-specific ones, suggesting that with
respect to the stimulatory mechanism replication was not
linked to recombination. Furthermore, the RARa bcr fragment
is missing a p53 consensus sequence and was not stably complexed
by wtp53 in gel shift experiments. Thus, the recognition
of the RARa DNA sequence was not the initial cause of
recombination enhancement by p53.
Notably, within the RARa bcr fragment, two topoisomerase I
recognition sequences (A/TGATG) are present (Figure 1B).
When we applied camptothecin on the parental LLC-
MK 2(neo) line, we monitored a rise specifically in the RARa
bcr-dependent recombination frequency, as was expected for
DNA breakage induced by topoisomerase I inhibition. However,
in LLC-MK2(p53her) cells, we measured the same
recombination frequency increase with and without camptothecin
treatment. This indicated that the combination of
camptothecin and wtp53 did not surpass the recombinationstimulatory
effect by camptothecin alone, suggesting an epistatic
pathway underlying recombination enhancement by
topoisomerase I inhibiton and wtp53. In this context, we consider
a recent report by Soe and colleagues (41), which
describes that p53 stimulates the formation of a double cleavage
complex containing two topoisomerase I molecules. This
complex leaves behind a gap of ~13 nt that supports strand
exchange mediated by the second topoisomerase molecule
in vitro. Thus, both camptothecin and p53 stabilize covalent
DNA--topoisomerase I complexes and, therefore, may cause
DNA breakage and recombination through the same enzyme.
Conclusions
Taken together, we have identified an 0.4 kb MLL fragment
that markedly promotes ectopic recombination in cis. DNA
recombination between MLL bcr viruses can be induced by
topoisomerase II inhibition, which provides an experimental
model for the chromosomal translocations leading to
treatment-related ALL. The inhibitory effect of wtp53 directed
towards these cancerogenic recombination events emphasizes
the importance of p53 in restraining malignant progression by
the surveillance of recombinative repair (15,16). In this work,
we also discovered that p53 up-regulates recombination at a
distinct sequence, namely at the RARa bcr, and our data
indicate mechanistic links to stable complex formation with
DNA-bound topoisomerase I. Therefore, we speculate that p53
maintains genomic stability not only by controlling the homology
length (15), but also by promoting homology-directed
repair after the generation of certain types of DNA lesions
and subsequent topoisomerase I cleavage complex formation
(41). Damage-specific processing might also explain why
Schiestl and co-workers (51) measured an increase of the
recombination frequencies in p53‡/‡ but not in p53 /
mice after X-ray treatment, whereas the benzo[a]pyreneinduced
recombination stimulation was smaller in p53‡/‡ as
compared with p53 / mice. Differential activation of p53 by
distinct lesions may also underlie the fact that in vivo the HRregulatory
activity of p53 is developmentally regulated (52).
Consistently, p53 is required for an irradiation-induced bypass
pathway, to substitute for V(D)J recombination in scid mice,
but counteracts spontaneous bypass rearrangements (53,54).
Specific recruitment might coordinate the different repairrelated
activities of wtp53, as suggested by the tightly regulated
interactions of wtp53 with topoisomerase I in response to
DNA damage (39). However, both wtp53 and mutant p53
interact with topoisomerase I (38), and failure to suppress
aberrant recombination coupled to the remaining capacity to
stimulate DNA exchange would be expected to generate a
gain-of-function phenotype in recombination, which could
explain the rise in gene amplification observed with some
mutant p53 proteins (55,56).
Acknowledgements
We are grateful to Dr Anne Dejean, Institut Pasteur, for the l phage containing
the RARa bcr locus, and to Prof. Dr B.Vogelstein, John Hopkins Oncology
Center, Baltimore, for pSK-45-13-2-PyCAT. We thank Dr Christine
Dudenhoffer and Evelyn Bendrat for help with mutant p53 and SV40 virus
preparation. This work was supported by grants 10-1281-Wi and 10-1907-Wi 2
from the Deutsche Krebshilfe.
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Received October 2, 2003; revised December 22, 2003;
accepted January 14, 2004
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