Loss of DNA Polymerase Z Causes Chromosomal Instability in Mammalian Cells

Research Article

John P. Wittschieben,1 Shalini C. Reshmi,2 Susanne M. Gollin,2 and Richard D. Wood1

1Department of Pharmacology, University of Pittsburgh Medical School and University of Pittsburgh Cancer Institute and 2Department of Human Genetics, University of Pittsburgh Graduate School of Public Health and University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania

Abstract chemical adducts derived from exogenous sources. A DNA Rev3L encodes the catalytic subunit of DNA polymerase Z polymerase with relaxed template specificity can function to (pol Z) in mammalian cells. In yeast, pol Z helps cells bypass the damaged base at a cost of increased mutation bypass sites of DNA damage that can block replication frequency. The most remarkable example of such an enzyme is the DNA polymerase (pol ). In the yeast Saccharomyces enzymes. Targeted disruption of the mouse Rev3L gene ~ ~ causes lethality midway through embryonic gestation, and cerevisiae, pol ~ is composed of catalytic Rev3 and accessory Rev7 Rev3LÀ/À mouse embryonic fibroblasts (MEFs) remain in a subunits (6, 7). The ability to extend mispaired termini may be the quiescent state in culture. This suggests that pol Z may be most unusual and biologically relevant catalytic activity of this necessary for tolerance of endogenous DNA damage during polymerase. Yeast pol ~ can efficiently extend termini left by normal cell growth. We report the generation of mitotically various DNA polymerases opposite a variety of types of different active Rev3LÀ/À MEFs on a p53À/À genetic background. DNA damage (8–14). Rev3L null MEFs exhibited striking chromosomal instability, The absence of REV3 in yeast causes a large decrease in the g with a large increase in translocation frequency. Many number of UV light–, -ray-, and methyl methanesulfonate (MMS)– complex genetic aberrations were found only in Rev3L null induced base pair and frameshift mutations (7). A substantial cells. Rev3L null cells had increased chromosome numbers, number of spontaneous mutations are also dependent on pol ~ and most commonly near pentaploid, and double minute presumably result from participation of the enzyme in bypass of chromosomes were frequently found. This chromosomal endogenous oxidative and hydrolytic damage (15, 16). Pol ~ is also instability associated with loss of a DNA polymerase activity sometimes involved in recombinational repair of strand breaks, in in mammalian cells is similar to the instability associated a manner yet to be defined. During recombinational repair of a with loss of homologous recombination capacity. Rev3L null directed double-strand break in budding yeast, point mutations MEFs were also moderately sensitive to mitomycin C, methyl can arise in the vicinity of the recombination event, and these methanesulfonate, and UV and ;-radiation, indicating that depend on functional Rev3 (17). mammalian pol Z helps cells tolerate diverse types of DNA Human and mouse REV3L are 353-kDa proteins with DNA damage. The increased occurrence of chromosomal trans- polymerase domains and a sequence required for interaction with f locations in Rev3LÀ/À MEFs suggests that loss of Rev3L REV7 but have a region of 1,400 amino acids not found in yeast expression could contribute to genome instability during REV3 (18–20). REV3L antisense experiments in HeLa cells show neoplastic transformation and progression. (Cancer Res 2006; that mammalian DNA pol ~ participates in DNA damage–induced 66(1): 134-42) mutagenesis (21). The frequency of UV-induced Hprt mutants is also much reduced in fibroblasts from a Rev3L antisense mouse Introduction model, and it seems that most UV-induced mutations in mammals are dependent on pol ~ (22, 23). The misinsertion and/or extension y Sites of DNA damage can block synthesis by DNA polymerases activities of pol ~ may also function during hypermutation of q and , the enzymes responsible for rapid, high-fidelity replication antibody V genes (22, 24) and in the mutator phenotype induced by of most of the eukaryotic genome (1). Stalled replication forks can hepatitis C virus (25). be reactivated by a specialized group of DNA damage bypass In these cell and animal antisense suppression models, some polymerases that function to insert bases across from damaged detectable expression of REV3L remains. For a definitive investi- bases, so that the DNA replication apparatus can proceed (2, 3). gation of pol ~ function, we and others generated mouse knockout Many types of DNA adducts may be bypassed by one or more of models. Inactivation of Rev3L causes embryonic lethality during these polymerases. The switch from a stalled DNA polymerase to midgestation (26–28). Tissues in many areas of homozygous null one capable of bypass is controlled at least in part by the embryos are disorganized with significantly reduced cell density, ubiquitination state of the proliferating cell nuclear antigen (4, 5). suggesting a requirement for bypass of endogenous DNA damage Some types of DNA damage are especially challenging to bypass, by pol ~. It may be relevant that oxidative damage to DNA could such as apurinic sites where a base has been lost, or bulky occur with increasing frequency during early midgestation due to the metabolic switch to oxidative phosphorylation that occurs at this point in development (29, 30). Note: The cytogenetic studies were carried out in the University of Pittsburgh À/À Cancer Institute Cytogenetics Facility. We report here that viable Rev3L mouse embryonic Requests for reprints: Richard D. Wood, Hillman Cancer Center, Research fibroblasts (MEFs) could be generated by inactivating p53 function. Pavilion, Suite 2.6, 5117 Centre Avenue, Pittsburgh, PA 15213. Phone: 412-623-7766; Fax: A chromosome analysis and study of resistance to DNA damage 412-623-7761; E-mail: [email protected]. I2006 American Association for Cancer Research. show that pol ~ plays an important role in chromosome stability in doi:10.1158/0008-5472.CAN-05-2982 mammalian cells.

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Materials and Methods karyotyping analysis, new coded slides were prepared using cells from an earlier passage than for G-band analysis. Chromosomes were hybridized to Generation of MEFs and cell growth. The Rev3L null allele inactivates 24-color, combinatorially labeled SkyPaint probes (Applied Spectral Imaging, REV3L by replacing two exons encoding the conserved DNA polymerase Carlsbad, CA) as described (34). Chromosome and probe denaturation, family B motif V and part of motif I with a promoterless IRES cassette hybridization, washing, and staining procedures were done as specified by containing a fusion of the lacZ and neomycin resistance genes (28). The p53 the manufacturer’s protocol. Fields of well-separated metaphase chromo- null allele originates from the disruption generated in the Donehower somes were captured and analyzed with the SkyVision I system (Applied laboratory (31). After timed matings between Rev3L+/À;p53+/À and +/À +/À +/À À/À Spectral Imaging). The aberration analysis was scored before decoding the Rev3L ;p53 or Rev3L ;p53 mice of B6;129 background, embryos genotype and was done in two parts as described in Results. were isolated on days E10.5-12.5. DNA from embryo yolk sacs and cultured To quantify micronuclei, 5 Â 105 cells per MEF line (passages 13-18) were cells was prepared using the Qiagen (Valencia, CA) DNeasy Tissue kit and seeded onto 15-mm coverslips in six-well dishes. Cells were fixed in 4% genotyped by PCR (28). Individual embryos were dissociated and placed paraformaldehyde (pH 7.0) and permeabilized with 1% Triton 100-X for into culture as tissue explants in a humidied 5% CO2 incubator. Initial 10 minutes at room temperature. Slides were washed with PBS and mounted culture medium was a 1:1 mixture of DMEM with 4.5 g/L glucose and Ham’s using Vectashield with 4V,6-diamidino-2-phenylindole (DAPI; Vector Labo- F12 (both from Life Technologies, Gaithersburg, MD), supplemented with ratories, Burlingame, CA) to stain the DNA. DNA from the indicated number 1% nonessential amino acids, 1% glutamine, and 10% fetal bovine serum of cells was analyzed using the Â100 objective of an Olympus Provis (FBS; Myclone, Logan, UT). AX70 microscope with reflected light fluorescence. DAPI-stained micro- À/À À/À MEFs from a single Rev3L ;p53 embryo began to proliferate nuclei were scored only if completely separated from the nucleus, as distinct following 3 months of occasional partial media changes and were then from nuclear blebs. Results are expressed as the frequency of micronuclei +/+ +/À passaged without a notable growth defect. Rev3L and Rev3L control (%) relative to the total number of interphase nuclei scored. MEF lines with p53À/À backgrounds were derived from the same litter of embryos as the Rev3L null line. Immortalized cell lines were established from spontaneously transformed clones following continuous passaging, Results and cell genotypes were confirmed by PCR (28, 32). Following immortal- Generation of proliferating MEFs lacking functional pol Z. ization, cells were cultured in DMEM (Mediatech, Herndon, VA; with Disruption of the mouse Rev3L gene causes lethality during 4.5 g/L glucose) with 10% FBS, 1% glutamine, 100 units/mL penicillin, and À/À 2 midgestation days E9.5-E12.5. Rev3L embryos from this 100 Ag/mL streptomycin in 75-cm flasks in 10% CO2. The growth rate of immortalized Rev3LÀ/À embryonic fibroblasts and a developmental stage were isolated and placed into culture to Rev3L+/+ control was measured by plating 4 Â 104 cells in six-well plates and derive MEFs. In repeated attempts, we and others found that À/À counting cell numbers over a period of 5 days. The experiment was repeated MEFs that are Rev3L but have an otherwise normal genetic thrice with cells from passages 12 to 17, and for each experiment, triplicate background, divide very poorly, if at all, in vitro (26–28, 35). wells were washed, trypsinized, and counted individually using a Altering embryo age, culture medium, and MEF culture method hemacytometer. Cell viability was determined by trypan blue exclusion, did not result in cells that could be passaged. In each case, and cell population doubling times were calculated using counts from days Rev3LÀ/À fibroblasts entered a quiescent state with cell death 2 and 4, during the period of exponential cell growth. occurring over weeks to months. DNA damage sensitivity. Cytotoxic and cytostatic effects of DNA Developmental and cellular proliferation defects due to loss of damage were assayed by measuring ATP levels. ATP is present in all DNA repair genes have been ‘‘rescued’’ to different extents by metabolically active cells and decreases rapidly upon necrotic or apoptotic cell death. Cells were grown in phenol-free DMEM plus 10% FBS in a 10% simultaneously removing p53 (36–38). In contrast, we did not detect any effect of removal of p53 for development of Rev3LÀ/À CO2 atmosphere. Cells used for these experiments were from passages 13 to 23. The ATPlite1step (Perkin-Elmer, Boston, MA) luminescence system embryos, a finding also reported by other groups (35, 39). +/À +/À and a Perkin-Elmer TopCount luminometer were used to determine cell Rev3L ;p53 mice were intercrossed and embryos from days viability. To ascertain the suitability of this assay for DNA damage sensitivity E10.5-12.5 were examined. Of the Rev3LÀ/À;p53À/À embryos studies using MEFs, a range of 125 to 50,000 cells per well were seeded in identified, four were resorbing (inviable) and four were viable. The quadruplicate, and ATP levels were quantified 5 hours later. The latter embryos did not develop further than their Rev3LÀ/À;p53+/À or relationship between cell number and luminescent signal was linear from Rev3LÀ/À;p53+/+ littermates and were developmentally impaired +/+ À/À 125 to 20,000 cells per well for both Rev3L and Rev3L MEF cell lines, relative to Rev3L+/+ and Rev3L+/À controls of any p53 genotype. with low variance and a <1% backgound signal. This confirmed the To determine if loss of p53 would assist the ability of Rev3L precision of this method and guided the optimal cell seeding density and null cells to divide in vitro, 39 embryos from crosses between ATP quantification time to remain in linear range. The cell sensitivity assays +/À +/À +/À À/À A Rev3L ;p53 and Rev3L ;p53 mice were isolated and were done as follows: 1,250 cells per well were seeded (100 L) in +/+ +/À quadruplicate in black 96-well plates (Perkin-Elmer) in DMEM + 10% FBS. cultured. Rev3L and Rev3L fibroblasts proliferated immedi- Twenty-four hours later, cells were treated with the indicated concen- ately and similarly. Embryonic fibroblasts from a single E10.5 À/À À/À trations of mitomycin C (MMC; Sigma, St. Louis, MO) or MMS (Sigma) for Rev3L ;p53 mouse embryo placed in culture began to 18 hours. Cells were UV and g-irradiated in complete medium at a dose rate proliferate after 3 months. Cell division was without delay from of 0.7 J/m2/s from a UV-C source and at 19.66 Gy/h from a 137Cs source, the time of first passage, and the Rev3L null MEFs had a respectively. Cells were washed with complete medium following treatment, characteristic spindle-shaped, fibroblast-like morphology. The fresh medium was added, and the cells were then grown for 2 days at which Rev3L null genotype was confirmed (Fig. 1A). Cells in one of four time untreated controls were 80% to 90% confluent. Results were expressed Rev3LÀ/À;p53+/À embryos placed into culture showed some mitotic as the number of cells in treated wells relative to untreated control wells activity during 8 months of partial medium changes, but this (% control). IC values were defined as the concentration or dose resulting 50 eventually ceased. Rev3L+/À and Rev3L+/+ MEFs that were p53À/À in 50% growth inhibition. were also obtained from the same litter of embryos and these were Cytogenetics and micronucleus formation. Cells were treated with À/À 0.5 Ag/mL colcemid for 2 hours followed by harvesting and trypsin-Giemsa passaged along with the Rev3L cells until spontaneous banding (33). Five individual metaphase cells from each coded cell line immortalization occurred. À/À were captured digitally, and composite karyotypes were prepared using the We hypothesized that Rev3L MEFs might have an increased CytoVision Ultra System (Applied Imaging, Santa Clara, CA). 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sensitivities of the Rev3L null cells compared with Rev3L wild-type cells as follows: MMC, 5.0-fold; UV, 3.7-fold; g irradiation, 2.3-fold; MMS, 1.8-fold. Similar absolute and relative sensitivity of the Rev3L null MEFs and a second wild-type control line to MMC, MMS, and g irradiation was observed when experiments were done in six-well plates, and cell numbers were determined using a hemacytometer. Microscopic examination revealed that both Rev3L null and control cells displayed a classic increased size response following g-irradiation. These results indicate that mammalian pol ~ functions in the tolerance of several diverse types of DNA damage. Increased chromosome number, fusion, and fragmentation in Rev3LÀ/À MEFs. We next looked for evidence of chromosomal instability associated with Rev3L deletion. Chromosome spreads were stained with Giemsa, and individual metaphases were counted for total chromosome numbers (Table 1). All cell lines had undergone polyploidization, an event commonly observed during establishment of MEF cell lines (41). The numbers of chromosomes were increased in the Rev3LÀ/À;p53À/À cells (range, 78-189) relative to the Rev3L+/À;p53À/À and Rev3L+/À;p53+/+ control lines (ranges, 56-129 and 61-121). The majority of Rev3L null cells were near pentaploid (90-107 chromosomes), whereas the majority of control MEFs were hypotetraploid (69-76 chromosomes). Hypotetraploidy is the usual state of spontaneously immortalized MEFs. Karyotyping revealed significantly increased spontaneous chro- À/À Figure 1. Establishment and growth of a Rev3L MEF cell line. A, PCR mosomal instability in Rev3LÀ/À;p53À/À cells (Table 2; Fig. 3A). strategy used to genotype Rev3L + and Rev3LÀ alleles. Two essential DNA polymerase active site exons are removed in the Rev3LÀ allele and replaced There were increased numbers of fused/translocated chromo- with a lacZ-neor (h-geo) cassette. Common primer F1 together with (+) allele R1 somes, chromosomes with terminal deletions, and rearranged and (À) allele R2 primers generates 457-bp wild-type and 734-bp targeted mutant PCR products, respectively. For Rev3LÀ/À cells, PCR results of ‘‘marker’’ chromosomes not identifiable by G-banding. For all three passages 2 and 7 are shown. Genotypes of Rev3L +/+ and Rev3L +/À cells from categories, the increase was 4- to 5-fold over control cell lines / the same litter of embryos are flanking the Rev3LÀ À cells. A single passage for (Table 2). The additional chromosomes found in the Rev3L null line each is shown. B, growth curves for mouse embryonic fibroblasts, either Rev3L +/+;p53À/À (o)orRev3LÀ/À;p53À/À (.). Equal cell numbers (4 Â 104 per were divided approximately equally between whole, intact well) were plated in six-well plates, and for each experiment, triplicate wells chromosomes and those that were rearranged or had terminal were counted over a period of 5 days. Points, mean of three independent deletions (9.2 and 11.2 average events per metaphase, respectively). experiments; bars, SE. replication. Cell growth of Rev3LÀ/À and Rev3L+/+ embryonic fibroblasts was measured (Fig. 1B). During exponential growth, the population doubling time of Rev3LÀ/À embryonic fibroblasts was slightly increased relative to a Rev3L+/+ fibroblast line (19.1 versus 17.2 hours). The Rev3L null cells routinely had a higher plating efficiency and/or lower lag time before entering exponential growth. Likewise, the cell density at confluence of Rev3LÀ/À;p53À/À MEFs was always higher than control lines (Fig. 1B; data not shown). Viability of the cells during the growth and drug sensitivity experiments shown below ranged from 86% to 97% for the Rev3L null cells and 91% to 95% for the control cells. Sensitivity of DNA pol Z null cells to DNA-damaging agents. The Rev3L null MEF line was used to determine how a lack of DNA pol ~ affects growth and survival after treatment with DNA- damaging agents. Relative cell survival was determined by measuring total ATP levels, which is a direct measure of viable cell number. The assay has a wide dynamic range and low background and was validated for MEFs as described in Materials and Methods. The Rev3LÀ/À MEFs were tested for their sensitivity to UV light, g-irradiation, MMS, and MMC (Fig. 2). The mutagenicity and cytotoxicity of the former three DNA-damaging agents has been extensively studied with yeast rev3D strains (7). MMC was tested Figure 2. Sensitivity of DNA pol ~ null cells to DNA-damaging agents. Equal because it can introduce interstrand cross-links into DNA. rev3- numbers of Rev3LÀ/À (.) and Rev3L +/+ (o) were plated in quadruplicate in mutant yeast cells are hypersensitive to photoactivated psoralen, 96-well plates. Cells were exposed to (A) MMC, (B) MMS, (C) UV-C, and (D) g-irradiation as described in Materials and Methods. After 2 days, cytotoxicity which can produce such cross-links (40). The ratio of the IC50 values was determined by quantification of ATP levels in treated and untreated cells. for these DNA-damaging agents provides a measure of the relative Points, mean of three independent experiments (% untreated control); bars, SE.

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+/+ Table 1. Chromosome numbers in Rev3L mutant MEFs increase in the number of micronuclei relative to control Rev3L ; p53À/À cells (Fig. 3B). Compared with Rev3L+/+ cells, more Rev3LÀ/À cells had multiple micronuclei, and both nuclei and micronuclei MEF Passage Range Mean Median Modal À/À À/À cell line karyotypes from the Rev3L ;p53 MEFs showed greater size heterogeneity. Futher, three anaphase bridges were observed in the Rev3L null Rev3L+/À;p53+/+ 14 56-129 77 73 73 cells, whereas none were seen in the control. Rev3L+/À;p53À/À 12 61-121 75 72 69, 74 Increased translocation events in the absence of DNA pol Z. Rev3LÀ/À;p53À/À 12 78-189 110 101 91, 98, 103, 115 Spectral karyotyping was used to characterize and quantify the rearrangements occurring in earlier passage cells in more detail. Spectral karyotyping enables unequivocal definition of rearrange- NOTE: Chromosome spreads were stained with Giemsa, and total ments on a chromosome-by-chromosome basis, and our analysis chromosome numbers were counted for 25 individual metaphases of concentrated on the detection and quantification of aberrations each cell line. Statistical averages are shown. Based on the distribution rather than cellular karyotyping. Robertsonian-like translocations of ploidies for all three cell lines, it is likely that the two to three (or centric fusions), isochromosomes (see Fig. 4 legend for metaphases at the high end of the range for each line resulted from definition), and mouse long arm translocations were found in failed cytokinesis of cells with chromosome numbers at the low end of À/À +/À the range. both Rev3L and control Rev3L MEFs (Fig. 4A and B). Three different classes of aberration were found only in the Rev3L null cells. These were dicentrics, insertions, and compound isochromosome/translocation events (Fig. 4B). Marker chromosomes were loosely divided into those larger than Both the total number of all aberrations and the total number of the smallest mouse chromosome (chromosome 19) and those different (nonclonal) aberrations were increased 4- to 5-fold in smaller than chromosome 19. The small marker chromosomes are Rev3LÀ/À MEFs (Table 3). This is similar to the increase in numbers similar to human small supernumerary marker chromosomes, of aberrant events as determined by G-banding. Certain types of which usually originate from the five human acrocentric chromo- aberrations were specifically enhanced in Rev3L null cells. somes (42). One third of the total marker chromosomes for both Aberrations occurring along the chromosome long arm were Rev3LÀ/À;p53À/À (13 total) and Rev3L+/À;p53À/À (3 total) cells increased 15-fold in Rev3L null cells (the sum of translocations, appeared in this category and were mostly heterochromatic (darkly isochromosomes plus translocations, insertions, and dicentrics). staining bands). These may arise following the creation of dicentrics The largest element of this increase is the number of translocation between telocentric chromosomes, which subsequently break events. Numbers of events involving the short arm and centromere within the q arm close to one centromere. This confers stability (isochromosomes and Robertsonian-like translocations) were to both the large chromosome and the small marker. Ten of the equivalent in Rev3LÀ/À and Rev3L+/À MEFs. 25 Rev3LÀ/À;p53À/À cells had very small chromosome fragments All chromosomes except for 6, 11, and Y were observed in with the characteristic dumbbell or bilobed shape of double rearrangements; 10 different chromosomes were involved in three minutes (Fig. 3A). These were not included in the chromosome to seven events each, whereas six chromosomes were involved in count and were not seen for either of the control MEFs. Ring or one to two events (Table 4). The spectral karyotyping analyses multiradial chromosomes were not observed in the Rev3L null cells. were completed in two equally sized series with cells from The chromosome instability of Rev3LÀ/À;p53À/À cells was also different passages. For the Rev3L null cells, only one aberration examined by a noncytogenetic method. Micronuclei can form from identified in the first series [der(2)t(1;2)] was found in the second DNA fragments that are not incorporated into daughter nuclei group, indicating that the chromosomal instability is a continuous because they lack a functional centromere or by fragmentation of process occurring cell by cell and dispersed throughout the anaphase bridges (43). The Rev3LÀ/À;p53À/À cells had a >2-fold mouse genome.

Table 2. Summary of G-band karyotype analysis

MEF cell line Mean chromosome Loss Gain Marker Terminal deletions Additions/translocations no., karyotyped cells chromosomes

Rev3L+/À;P53+/+ 77 7.4 4.2 0.2 0 0.2 Rev3L+/À;p53À/À 75 9.2 1.8 1.8 0.6 0 Rev3LÀ/À;p53À/À 92 8.6 9.2 7.8 2.4 1

NOTE: Five G-banded metaphases were completely karyotyped. All results are expressed as the mean number of each category per metaphase. The numbers of intact chromosomes for each autosome and sex chromosome were counted for each karyotype. The ‘‘loss’’ and ‘‘gain’’ columns refer to average net variations in whole chromosome numbers from a 4N chromosome number. Marker chromosomes are defined as chromosome rearrangements not identifiable by G-banding. Both of the control lines lost more chromosomes than they gained. This, together with the low total numbers of rearranged chromosomes and those with deletions (last three columns), accounts for their net hypotetraploid mean numbers [80 À loss + (gain + markers + deletions + fusions)]. The Rev3LÀ/À;p53À/À cells gained slightly more intact chromosomes than they lost. This, and the greater number of chromosomes with rearrangements and deletions, comprises the near-pentaploid mean number for the karyotyped Rev3LÀ/À;p53À/À cells.

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required to help cells tolerate endogenous DNA damage during mammalian development. Similarly, cells from Rev3L null embryos do not proliferate in vitro but remain quiescent with cell death occurring over time (26–28, 35). The null Rev3L cells from midgestation embryos may have accumulated levels of unrepaired endogenous DNA damage that activate DNA damage checkpoints and preclude cell division in vitro. As reported here, cells from a Rev3LÀ/À;p53À/À embryo began dividing rapidly after 3 months in culture. Dividing cells from one of four Rev3LÀ/À;p53+/À embryos were observed after an even longer period (6-8 months) but ceased division before being passaged. Mitotic activity of Rev3LÀ/À cells with a p53 wild-type background was never observed, even after extended culture periods. Most likely, a p53-dependent checkpoint is only partly responsible for the arrested division observed in cultured Rev3LÀ/À cells. A similar conclusion was reached by Zander and Bemark, who also generated proliferating Rev3L null MEFs in a p53 mutant background (44). The population doubling time of our Rev3LÀ/À embryonic fibroblasts was f11% longer than a Rev3L+/+ control, whereas the plating efficiency and saturation density were higher for the Rev3L null cells. Vertebrate Rev3-null cells capable of cell division were first reported for the gene-targeted chicken lymphocyte DT40 cell line, and a comparable f12% increase in cell cycle time was found (45). p53 expression is not detectable in DT40 cells (46), emphasizing that checkpoint and apoptosis functions must be compromised in vertebrate cells for in vitro cell division to occur in the absence of pol ~. The isolation of mitotically active Rev3L null Figure 3. Chromosomal instability in Rev3LÀ/À MEFs. A, chromosomal instability in a representative G-banded karyotype of a Rev3LÀ/À;p53À/À metaphase. A wide range of whole chromosome gains and losses are seen (e.g., chromosomes 9 and 12). Two translocations involving chromosomes 7 and 17 (open arrows). A chromosome 19 carrying a deletion (closed arrow). Both large and small supernumerary marker chromosomes, as well as double-minute chromosomes (dmin), are placed between mouse chromosome 19 and the sex chromosomes. One marker contains a chromatid break (chtb). The increase in both structurally normal and abnormal chromosomes leads to a total chromosome number of 95 in this metaphase. The Rev3LÀ/À;p53À/À cell line is derived from a male embryo. B, micronucleus formation in Rev3L +/+;p53À/À and Rev3LÀ/À;p53À/À cells. The frequency of spontaneous micronucleus formation in Rev3L +/+ and Rev3LÀ/À interphase nuclei was determined by DAPI staining. Sample number was >1,400 cells for each line.

The distribution of chromosome aberrations is not entirely random. Chromosome 2 was involved in 11 long arm aberrations (Table 4), a number larger than expected even taking chromosome size into account. This may indicate the presence of one or more breakage or recombination hotspots on mouse chromosome 2. For each of the four der(2) translocations (involving chromosomes 1, 10, 15, and 17), a derivative translocation involving each of the partners and chromosome 2 was detected, suggesting that reciprocal Figure 4. Spectral karyotyping analysis of DNA pol ~ null cells reveals details of exchanges occur. A possible reciprocal event involving chromo- genome instability. A, types of structural chromosome aberration found in control somes 3 and 19 was more complex (Fig. 4B). Based on chromosome Rev3L +/À MEFs: isochromosome (i), Robertsonian-like translocations (rob), and translocations (der). Cells analyzed were passages 8 and 9. B, these three length, it was unambigous that seven of the translocations (involving types of events were also found in Rev3LÀ/À MEFs (top). Examples of six different chromosomes) in Rev3L null cells involved only a partial aberrations found only in Rev3LÀ/À MEFs (bottom): dicentrics (dic), insertions chromosome as one of the exchange partners. This shows that (ins), and combined isochromosome/translocation events (i and t). The der(19)ins(3;19) and i(3)+t(3;19) abnormalities are possible products of a rearrangements occur within the chromosome long arm in Rev3L reciprocal translocation event. Rev3LÀ/À cells analyzed were passages 7 and 9. null cells and correlate with the terminal chromosome deletions Mouse chromosomes are telocentric containing essentially only a long arm, with a short arm composed solely of centromeric and telomeric repeat sequences. found by G-banding. Two types of defect can occur to generate metacentric (biarmed) chromosomes. Isochromosomes may result from DNA breakage within the short arm or Discussion centromere followed by post-replication reunion of the sister chromatids to generate a chromosome with two copies of the long arm flanking the now Mammalian cells can proliferate rapidly in the absence of metacentric centromere (72). Interchromosome fusions involving the mouse Rev3L. The absence of Rev3L in the mouse causes impaired short arm (from the telomere to the centromere) produce metacentric chromosomes (Robertsonian-like translocations). The exchange usually involves embryonic and extraembryonic tissue development, leading to the short arm of both participants, but a Robertsonian-like configuration may also lethality during midgestation. This suggests that pol ~ may be be created between one short and one long arm.

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sis doubles chromosome content. This lessens restrictions on the Table 3. Loss of DNA pol ~ causes elevated translocation frequency types of chromosome abnormalities that can accumulate in cells. The Rev3L null cells were near pentaploid due to gain of whole Rev3L+/À;p53À/À Rev3LÀ/À;p53À/À chromosomes and aberrant chromosomes. The gain of complete chromosomes may be caused by mis-segregation of chromatids Total no. chromosomes 1,081 1,110 that are not fully replicated and separated. The incidence of analyzed chromosomes with rearrangements or terminal deletions was 4- to Total no. aberrations 10 56 5-fold greater in Rev3L null cells than in the control lines. Rev3À/À Total no. different 9 (0.8%) 37 (3.3%) DT40 cells had a 3-fold increase in the frequency of spontaneous (nonclonal) aberrations* aberrations was found (45). These were breaks and gaps rather Robertsonian-like 44than exchanges. MEFs may be relatively more proficient at DSB translocations repair by nonhomologous end-joining (NHEJ) than are DT40 cells. Isochromosomes (iso) 3 2 Small chromosome fragments resembling double minutes were iso + der 02 f (complex translocations) found in 40% of the Rev3L null cells and not in control cells. der (translocations) 2 25 Different models for the origin of these extrachromosomal acentric Insertions 0 2 species all postulate DNA breaks as an essential step (55). Dicentrics 0 2 A form of multicolor fluorescence in situ hybridization known as COBRA was used to examine metaphases from Rev3LÀ/À embryos, and the incidence of translocations seemed to be increased several- *The numbers of nonclonal (unique) aberrations for each line is the fold (35). The total numbers of rearrangements found were low, sum of the aberration types shown below this total. The analysis was and different categories of translocations were not reported. We done in two series using passages 8 and 9 for Rev3L+/À;p53À/À cells À/À À/À did spectral karyotyping analysis to identify and quantify the and passages 7 and 9 for Rev3L ;p53 cells. For the Rev3L null different types of chromosome rearrangements occuring in Rev3L cells, the majority of aberrations in both cases were translocations null MEFs. The most remarkable finding is the order of magnitude (70% and 85%). increase in the number of translocations in Rev3L null cells relative to control Rev3L+/À cells. The well-dispersed distribution of translocations among the mouse autosomes indicates that the MEFs confirms that pol ~ is not essential for chromosome increase in translocation frequency is not due to recurrent replication in vertebrates. As discussed below, it is however, breakage fusion bridge cycles but is instead due to random required for genome stability in mammalian cells. genomic instability. Other indications for increased chromosomal Generalized sensitivity of pol Z–disrupted cells to DNA- breakage in the null cells are the appearance of insertions, which damaging agents. The modest sensitivity of Rev3LÀ/À MEFs to may involve multiple DNA breaks for their formation, and several different DNA-damaging agents indicates that pol ~ compound translocation/isochromosome events. These compound function increases tolerance to a broad range of DNA adducts. events most likely arise from sequential rearrangements. Rev3L null MEFs are relatively more sensitive to mitomycin C and Chromosome breakage in Rev3L null cells. The high UV-C irradiation than g-irradiation and MMS. This suggests that in frequency of DNA rearrangements in Rev3L null cells suggests MEFs, there are more redundant mechanisms of tolerance to that pol ~ is required to prevent multiple double-strand breaks ubiquitous endogenous types of DNA damage (oxidative base from occurring in the mammalian genome. In the absence of this lesions and base methylations) than to less frequent DNA lesions enzyme, replication forks stalled at sites of DNA damage may originating from environmental sources. Other specialized DNA actively or passively collapse, leading to DNA double-strand breaks. polymerases are capable of synthesis at sites of simple base Homologous recombination and NHEJ repair pathways are damage or AP sites (47). DNA pol ~ may be more uniquely able to functional in these cells and will rapidly repair DNA breaks, both insert and/or extend bases opposite the larger, more helix- correctly and incorrectly (56). Frequent reciprocal translocations distorting lesions introduced by mitomycin C and UV-C radiation. occur in mouse embryonic stem cells when there are two double- These results are generally consistent with data on sensitivity to strand breaks on different chromosomes (57). DNA damage damaging agents in other mutant organisms and Although translocations were significantly elevated, chromosome cells lacking pol ~. Yeast rev3 mutant cells are moderately sensitive exchanges involving telomeric and centromeric repeats in Rev3LÀ/À to cross-linking agents and UV and mildly sensitive to g-irradiation and Rev3L+/À MEFs were equivalent. In contrast, chromosomes from and MMS (12, 48–50). Yeast REV3 null cells are not hypersensitive telomerase null cells with shortened telomeres form increased to the oxidizing agents hydrogen peroxide and menadione (51). numbers of p-p arm fusions (58). MEFs from NHEJ null mutants Neurospora crassa and Arabidopsis thaliana cells disrupted for (Ku70, Ku80, DNA-PK, and Lig4) have a high incidence of chromosome Rev3 also follow this sensitivity pattern to these same agents and chromatid fragmention, and the increased numbers of (52, 53). DT40 cells deleted for Rev3 are sensitive to these four types Robertsonian-like translocations in all these mutants indicate that of DNA damage, with a notable difference being the pronounced NHEJ functions to repair breaks occurring within the short arm of sensitivity of these cells to cisplatin (45). Independently isolated mouse chromosomes (59–61). Fusions involving short arms comprise Rev3LÀ/À;p53À/À MEFs are modestly sensitive to both cisplatin and about half of the total translocations found in these NHEJ null cells, UV radiation (44). A REV3L antisense knockdown cell line, which which is clearly different than the pattern in pol ~ null cells. retains a low level of REV3L expression, is mildly sensitive to Cytogenetic studies of fibroblasts from homologous recombina- cisplatin and has little or no UV sensitivity (21, 54). tion-deficient mice show more similarity to the results presented Chromosomes in Rev3L null embryonic fibroblasts are here for Rev3L null MEFs. Chromosome rearrangements involving structurally unstable. Tetraploidy arises when aberrant cytokine- the chromosome long arm are the most common aberration in www.aacrjournals.org 139 Cancer Res 2006; 66: (1). January 1, 2006

Table 4. Chromosome aberrations found in Rev3L(+/À) and Rev3L(À/À) MEFs

i rob t dic ins i and t

Rev3L+/À MEF aberrations i(1) rob(6;15) der(2)t(2;7) i(7) rob(6;7) der(5)t(5;16) i(18) rob(7;15), rob(12;19) Rev3LÀ/À MEF aberrations i(1) rob(2;5) der(1)t(1;2)9) dic(2;5) der(10)ins(16;10) i(3)+t(3;19) i(13) rob(4;14) der(1)t(1;5) dic(17) der(19)ins(3;19) i(13)t(13;14) rob(4;5) der(1)t(1;5;16) rob(11;15) der(2)t(1;2) der(2)t(2;10) der(2)t(2;15) der(2)t(2;17) der(3)t(2;3) der(3)t(3;10) der(4)t(X;4) der(5)t(2;5) der(8)t(8;10) der(9)t(9;18) der(10)t(2;10) der(10)t(10;17) der(12)t(12;14) der(14)t(7;14) der(15)t(2;15) der(15)t(15;19) der(16)t(10;16) der(16)t(14;16) der(17)t(2;17) der(19)t(8;19) der(X)t(X;15) der(X)t(X;1

NOTE: The events summarized in Table 3 are listed. Telomere sequence may or may not be present at fusion point. Breakpoints are unknown. Due to the nature of this analysis, reciprocal events may only be inferred. Abbreviations: i, isochromosome; rob, Robertsonian-like translocation. t, translocation; der, derivative events; dic, dicentric; ins, insertion; i and t, combined isochromosome and translocation events.

Rad51d- and Xrcc2-deficient cells. Loss of RAD51D protein results backup mechanisms are not sufficient to prevent chromosomal in a 24-fold increase of chromosome exchanges with a 6-fold instability, however, when DNA replication stalls at other genome increase in end-to-end fusions (62). Immortalized Xrcc2À/À MEFs sequences. have a 100-fold increase in chromosomal rearrangements com- Implications for tumorigenesis. Human REV3L maps to pared with Xrcc2+/+ MEFs but no difference in the number of end- chromosome 6q21. This region of 6q has long been postulated to to-end fusions (63). A possible explanation for the relative contain multiple tumor suppressor genes. Chromosome deletions of difference is that RAD51D associates with telomeres, whereas 6q21 have most often been reported for hematopoietic neoplasms, Xrcc2 does not (62, 64). The similar distribution of aberrations in including acute lymphoblastic leukemia, T-cell lymphoma, and immortalized Rad51d, Xrcc2, and Rev3L null MEFs may reflect the gastric high-grade large B-cell lymphoma (65–67). Some lymphomas fact that both homologous recombination and translesion derive from aberrant somatic hypermutation. As pol ~ may normally synthesis are pathways that help cells tolerate DNA damage function in this process, and there is an increased incidence of encountered by replication forks. In the absence of either pathway, translocations in the absence of Rev3L, it is possible that loss or broken DNA ends may form with increased frequency and be dysregulation of this polymerase may sometimes be a formative joined to nonhomologous chromosomes. event for such cancers. Some cases of chronic lymphocytic leukemia, The equal frequency of Robertsonian-like translocations and non-Hodgkin’s lymphoma, and diffuse large B cell lymphoma may isochromosomes in Rev3LÀ/À and Rev3L+/À cells indicates that loss originate from hypermutating B cells, and all three have been found of pol ~ does not significantly affect exchanges at telomeres and to have frequent deletions of 6q21 (68–70). Furthermore, the REV3L centromeres. Either DNA pol ~ does not function during replication gene is entirely within the FRA6F fragile site, and the 3V region of of telomeric and centromeric repeat sequences, or other DNA human REV3L overlaps one of two breakage hotspots (71). Deletion translesion bypass polymerases and DNA repair pathways are able breakpoints within this 1.2-Mbp fragile site occur in many blood and to perform compensatory functions when pol ~ is absent. Any such solid tissue cancers.

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Compromising the function of pol ~ could have significant Acknowledgments consequences for carcinogenesis. Human chromosome instability Received 8/21/2005; revised 10/14/2005; accepted 10/25/2005. syndromes and the transgenic mice generated to model them are Grant support: NIH grants CA098675 (R.D. Wood) and P30 CA47904 (R.B. often prone to tumorigenesis. As chromosome translocations can be Herberman). associated with oncogene activation, their increased occurrence in The costs of publication of this article were defrayed in part by the payment of page À/À charges. This article must therefore be hereby marked advertisement in accordance Rev3L MEFs implies that Rev3L null cells could have increased with 18 U.S.C. Section 1734 solely to indicate this fact. oncogenic potential. Loss of Rev3L expression, particularly after loss We thank Christine Saunders and the Cancer Research UK Cell Production team for expert assistance with embryo culture, members of our laboratory and Drs. of p53 function, might occur during neoplastic progression and Laura Niedernhofer, Robert Sobol, Karen Vasquez, and Christopher Bakkenist for contribute to the genome instability inherent in many tumors. discussion.

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John P. Wittschieben, Shalini C. Reshmi, Susanne M. Gollin, et al.

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