Role of Postreplication Repair in Transformation of Human Fibroblasts to Anchorage Independence1

(CANCER RESEARCH 51. 2960-2964. June 1, 1991] Role of Postreplication Repair in Transformation of Human Fibroblasts to Anchorage Independence1

Jayne C. Boyer,2 William K. Kaufmann, and Marila Cordeiro-Stone1 Curriculum in Toxicology Â¡J.C. B., H'. K. K., M. C-S.J, Department of Pathology Â¡H'.K. A'.. M. C-S./, and Lineberger Comprehensive Cancer Center (W. K. K., M. C-S./, The University of North Carolina at Chapel Hill, Chapel Hill. North Carolina 27599-7525

ABSTRACT the cells of the variant class of XP do not exhibit a deficiency in excision repair of UV-induced DNA damage (6, 7). Fibro Cellular capacity for postreplication repair (PRR) and sensitivity to blasts from these individuals appear to exhibit a defect or transformation to anchorage independence (AI) were quantified in normal deficiency in the replication of UV-damaged DNA templates foreskin and \eroderma pigmentosum (\P) variant fibroblasts after treatment with UV or benzo(â€žvaluesof 4.3 .Im' and 0.14 MMfor express a reduced capacity for PRR of UV-damaged DNA, but the inhibition of DNA synthesis in active replicons by UV and BPDE-I, they display the same ability as do normal cells to replicate respectively. \P variant cells (CRL1162) exhibited corresponding I),, DNA containing BPDE-I adducts (10, 11). XP variant fibro values of 1.5 .I/in and 0.16 ;UM.The increased sensitivity to inhibition of blasts are only slightly more sensitive than are normal cells to DNA replication by UV in these XP variant fibroblasts (2.9-fold greater the killing action of UV radiation (10, 12) but have been found than normal) was mirrored by an enhanced frequency of transformation to be hypermutable (12, 13) and hypertransformable by UV to AI. \P variant fibroblasts (CRL1162) were 3.2 times more sensitive (14). On the other hand, mutation frequencies induced in XP to transformation to AI by UV than were the normal foreskin fibroblasts. variant cells by exposure to BPDE-I were similar to those As predicted by the PRR studies, both cell types exhibited similar observed in normal cells (6). These results suggest that XP frequencies of AI colonies induced by BPDE-I. Apparent thresholds were observed for induction of AI by UV (normal fibroblasts, 2.7 J/m2; XP variant fibroblasts lack an error-free pathway of PRR of UV- variant fibroblasts, 0.3 J/m2) and BPDE-I (both, 0.05 JÃ•M).Dosesof UV induced lesions that is present in normal cells. However, this and BPDE-I above these thresholds produced proportional increases in pathway in normal cells appears to be ineffective in bypassing the inhibition of DNA replication in operating replicons and in the induced BPDE-I-induced DNA lesions (10). Thus, one would expect frequency of anchorage-independent colonies. At doses of UV and BPDE- that normal fibroblasts and XP variant cells should be equally I that produced the same degree of inhibition of DNA strand growth, sensitive to transformation to AI by BPDE-I. In this paper, we BPDE-I induced a greater number of cells capable of anchorage-inde present experimental data that confirm this prediction. Thus, pendent growth than did UV in both normal and XP variant fibroblasts. cellular sensitivity to transformation to AI by the carcinogens UV and BPDE-I appears to be quantitatively associated with INTRODUCTION sensitivity to inhibition of DNA strand growth caused by car cinogen-induced damage to the DNA template. Replication of carcinogen-damaged DNA is believed to result in mutations and chromosomal aberrations that may contribute MATERIALS AND METHODS to carcinogenesis (1, 2). Prereplicative repair of such damage reduces cellular risk of transformation (3). Cells from individ Cell Culture Conditions. Normal human fibroblasts were derived from uals who exhibit enhanced sensitivity to the development of neonatal foreskin (NHF1) and established in culture according to cancers are valuable tools for the examination of the roles of published methods ( 15), or they were obtained from the Human Genetic DNA repair and replication in mutagenesis and carcinogenesis. Mutant Repository, Camden, NJ (GM3348, skin biopsy of a clinically Individuals with the XP4 syndrome are affected by a genetic unaffected 10-yr-old male). XP variant fibroblasts were obtained from predisposition to sunlight-induced skin cancer (4). Cells of the American Type Culture Collection (CRL1162, XP strain designa tion XP4BE) or as a generous gift from Dr. Seiji Rondo (XP42TO). individuals expressing the classical form of XP are deficient in Cell stocks were grown in plastic tissue culture dishes, at 37Â°Cin a the operation of the nucleotidyl DNA excision repair pathway humidified atmosphere of 5% CO2, in Ham's FIO medium supple which removes DNA damage induced by UV (5). In contrast. mented with 15 to 20% fetal bovine serum and either penicillin (100 units/ml) plus streptomycin (100 /jg/ml) or gentamicin (50 Mg/ml). Received 12/17/90; accepted 3/20/91. The costs of publication of this article were defrayed in part by the payment Cells were subcultured 2 to 3 times/wk to maintain logarithmic growth of page charges. This article must therefore be hereby marked advertisement in and used between passages 1 and 18. The colony-forming efficiency on accordance with 18 U.S.C. Section 1734 solely to indicate this fact. plastic ranged from 10% to 15% for the XP variant cells and from 25% ' This work was supported by Institutional Grant IN-15-2 from the American Cancer Society and USPHS Grant CA42765 from the National Cancer Institute. to 40% fortheNHFl cells. 2Current address: Laboratory of Molecular Genetics. National Institute of Treatment with UV and BPDE-I. Cells to be irradiated with UV were first rinsed with warm (37Â°C)HBSS. A short-wave UV lamp emitting Environmental Health Sciences. Research Triangle Park, NC 27709. 3To whom requests for reprints should be addressed. mostly 254-nm radiation was used to irradiate cells. The incident 4The abbreviations used are: XP. xeroderma pigmentosum: AI. anchorage independence: BPDE-I. bcnzo(a)pyrene-diol-epoxide I: HBSS. Hanks' balanced fluence rate was 0.23 to 1.35 J/nr/s as determined with a UV radi salt solution; NHF1, normal human fibroblast strain derived from foreskin; PRR. ometer (UV Products, Inc., San Gabriel, CA). For treatment with postreplication repair. BPDE-I, culture medium was removed, and cells were rinsed twice with 2960

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1991 American Association for Cancer Research. POSTREPLICATION REPAIR AND CELI. TRANSFORMATION warm HBSS and then covered with 5 ml of the same solution. An from the treated cell frequencies. Thus, 46 - 13 = 33 colonies per 5 x aliquot of 25 /il of anhydrous dimethyl sulfoxide containing varying IO4 normalized cells were induced by UV. Because S-phase cells are concentrations of BPDE-I was added, and cultures were incubated at believed to be most at risk during carcinogen treatment (21) and because 37T for 10 min. The BPDE-I-containing solution was then removed, the number of S-phase cells observed in logarithmically growing cul and the cells were rinsed once with warm HBSS and refed with fresh tures of XP variant fibroblasts (15 to 45%) was routinely lower than medium containing serum. The concentration of the stock solution of that observed in cultures of foreskin fibroblasts (35 to 60%). the BPDE-I was determined from absorbance readings at 345 nm (extinc frequency of soft agar colonies was also normalized to the percentage tion coefficient of 48,600 cnr/mol), and the activity was determined of cells in S phase at the time of carcinogen treatment. The frequency using the method of MacLeod and Lew (16). of S-phase cells in culture was determined by autoradiography (22) in Inhibition of DNA Replication. Effects of UV and BPDE-I on DNA parallel cultures pulse labeled with pHjthymidine and cytocentrifuged strand growth were measured using the method of Kaufmann and onto microscope slides. Cleaver (9). Briefly, cells from logarithmically growing cultures were seeded at 4 x IO5cells per 100-mm dish and incubated with 5 nCi/ml of [14C]thymidine (ICN Pharmaceuticals, Inc., 50 Ci/mol) for 2 days to RESULTS label DNA uniformly. One to 2 h before carcinogen treatment, the The objective of this study was to test the hypothesis that medium was replaced with fresh unlabeled medium. Fibroblasts were then treated with UV or BPDE-I as described above. After treatment, cellular sensitivity to inhibition of DNA replication by carcin cells were incubated at 37Â°Cin reserved medium for 30 min and then ogen-induced template damage was predictive of sensitivity to pulse labeled with 50 ^Ci/ini of |'H]thymidine (ICN Pharmaceuticals, transformation to AI. Therefore, the effects of UV and BPDE- Inc.. 40 to 50 Ci/mmol) for 15 min. Cells were harvested, and the size I on DNA replication and transformation were compared in distribution of nascent DNA was analyzed in alkaline sucrose gradients normal and XP variant fibroblasts. Fig. 1 depicts the inhibition (9, 17). The sum of the incorporation of'H cpm into large intermediates of DNA strand growth by UV and BPDE-I. These results were of DNA replication (5 x IO7 to 2 x 10s dallons), after normalization generated from sedimentation profiles in alkaline sucrose gra to the number of cells added to each gradient (I4C cpm). was expressed dients as described in "Materials and Methods." We determined as the percentage of the same sum measured in control cells (10). DOvalues representing the increment of carcinogen dose that Anchorage Independence Assay. Assays were conducted according to reduced to 37% of control the amount of [3H]thymidine incor the methods of McCormick et al. (18, 19). Briefly, 2 to 3 x 10' cells were seeded into 100-mm dishes and treated 20 to 24 h later with poration into large intermediates of DNA replication. XP var iant fibroblasts displayed an enhanced sensitivity to UV-in- carcinogen as described above. Treated cultures were refed with fresh medium and maintained in logarithmic growth by subculturing at a 1:3 duced inhibition of DNA strand growth when compared with (XP variant cells) or 1:10 dilution (NHF1 cells). After the optimal normal cells (Fig. 1/1). The calculated Dn value for foreskin expression period of 5 population doublings (Refs. 14 and 18; Footnote fibroblasts (NHF1) was 4.3 J/m2. We have included in Fig. 1 5), 5 x IO4cells per 60-mm dish were seeded into 3 ml of 0.33% Noble our results in this assay previously published (10) for CRL1162 agar (Difco) over a 3-ml base layer of 0.5% agar in Ham's FIO (Dn = 1.5 J/m:). Other previously published Dn values for UV- supplemented with 7% (NHF1 ) or 20% (XP variant) fetal bovine serum induced inhibition of DNA strand growth were: normal skin (Hyclone). The serum concentration was adjusted to produce about 10 fibroblasts, GM3348 (DH= 6.3 J/m2); foreskin fibroblasts, HS1 to 20 background colonies per 5 x IO4cells seeded. In order to achieve (D0 = 4.1 J/m2); XP variant fibroblasts, GM2359 (Da = 2.0 to this frequency of background colonies when using XP variant fibro- 2.4 J/m2); and XP30RO (Dâ€ž= 1.9 J/m2). Another XP variant blasts, the top layer of the agar was also supplemented with epidermal growth factor (10 ng/ml). insulin (2 Mg/ml), and putrescine (50 MM).In fibroblast strain, XP42TO, exhibited a sensitivity to UV (D0 = 2.6 J/m2) which was very similar to that observed with the XP some experiments with XP variant cells, hydrocortisone (10 Mg/ml) was also used in the top agar layer (20). It was found to increase the variant fibroblasts listed above. As illustrated in Fig. IB, DNA size of the colonies, but had no effect on their frequency (data not strand growth in both normal and XP variant cells was equally shown). An aliquot of the agar suspension was diluted with Ham's FIO inhibited by BPDE-I (NHF1, DH= 0.14 MM;CRL1162, A> = containing 15% fetal bovine serum to determine the colony-forming 0.16 MM)-The Dâ€žvalues that were previously published for efficiency of these cells on plastic (10, 18, 19). Agar dishes (8 per dose BPDE-I-induced inhibition of DNA strand growth (10) were of carcinogen used) were fed 1 ml of medium containing the appropriate greater than those presented here. This is because the doses of concentration of serum and supplements on the first day after seeding BPDE-I used in the previous experiments were not corrected and 0.5 ml every 7 days thereafter. When colonies reached a size of 50 Mmof larger (4 to 6 wk after seeding), cells were stained by adding 1 for the loss of activity due to hydrolysis of the diol-epoxide ml of a solution of nitroblue tetrazolium (1 mg/ml) and fi-NADH (0.5 (16). mg/ml) per 60-mm dish, followed by incubation overnight at 37Â°C.The For quantitation of the induction of cell transformation, blue-stained colonies were counted microscopically. The colony counts carcinogen-treated cell populations were assayed for colony were normalized to the relative plating efficiencies on plastic of the formation in soft agar. The doses of carcinogen that were aliquot of cells taken from the top agar cell suspension. This normali selected produced 30 to 90% reduction of cell colony formation zation corrected for artefacts which may have resulted from either heat- efficiency on plastic (10). No differences in the appearance or induced cell death (i.e., during the suspension of cells into molten agar) size of XP variant versus normal fibroblast colonies were seen or from the seeding of dead or nonproliferative cells that had persisted during passaging of the original carcinogen-treated cultures. For ex in the AI assay. Both cell types exhibited a linear dose-response ample, in one experiment in which cells were treated with a dose of UV relationship for transformation to anchorage independence by that killed 70% of the population, 41 colonies were produced per 5 x UV and BPDE-I (Fig. 2). The slopes of the lines representing 10' cells seeded in soft agar. These same cells exhibited an 88% relative UV-induced growth in soft agar. normalized to cells in S phase colony-forming efficiency on plastic at the time of the AI assay. This at the time of carcinogen treatment, were 33.6 and 10.4 colo gives a normalized frequency of 41/0.88 = 46 colonies per 5 x IO4 nies/50,000 cells per J/m2 for UV-treated XP variant and cells. Control cells were sham irradiated or treated with dimethyl normal fibroblasts. respectively (Fig. 2A). This 3.2-fold differ sulfoxide, cultured in parallel, and plated in soft agar for the determi ence in slope was found to be statistically significant (P < 0.05) nation of the frequency of background colonies. In this example, the background frequency of 13 colonies per 5 x IO4cells was subtracted by analysis of variance with linear regression for multiple y observations at given x values (23). The same analysis of the 5 Unpublished results. lines representing BPDE-I-induced growth in soft agar by the 2961

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5 IO I5 O IO 0.20 030 040 UV (J/m2) BPDE-I l Fig. I. Effect of IJV and BPDE-I on DNA strand growth. Cells were seeded at 4 x IO5cells per 100-mm plate and incubated with |'4C]thymidine for 2 days to label DNA uniformly. One to 2 h before carcinogen treatment, the medium was replaced with fresh unlabeled medium. Fibroblasts were treated with UV (A) or BPDE-I (fi) in log phase as described in "Materials and Methods." Cells were incubated after treatment in reserved medium for 30 min at 37*C and then pulse labeled with 50 nCi/ml of ['H]thymidinc for 15 min. Cells were harvested, and the size distribution of nascent DNA was analyzed in alkaline sucrose gradients. The sum of the incorporation of JH cpm into large intermediates of DNA replication (5 x IO7 to 2 x 10* dallons), after normalization to the number of cells added to each gradient (UC cpm). was expressed as the percentage of the same sum measured in control cells. In A, each point represents the average of I to 5 determinations at the indicated UV fluences. Different symbols represent experiments performed with different cell types: â€¢,normal fibroblasts (NHF1. 5 experiments. 4 or 5 fluences of UV in each); O. XP variant (CRLI162. 2 experiments. 4 fluences of UV in each). The data for CRL1162 have been published (10). but are included here for comparison. In B, each point represents the average of I to 3 determinations at the indicated BPDE-1 concentrations. Different symbols represent experiments performed with different cell types: â€¢.normal fibroblasts (NHF1. 4 experiments. 5 concentrations of BPDE-I in each): O. XP variant fibroblasts (CRLI 162, 2 experiments. 5 concentrations of BPDE-I in each). For simplicity, only one regression line was drawn through the data points. However, Do values were determined for each cell strain independently. Bars, SD. two cell types (Fig. 2B) showed that the slopes were not signif (reviewed in Ref. 2). XP variant cells exhibit defective PRR icantly different (P> 0.50). In these experiments, the foreskin after exposure to UV (8-11, 27) and were used to study the fibroblasts, NHF1, the normal dermal skin fibroblasts, relationship between cellular capacity for PRR and the sensitiv GM3348, and two XP variant fibroblast strains, CRLI 162 and ity of human fibroblasts to induction to anchorage XP42TO, exhibited similar BPDE-I-induced frequencies of soft independence. agar growth. Thus, XP variant fibroblasts (CRL1162) exhibited Low doses of UV or BPDE-I, which are known to inhibit an approximately 3.2-fold increased sensitivity to transforma primarily replicÃ³n initiation (9, 17), failed to induce anchorage tion to AI by UV as compared with normal cells, but normal independence in human fibroblasts. However, higher doses of sensitivity to BPDE-I-induced growth in soft agar. these carcinogens, which inhibited DNA synthesis in operating It was also observed that there was a threshold for induction replicons by more than 45%, caused a dose-dependent induction of AI by UV and BPDE-I (Fig. 2). The threshold fluences for of growth in soft agar. Down-regulation of replicÃ³n initiation XP variant and normal cells treated with UV were 0.3 J/m2 is believed to be a protective response that provides time for and 2.7 J/m2, respectively. Both cell types exhibited a threshold the repair of DNA lesions prior to replication (2, 25, 28, 29). dose of about 0.05 MMBPDE-I. Thus, it is not surprising that anchorage-independent colonies As a final element in this analysis, the induction of AI were not induced by doses of carcinogen that only inhibit colonies was expressed as a function of the level of inhibition replicÃ³n initiation. Higher doses, however, interrupted strand of DNA replication by template lesions (Fig. 3). Doses of growth and caused gaps in the daughter-strand DNA. We carcinogen that inhibited replication to the same degree in presume that it is in the process of overcoming the blockage of normal and XP variant cells produced similar frequencies of the replication machinery at the site of DNA damage and/or soft agar. When doses of UV and BPDE-I were normalized to eliminating gaps in daughter DNA through pathways of PRR their effects on DNA strand growth, BPDE-I-induced DNA that mutations are produced and transformation is induced. damage appeared to produce more AI colonies than did UV- Other researchers have successfully transformed human fibro induced damage, in both normal and XP variant fibroblasts. blasts to AI with UV (14) and BPDE-1 (30). The frequencies of UV- and BPDE-I-induced AI presented in this paper compare favorably with those published by these other authors, who also DISCUSSION observed threshold doses of carcinogen below which AI was not The carcinogens UV and BPDE-I have been shown to inhibit observed. DNA synthesis by two mechanisms: down-regulation of repli Based on A> values for UV-induced inhibition of DNA rep cÃ³ninitiation and inhibition of chain elongation (9, 17, 24, 25). lication, the XP variant fibroblasts (CRLI 162) expressed ap Carcinogen lesions in the DNA template interfere with the proximately 34% of normal PRR activity, or a 2.9-fold higher polymerization step and may lead to the production of gaps in sensitivity to UV than the foreskin fibroblasts (NHF1). After the lagging-strand DNA by interrupting the synthesis of Oka- BPDE-I treatment, however, both of these cells displayed sim zaki fragments (26). Several pathways of PRR have been pro ilar PRR activities. The enhanced sensitivity to inhibition of posed that may facilitate the replicative bypass of template replication by UV was mirrored in XP variant fibroblasts by an lesions and/or the sealing of gaps in daughter-strand DNA enhanced frequency of transformation to AI. XP variant cells 2962

200 were 3.2 times more sensitive to the induction of AI by UV than were the normal fibroblasts. Both cell types exhibited similar frequencies of AI colonies induced by BPDE-I. We have previously reported that, in normal fibroblasts, approximately 5 cyclobutane pyrimidine dimers per replicÃ³n are needed to inhibit replication to 37% of control levels, whereas only 1 or 2 dimers produced the same effect in XP variant cells (10). In the same analysis, it was found that, in both cell types, just 1 or 2 BPDE-I-DNA adducts per replicÃ³n inhibited replication to 37% of control levels. From the data presented here and the lesion frequencies needed to inhibit replication (10), it appears that normal cells may be endowed with a mechanism to bypass a majority of UV photoproducts without significant blockage of replication forks (Fig. IA) and in a error-free manner (Fig. 2A). This would be in contrast to 200 the error-prone PRR pathway(s) that may be used to bypass â€¢NHFI â€¢GM3348 UV lesions in XP variant cells, a minority of UV photoproducts o CRLII62 w 150 in normal fibroblasts, and BPDE-I-induced adducts in both cell ~a &XP42TO types. The occurrence of error-free and error-prone pathways O of PRR can be inferred from the diverse types of genetic S 100 alterations that result from replication of damaged templates. For example, sister chromatid exchanges may be derived from o homologous recombination which is a putative error-free proc O 50 ess and may play a role in the repair of gapped DNA (31, 32). Deletions and base substitution mutations are expected to arise from the operation of error-prone PRR pathways. Furthermore, 010 020 our definition of PRR would also include the correction of BPDE-I ( replication errors that might be incurred by the eukaryotic DNA Fig. 2. Induction of anchorage independence by UV and BPDE-I. Fibroblasts polymerases (33). were treated with UV (A) or BPDE-I (A) as described in "Materials and Methods." One model which may account for the data holds that XP Cells were suspended in agar after being cultured for approximately 5 population variant cells lack a factor that prevents the interruption of doublings. Colonies were enumerated after 4 to 6 wk of growth in agar. Colonies per 50,000 cells seeded were normalized to the colony-forming efficiency on polymerization at DNA sites containing pyrimidine dimers (i.e., plastic at the time of agar suspension and to the percentage of S-phase cells at permits error-free read-through by DNA polymerases). This the time of carcinogen treatment. Background colonies from sham-irradiated (A) and solvent-treated (B) cultures were subtracted to estimate the number of factor, by binding DNA sequences containing dimers, may colonies per 50.000 cells that were induced per dose of carcinogen. Closed symbols change the conformation of the DNA such that coding infor denote fibroblasts derived from normal individuals, and open symbols represent mation can be regained, allowing fast and error-free bypass. A strains of XP variant fibroblasts. A: â€¢.NHFI

60 3), similar slopes were observed for normal and XP variant fibroblasts. Thus, at equal levels of inhibition of DNA strand 00 growth, similar frequencies of AI were observed in both cell â‚¬9 40 types. This suggests that, in either normal or XP variant fibro blasts, once replication becomes blocked at a DNA lesion, there 20 exists the same probability of producing a mutation that will lead to the AI phenotype. When compared to UV, the higher level of BPDE-I-induced AI per increment of inhibition of DNA 100 50 10 DMA Strand Growth strand growth in human fibroblasts may be related to a slower (Percent of Control) rate of repair or to a greater mutagenic potency of BPDE-I- Fig. 3. Relationship between inhibition of DNA strand growth and induction induced DNA adducts than UV-induced photoproducts. of anchorage independence. Normal and XP variant fibroblasts were treated with UV (-â€¢â€¢-. NHFI; -â€¢-, CRLI162) or BPDE-I ( . NHFI; , CRLI162). Doses of carcinogen that induced AI (Fig. 2) were expressed as the ACKNOWLEDGMENTS observed level of DNA strand growth (relative to untreated controls) at the respective doses (Fig. 1). The lines were drawn only within the range of carcinogen We are grateful to Dr. Sciji Kondo from the Department of Derma doses at which AI was measured. tology, School of Medicine, Tokyo Medical and Dental University, 2963

Japan, for the generous gift of the fibroblast strain designated XP42TO. Hanawalt (eds.). DNA Repair: A Laboratory Manual of Research Procedures, pp. 501-522. New York: Marcel Dekker. Inc.. 1981. We thank Dr. Lester Lee for his advice and assistance with the statistical 16. MacLeod, M. C.. and Lew. L. A rapid spectrophotometric assay for the analysis of the transformation data and Sandra J. Wilson who provided integrity of diol epoxides. Carcinogcnesis (Lond.). 9: 2133-2135. 1988. valuable technical assistance. 17. Kaufmann. W. K., Boyer, J. C., Smith. B. A., and Cordeiro-Stone, M. DNA repair and replication in human fibroblasts treated with (Â±)-r-7,/-8-dihy- droxy-/-9.10-epoxy-7.8.9.10-tetrahydrobenzo(a)pyrene. Biochim. Biophys. Ada. Â«24:146-151. 1985. REFERENCES 18. McCormick. J. J., Kateley-Kohler, S.. and MÃ¤her.V. M. Factors involved in quantitating induction of anchorage independence in diploid human fibro 1. Grisham. J. W., Kaufmann. \V. K., and Kaufman. D. G. 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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1991 American Association for Cancer Research. Role of Postreplication Repair in Transformation of Human Fibroblasts to Anchorage Independence

Jayne C. Boyer, William K. Kaufmann and Marila Cordeiro-Stone

Cancer Res 1991;51:2960-2964.

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