Defective Postreplication Repair in Xeroderma Pigmentosum Variant Fibroblasts1

[CANCER RESEARCH 50. 2593-2598, May I. 1990] Defective Postreplication Repair in Xeroderma Pigmentosum Variant Fibroblasts1

Jayne C. Boyer, William K. Kaufmann, Bruna P. Brylawski, and Mania Cordeiro-Stone2

Curriculum in Toxicology fj. C. B., W. K. K., M. C-S.J, Department of Pathology [W. K. K., B. P. B., M. C-S.J, and Uneberger Cancer Research Center [W. K. K., M. C-S.J, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7525

ABSTRACT as the growth of nascent DNA strands in carcinogen-damaged cells from abnormally small molecular weights to larger sizes Postreplication repair (PRR) was quantified in normal human fibro- equivalent to parental DNA strands (7, 8, 10-12). The opera blasts and in xeroderma pigmentosum (XP) variant fibroblasts after treatment with UV or benzoja|pyrene diol epoxide-I (BPDE-I). PRR tion of PRR pathways can also be inferred from alterations in may be defined as the elimination of discontinuities in the daughter- the relative proportion of large nascent DNA molecules in the strand DNA and the replicative bypass of lesions in the DNA template. steady state distribution of intermediates of DNA replication Pathways of PRR reduce the number of DNA growing points that are (9). We have quantified PRR in normal human fibroblasts and blocked at template lesions and increase the rate of growth of nascent in XP variants by using velocity sedimentation to analyze DNA DNA on damaged templates. Rates of DNA synthesis and strand growth synthesis in logarithmically growing and synchronized cultures, were measured in solvent- and carcinogen-treated cells by velocity sedi after treatment with UV or BPDE-I. The results have allowed mentation analyses of radiolabeled nascent DNA in alkaline sucrose an estimation of the deficiency in PRR expressed in the XP gradients. Logarithmically growing normal fibroblasts displayed M>val variant cells after DNA damage by UV. The data also confirmed ues of 6.3 .l/nr and 0.37 JIMfor the inhibition of DNA synthesis in active that the DNA metabolic pathway that allows bypass of UV- replicons by UV and BPDE-I, respectively. Under identical conditions, the XP variant cells exhibited Da values of 1.5-2.0 J/m2 and 0.27-0.31 induced photoproducts is ineffective for bypass of BPDE-I MM.Pulse-chase experiments were performed in cells synchronized at adducts in normal fibroblasts (13). the beginning of the S phase. Normal and XP variant cells displayed inhibition of DNA strand growth by UV, with Da values of 21.6 and 7.0 J/m2, respectively. The Da values for the inhibition of DNA strand growth MATERIALS AND METHODS by BPDE-I were 0.85 JIMfor the normal cells and 0.62-0.79 JIMfor the Cell Culture Conditions and Synchronization Protocol. Normal human XP variant cells. The inhibitions of DNA replication by UV and BPDE- fibroblasts were obtained from the Human Genetic Mutant Cell Re I were also analyzed in terms of DNA lesion frequencies. Based on the pository (Camden, NJ) (GM3348, passage 6, skin biopsy from a clini Dn values for inhibition of DNA replication, we concluded that the XP cally unaffected 10-year-old male) or were derived from the foreskin of variant cells express maximally 25-33% of the total PRR activity ob a normal newborn (NHF1). XP variant fibroblasts were obtained from served in normal fibroblasts after UV treatment. Conceivably, this defi ciency in PRR activity results in the XP variant's increased risk of the Human Genetic Mutant Cell Repository (GM2359, XP strain designation XP115LO) or from the American Type Culture Collection cancers induced by sunlight, because XP variant cells and normal fibro (CRL1162, XP strain designation XP4BE). Cell stocks were grown in blasts are equally proficient in excision repair. Both normal and XP plastic tissue culture dishes, at 37Â°Cina humidified atmosphere of 5% variant fibroblasts, however, displayed similar PRR activities in response CO2, in Ham's FIO medium supplemented with 15% fetal bovine serum, to BPDE-I treatment. penicillin (100 units/ml), and streptomycin (100 /ig/ml). Cultures were subcultured 2 times a week to maintain logarithmic growth and were used between passages 4 and 15. Cultures were tested at the time these INTRODUCTION experiments were performed and were found to be free of Mycoplasma, either by the fluorescent stain method (14) or by using the Genprobe Mutations and chromosomal aberrations produced by the kit (Fisher Scientific Co.). The cells were synchronized by plating IO6 replication of carcinogen-damaged DNA are believed to con tribute to carcinogenesis (1). Patients with the disease XP3 cells/ 100-mm plate on day 1, feeding them on days 3 and 5, and then keeping the cultures undisturbed for 3 days (15). On day 8, cells were develop multiple skin cancers on sunlight-exposed areas of the replated at 3-4 x IO5 cells/60-mm plate in the presence of 2 Mg/ml body (2-4) and their cells in vitro are extremely sensitive to the aphidicolin and 0.08% DMSO. After a 24-h incubation, cells were mutagenic and transforming effects of UV (2). Carcinogenesis released from the aphidicolin block by washing 3 times with HBSS and by UV has been linked to a deficiency in excision repair of UV- refeeding with fresh medium. induced DNA photoproducts in patients with the classical form DNA Labeling and Treatment with UV and BPDE-I. When uniform of XP (5, 6). As a result of this deficiency, more lesions than labeling of parental DNA was desired, cultures were incubated in complete medium containing 5.0-10.0 nCi/ml [14C]thymidine (specific normal are present during replication of DNA in XP fibroblasts than in normal fibroblasts. In contrast, the XP variant pheno- activity, 50 Ci/mol; ICN Pharmaceuticals, Inc.), during logarithmic growth. Cell cultures were treated with either BPDE-I or UV and pulse- type appears to exhibit a defect in the ability to replicate DNA labeled with ['Hjthymidine as specified in the description of each containing UV-induced DNA lesions (7-9). Excision repair is experiment. For treatment with BPDE-I, culture medium was removed normal in these cells. Analysis of replication of carcinogen- and cells were rinsed once with warm HBSS and then covered with 5 damaged DNA in XP variant cells may help to elucidate the ml of the same solution. An aliquot of 25 Â¿ilofanhydrous DMSO, mechanisms of PRR. PRR may be defined as the elimination containing varying concentrations of BPDE-I, was added and cultures of daughter-strand gaps and the bypass of lesions in the DNA were incubated at 37Â°Cfor 10 min. The concentration of the stock template. PRR can be recognized in pulse-chase experiments solution was determined from absorbance readings at 345 nm (extinc tion coefficient, 48,600 cm2/mol). The BPDE-I-containing solution Received 7/17/89; revised 12/27/89. was then removed and the cells were rinsed once with HBSS warmed The costs of publication of this article were defrayed in part by the payment to 37Â°C.UV fluence rate was 0.23-1.35 J/m2/s from a short-wave UV of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. lamp emitting mostly 254 nm radiation. Fluence rate was measured 1This work was supported by PHS Grant POI CA42765. using a UV radiometer (UV Products, Inc., San Gabriel, CA). When ! To whom requests for reprints should be addressed. experiments included a period of chase, cell cultures were incubated 3The abbreviations used are: XP, xeroderma pigmentosum; BPDE-I. benzo- immediately after the pulse-labeling in medium supplemented with 10~4 [ajpyrene diol epoxide-I; DMSO. dimethyl sulfoxide: ESS. endonuclease-sensitive site; ELISA, enzyme-linked immunosorbent assay; HBSS, Hanks' balanced salt M thymidine and 10~5M deoxycytidine. solution; PRR, postreplication repair. Alkaline Sucrose Gradients. Procedures for cell harvesting, analyses 2593

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1990 American Association for Cancer Research. POSTREPLICATION REPAIR IN XP VARIANT FIBROBLASTS of distributions of sizes of labeled nascent DNA in alkaline sucrose IOr gradients, and molecular weight determinations have been described (15-17). Briefly, cells were scraped from plates and added directly to a lysis layer (I M NaOH and 0.02 M EDTA) on top of an alkaline sucrose gradient (36 ml, 5-20% sucrose in 0.4 M NaOH, 2.0 M NaCl, 0.01 M EDTA). These gradients were left under standard fluorescent illumi nation for 1 h and centrifuged in a Beckman SW28 rotor, at 25,000 rpm and 20Â°C,for4 or 5 h. Average molecular weights of labeled DNA were determined from the distributions of radioactivity in precalibrated gradients (15). Cytotoxicity Assay. Cells from exponentially growing cultures were plated into 100-mm dishes at cloning densities (500 to 1000 cells/dish, 3 to 10 dishes/dose), and treated with carcinogen 16 to 20 h later. Cultures were fed after 5 days and fixed and stained after 10 to 14 days. 4 6 10 Percentage of survival was determined from the ratio of the colony- UV(J/m2) forming efficiencies of treated and untreated cells. Kndonuclease-sensitive Site Assay. The method of van Zeeland et al. Fig. 1. Frequency of endonuclease-sensitive sites as a function of UV fluente. Human fibroblasts whose DNA had been prelabeled with [JH|thymidine were (18, 19) was used to estimate the frequency of UV-induced cyclobutane- irradiated with UV and then disrupted by two cycles of freezing and thawing. type pyrimidine dimers. In brief, cells whose DNA had been prelabeled After incubation in high salt buffer, the permeabilized cells were treated with M. with radioactive precursors were irradiated with UV and then disrupted luteus UV-DNA endonuclease at 37"C for 15 min. The samples were then analyzed by velocity sedimentation in alkaline sucrose gradients (see "Materials by two cycles of freezing and thawing. After an incubation in high salt and Methods"). Each symbol represents a separate experiment using normal skin buffer to dissociate chromatin proteins, the permeable cells were treated fibroblasts (GM3348. â€¢,A, x, â€¢),foreskin fibroblasts (NHFI, O, D), or XP with 5000-7500 units of Micrococcus luteus UV-DNA endonuclease variant fibroblasts (CRL1162, A). The line was drawn using linear regression (r (Applied Genetics Inc., Freeport, NY) per 50,000 cells, at 37Â°C,for 15 = 0.99) through all the data points (not including the origin), regardless of cell min. The sample was then lysed on top of a 5-20% alkaline sucrose type. Estimated slopes for individual cell strains (GM3348,0.95 ESS/10' daltons; NHFI, 0.80 ESS/10' daltons; CRL1162.0.85/10' daltons) were not significantly gradient and sedimented. and molecular weights were determined as different from the overall slope of 0.92 ESS/10' daltons. described above. ELISA. BPDE-I DNA adducts were measured using a competitive form of ELISA (20). DNA from human fibroblasts was prepared by lysing the cells for 2 h at 37Â°C,ina solution containing 100 HIMNaCl, IO mivi EDTA (pH 8.0), 0.5% sodium A'-lauryl sarcosinate (Sigma Chemical Co., St. Louis, MO), and 0.2 mg/ml proteinase K (E. Merck, Darmstadt, FRG). The lysate was dialyzed for 18 h against Tris-EDTA buffer (10 IHMTris-HCI, pH 7.5, with 1 HIMEDTA) at 4Â°Cand DNA was banded in CsCI gradients. Fractions containing DNA were pooled, concentrated, and dialyzed against Tris-EDTA buffer. The DNA sam ples were sheared by passing them through a 22-gauge needle 10 times. DNA concentration was determined by measurements of absorbance at 260 nm, and the samples were diluted to the optimal concentration of 50 Â¿ig/mlwÂ¡thphosphate-buffered saline (Grand Island Biological Co., 000 0.50 1.00 1.50 Grand Island, NY). ELISA assays were conducted with primary anti BPDE-K/jM) bodies elicited in rabbits against BPDE-I-DNA adducts. Goat anti- Fig. 2. Frequency of BPDE-I adducts in DNA as a function of BPDE-I rabbit IgG conjugated to alkaline phosphatase, andp-nitrophenyl phos concentration. The DNA from BPDE-1 treated human fibroblasts was purified and the frequency of BPDE-I adducts in the DNA was determined by ELISA (see phate were obtained from Sigma. "Materials and Methods"). Each symbol represents a separate experiment using normal skin fibroblasts (GM3348. â€¢,O),foreskin fibroblasts (NHFI, A), or XP variant fibroblasts (CRL1162, D). The line was drawn by linear regression (r = RESULTS 0.89) through all the data points (not including the origin), regardless of cell type. Estimated slopes for fibroblasts from normal individuals (GM3348 and NHFI. Quantification of Carcinogen-induced DNA Lesions. Carcin 123 fmol adducts/^g DNA per ,IM BPDE-I) and for XP variant cells (CRL1162, ogen-induced DNA lesions were quantified to facilitate com 121 fmol adducts//jg DNA per /IM BPDE-I) were not significantly different from the overall slope of 122 fmol adducts//ig DNA per /Ã•MBPDE-I. In order to parisons of the effects of each type of lesion on cell survival simplify the graph, only the average of two or three ELISA determinations per and DNA replication in the various human fibroblast strains. DNA sample in each experiment (three different doses) were illustrated above. Using the ESS assay, we observed a linear relationship (Fig. 1) between the fluence of UV used to irradiate cells and the number as proposed by MacLeod and Lew (29). However, by using of the most abundant photoproduct in DNA, the cyclobutane- anhydrous DMSO and freshly prepared solutions of BPDE-I type pyrimidine dimer. It was determined from the slope of the for each experiment, reproducible binding levels were achieved line that 0.92 dimers were produced per IO8 daltons of DNA (Fig. 2). Treatment of cells with BPDE-I produces one major per J/m2. Using either the ESS assay or thin layer chromatog- DNA adduci, Ãrans-(7R)-A/2-(10|7Â«,8l3,9la-trihydroxy-7,8,9,10- raphy, other laboratories have reported values ranging between tetrahydrobenzo[a]pyrene|-yl)-deoxyguanosine (30, 31), and 0.7 and 4.2 dimers per 10" daltons per J/m2 (7, 19, 21-28). small quantities of minor adducts (32). Although the ELISA Errors in the calibration of the UV source or the velocity assay has a high degree of sensitivity, the results of assays of sedimentation gradients may account for these variations. the same sample are sometimes variable. This problem was A similar linear relationship between concentration of circumvented by taking the average of several measurements of BPDE-I applied to the cultures and frequency of BPDE-I-DNA the frequency of BPDE-I adducts in the same DNA sample. adducts was observed. The data presented in Fig. 2 defined a Direct determination of adduction by a radiochemical method yield per //M BPDE-I of 122 fmol of adducts per ng DNA, yielded binding levels identical to those estimated with ELISA which represents an average of 12 BPDE-I adducts per IO8 (results not shown). daltons of DNA per ÃŸM.Inthese studies, we have not measured Cytotoxicity of UV and BPDE-I. Fig. 3 shows the effects of the concentration of active BPDE-I in our treatment solutions, UV and BPDE-I on cell survival in normal human and XP 2594

Table 1 Do values for cytotoxicity and inhibition of DNA replication in human fibroblasts Do as fluence of UV (J/mÂ¡)or concentration of BPDE-I GIM)Â°

Inhibition of DNA replicationA.

CarcinogenCellUV Normal*XP variantCRL1162GM2359BPDE-I

5 10 15 20 0 10 0.20 0.30 NormalXP UV (J/m2) BPDE-I (juM) variantCRL1162GM2359Cytotoxicity5.13.0NDf0.100.12NDLog-phasecells6.31.52.00.370.310.27Synchronizedcells21.67.0ND0.850.790.62'' Fig. 3. Cytotoxicity by UV and BPDE-I in exponentially growing cells. Col ony-forming efficiencies were determined 10-14 days after carcinogen treatment and are expressed as a percentage of colony-forming efficiencies of control cells. Inhibition of DNA replication, DQas Each point represents the average of 1-4 determinations at the indicated UV DNA lesions/replicon' fluence or BPDE-I concentration. A. Control cells were sham-irradiated. â€¢, cells2.3(5.1)'0.6(1.3)0.7(1.5)1.81.51.3Synchronized normal fibroblasts (GM3348, 11 experiments, 2-5 fluences of UV in each); D, B.CarcinogenUVBPDE-ICellNormalXP cells8.0(17.6)2.6 XP variant fibroblasts (CRL1162, 8 experiments, 2-6 UV fluences in each). B. Control cells were treated with the solvent (DMSO, 0.5% final concentration). varianlCRL1162GM2359NormalXP â€¢,normal fibroblasts (GM3348, 4 experiments, 2-5 concentrations of BPDE-I (5.7)ND4.13.83.0 in each); D, XP variant fibroblasts (CRL1162, 4 experiments, 4 or 5 concentra tions of BPDE-I in each). variantCRL1162GM2359Log-phase variant fibroblasts. Normal fibroblasts treated with UV exhib ited a shoulder region in the survival curve, which was not Â°Dais the increment of dose that reduces the parameter that is being measured detected in experiments with the XP variant cells (Fig. 3A). to 37% of its initial value and corresponds on average to one inactivating event Normal and XP variant fibroblasts treated with BPDE-I exhib per target. These values were determined by linear regression analyses of all data points obtained in the dose-response studies for each cell strain (Figs. 3-5). ited similar cell survival responses, with a small shoulder at low *GM3348. concentrations (Fig. 3B). From the slope of the linear portion ' ND, not determined. **Not included in Fig. 5. of these cytotoxicity curves, we estimated A> values, defined as ' The DQvalues are expressed as lesions (cyclobutane-type pyrimidine dimers the increment in carcinogen dose that reduced cell survival from or BPDE-1-DNA adducts) per 4 x IO7dallons of DNA, assuming a yield of 0.92 100% to 37% (one lethal hit/cell, on average). These values are dimers per 10' daltons per J/m2 (Fig. 1) and 12 BPDE-I adducts per 10" dallons per /iM. shown in Table \A. ^In parentheses is indicated the number of pyrimidine dimers per replicÃ³n, Inhibition of DNA Replication. The effects of UV and BPDE- assuming a yield of 2 dimers per 10*daltons per J/m2. I on DNA strand growth were measured using velocity sedi mentation analyses of DNA synthesized in logarithmically were indicated by different symbols in Fig. 4A, but only an growing and synchronized cell cultures. To determine the A> average regression line was drawn through all points. for inhibition of DNA strand growth in asynchronous cell The effects of the two carcinogens on the growth of nascent populations, the protocol of Kaufmann and Cleaver (9) was DNA molecules were also measured using synchronized cells used. Logarithmically growing cultures that had been uniformly labeled with ['"CJthymidine were treated with UV or BPDE-I (Fig. 5). One advantage of cell synchronization was to reduce the heterogeneity of the distribution of sizes of intermediates and then incubated for 30 min in nonradioactive culture me dium. These cells were pulse-labeled for 15 min with [3H]- of DNA replication, which facilitated measurements of the growth of nascent DNA in damaged cells (15). In these exper thymidine and harvested, and the size distribution of nascent iments, synchronized cells that had been uniformly labeled with DNA was analyzed in alkaline sucrose gradients. The sum of [14C]thymidine were pulsed with [3H]thymidine, then treated the incorporation of 3H cpm into large intermediates of DNA replication (M, 5 x IO7 to 2 x 10s), after normalization to the with carcinogen, and chased for 2 h in nonradioactive medium. number of cells added to each gradient (I4C cpm), was expressed The population of nascent DNA molecules was pulse-labeled prior to carcinogen treatment and, therefore, had the same as a percentage of the same sum measured in control cells treated with solvent only. The logarithm of these percentages initial distribution of sizes in control and treated cells. The was plotted against carcinogen dose (Fig. 4). Again, we esti average increase in the molecular weight of the nascent DNA mated DOvalues corresponding to the increment of carcinogen that was achieved after 2 h of growth in treated cells was dose that reduced DNA synthesis from 100% to 37% of control calculated and expressed as a percentage of control. In this values (Table IA). In normal fibroblasts, the A> values for the assay, the D0 values for inhibition of DNA strand growth by inhibition by UV and BPDE-I of DNA strand growth were 6.3 UV in normal and XP variant cells (CRL1162) were 21.6 and J/m2 and 0.37 ^M, respectively. In this assay, two different 7.0 J/m2, respectively. Both cell types responded similarly to strains of XP variant cells exhibited A> values of 1.5-2.0 J/m2 treatment with BPDE-I and the calculated A, values were 0.85 and 0.27-0.31 UM (Table \A). For comparison, results from Ã•Ã•Mforthe normal fibroblasts and 0.79 /Â¿M(CRL1162) and 0.62 equivalent experiments with UV, performed with XP variant MM(GM2359) for the XP variant cells (Table IA). cells and published previously (9), are also included in Fig. 4A. The DOvalues for the inhibition of DNA replication by UV Analyzed independently, they resulted in D0 values of 1.9 J/m2 and BPDE-I in human fibroblasts were also expressed as DNA (XP30RO) and 2.4 J/m2 (GM2111). Note that GM2111 is a lesions per replicÃ³n. These results are presented in Table \B different designation for the cells identified here as GM2359 and allow the direct comparison of the inhibition of DNA and also corresponds to XP115LO. For simplicity of display, replication by two carcinogens producing different DNA le the results obtained with different strains of XP variant cells sions. 2595

IO 15 20 UV U/m2) 020 040 BPDE-1 OuM) Fig. 5. Effect of UV and BPDE-I on DNA strand growth in synchronized fibroblasts. The parental DNA was prelabeled with |'4C]thymidine and cells were Fig. 4. Effect of UV and BPDE-I on DNA synthesis in active replicons. Cells were seeded at 400.000 cells/100-mm plate and incubated with 5 nCi/ml [UC]- synchronized by confluence-arrest and aphidicolin block, as described in "Mate rials and Methods." Cells were pulse-labeled for 10-15 min with 50 fiCi/ml [JH]- thymidine for 2 days to uniformly label the parental DNA. One to 2 h before thymidine at l h after release from aphidicolin block. After the pulse, cells were carcinogen treatment, the medium was replaced with fresh unlabeled medium. Fibroblasts Â»eretreated with UV (A) or BPDE-I (B) in logarithmic phase, as washed once with saline and UV irradiated (A) or treated with different concen trations of BPDE-I for 10 min (B). Following irradiation, cells were incubated in described in "Materials and Methods." Cells were incubated in reserved medium complete medium supplemented with 10~4Mthymidine and IO"5Mdeoxycytidine. for 30 min at 37'C and then pulse-labeled with 50 Â»iCi/ml['Hjthymidine Cells were harvested after 2 h elapsed from the beginning of the pulse and were for 15 min. Cells were harvested and the size distribution of nascent DNA applied to alkaline sucrose gradients. The increase in the ratio between the average was analyzed in alkaline sucrose gradients. The sum of the incorporation of molecular weights for the distribution of 3H-labeled molecules and the uC-labeled 'H cpm into large intermediates of DNA replication (M, 5 x IO7 to 2 x 10"). after normalization to the number of cells added to each gradient ("C cpm). was molecules for treated cells is presented as a percentage of the increase in the same ratio determined for the controls. For determination of average molecular weights expressed as the percentage of the same sum measured in control cells. A. Each in alkaline sucrose gradients, see Ref. 15. Each graph point represents the average point represents the average of 1-3 determinations at the indicated UV fluence. of 1-4 determinations at the indicated UV fluence (A) or BPDE-I concentration Different symbols represent experiments performed with different cell types: â€¢. GM3348 (3 experiments. 2-5 fluences of UV); D. CRLI162 (2 experiments. 4 (B). A. Different symbols represent experiments performed with different cell fluences of UV); A, GM2359 (2 experiments. 2-5 fluences of UV); C, GM211I types: â€¢,normal fibroblasts (GM3348, 5 experiments, 2-5 fluences of UV); and Q, XP variant cells (CRLI 162, 4 experiments, 2-5 fluences of UV). B. Different (same as GM2359. 2 experiments, 5 fluences of UV); and O. XP30RO (2 experiments. 5 fluences of UV). B. Each point represents the average of 1-5 symbols represent experiments performed with different cell types: â€¢,normal fibroblasts (GM3348, 2 experiments, 5 concentrations of BPDE-I); and D, XP determinations at the indicated BPDE-I concentrations. Different symbols rep variant cells (CRLI 162, 4 experiments, 3-5 concentrations of BPDE-I). resent experiments performed with different cell types: â€¢,GM3348 (6 experi ments. 1-5 concentrations of BPDE-I): D. XP CRLI 162 (2 experiments. 4 or 5 concentrations of BPDE-I): and A. GM2359 (6 experiments. 1-5 concentrations of BPDE-I). For simplicity, only one line was drawn through the data points for (Figs. 4 and 5, Table \A). The CRLI 162 XP variant strain also the two XP variant strains. However, all the results were analyzed for each cell displayed normal sensitivity to cytotoxicity by BPDE-I (Fig. 3) strain independently, as indicated in Table 1. and, in the hands of others (22), normal sensitivity to mutagenesis by BPDE-I. DISCUSSION In order to compare the effects of lesions produced by UV The XP variant phenotype has been defined on the basis of and BPDE-I directly, we measured the respective lesion fre defective replication of UV-damaged templates (7-9, 11, 12). quencies in DNA using the ESS assay and ELISA. D0 values We have attempted to quantify this phenotypic defect by meas were determined from the slopes of dose-response curves (Figs. urements of inhibition of DNA strand growth caused by UV 3-5, Table IA) and expressed as DNA lesions per mass of and BPDE-I (Figs. 4 and 5). Previous measurements of PRR DNA (4 x IO7 daltons) equivalent to one replicÃ³n of average capacity in the XP variant cell strain GM2359 and in the size (20 ^m). It is important to consider DNA damage in terms normal skin fibroblast GM3348 (13) have been confirmed in of lesion frequency because it is impossible to compare fluence this publication using another XP variant cell strain, CRLI 162. of a physical carcinogen with the concentration of a chemical Direct comparison of D0 values (Table 1) determined in dose- carcinogen (Table \B). D0values within a given assay permitted response studies indicated that these two strains of XP variant direct comparisons of PRR capacity among strains of normal fibroblasts were 3-4 times more sensitive to inhibition of DNA fibroblasts and XP variant cells. The Dâ€žvaluesthat have been strand growth by UV than was the normal fibroblast strain calculated in the same assay and expressed as lesions per (GM3348). The implication of these results is that pathways of replicÃ³nenabled us to compare PRR responses in two different PRR in normal fibroblasts facilitate the bypass of 66-75% of cell types after treatment with two different carcinogens. those DNA lesions that block strand growth in XP variant cells. The results in Table IB indicate that the D0 value for the Parallel studies of the inhibition of DNA replication by inhibition of DNA replication in logarithmically growing XP BPDE-I in normal and XP variant fibroblasts indicated that variant cells (1.5-2.0 J/m2) corresponded to 0.6-0.7 dimers/ both cell types displayed similar capacities for PRR after DNA replicÃ³n. It seems incongruous that an average of less than 1 damage by this chemical carcinogen. These results are in ap dimer/replicon would be sufficient to inactivate 67% of the parent disagreement with those previously published by Waters DNA synthesis within active replicons. Therefore, it appears et al. (21), who studied DNA replication in human fibroblasts that either the size of an average replicÃ³n in human cells may after treatment with UV and the trans (I) and cis (II) diaster- be larger than 20 urn or our ESS assay may have underestimated eomeric forms of BPDE. In their study, another strain of XP the yield of UV-induced pyrimidine dimers. We consider the variant fibroblasts exhibited substantially increased sensitivity second possibility as more plausible, because we have been able to inhibition of DNA replication by UV and BPDE-II but, to a to reproduce in our laboratory D0 values for the inhibition of lesser degree, an increased sensitivity to higher concentrations DNA replication obtained by others. For instance, the results of BPDE-I than used in our studies. The GM2359 (XP115LO) published by Kaufmann and Cleaver (9) yield a D0 value of 2.4 and CRLI 162 (XP4BE) XP variant strains that we have studied J/m2 for GM2359 (designated GM2111 in Ref. 9), which is in detail do not appear to be appreciably hypersensitive to close to the value of 2.0 J/m2 estimated in this publication for BPDE-I, in spite of their established hypersensitivity to UV the same cell strain. This biological dosimetry indicates that 2596

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1990 American Association for Cancer Research. POSTREPLICATION REPAIR IN XP VARIANT FIBROBLASTS the measurements of UV fluence in our laboratory does not and transformation by UV displayed by XP variant cells appears deviate significantly from determinations elsewhere. Measure to be quantitatively linked to their deficiency in PRR. It has ments of dimer yields by thin layer chromatography has re been proposed that nonpairing lesions in damaged DNA block vealed a value of 2 dimers per IO8 daltons per J/m2 (22). This DNA polymerase and that error-prone mechanisms of bypass value is also in agreement with those determined by the ESS of such lesions may represent pathways leading to mutagenesis assay and published by Lehmann et al. (7), Waters et al. (21), (reviewed in Ref. l). The blocked replication sites may also and Fugiwara and Satoh (24). Assuming that our ESS values represent sites that are susceptible to breakage and are respon are underestimated, then the values in Table IB should be sible for clastogenesis (36). One possible explanation for the corrected by a factor of 2.2 (numbers in parentheses). Therefore, decreased PRR capacity of XP variant fibroblasts is that these we are led to conclude that the same effect on DNA replication cells lack a specific pathway of PRR (1) that is present in is produced per damage site in XP variant cells treated with normal cells. Such a pathway allows normal cells to readily UV or BPDE-I. Each pyrimidine dimer or BPDE-I adduci bypass UV-induced photoproducts without error. The nature appears to block DNA replication in XP variant cells. Normal of this pathway is unclear at the moment. However, caffeine- fibroblasts, on the other hand, appear to express a mechanism sensitive and caffeine-resistant pathways of PRR have been for rapid bypass of UV-induced lesions which is not expressed described in human cells, and XP variant cells appear to be in XP variant cells. This mechanism facilitates rapid bypass of deficient in the caffeine-resistant pathway (7, 8). two of three pyrimidine dimers but does not appear to facilitate the bypass of BPDE-I adducts in normal fibroblasts. ACKNOWLEDGMENTS We caution about assigning biological significance to the numerical differences between D0 values determined in the We thank Dr. Ann Ganesan for guidance and the T4 endonuclease assays with synchronized and asynchronous cell cultures. In the V used during the implementation of the ESS assay in our laboratory. assay performed with synchronized cells, excision repair occur We gratefully acknowledge the generous gift of the aphidicolin from the Natural Products Branch of the National Cancer Institute. ring during the chase period of 2 h should eliminate 4 times more lesions than during the post-treatment incubation of 30 min in the assay with logarithmically growing cells. Because REFERENCES both normal and XP variant cells are equally proficient in 1. Kaufmann. W. K. Pathways of human cell post-replication repair. Carcino- excision repair, the relative differences in PRR activity remains genesis (Lond.), 10: 1-11, 1989. the same in both assays. However, it is also possible that the 2. Cleaver, J. E. Xeroderma pigmentosum. In: J. V. Stanbury, J. B. Wyngaar- den, D. S. Fredrickson, J. L. Goldstein, and M. S. Brown (eds.). 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Jayne C. Boyer, William K. Kaufmann, Bruna P. Brylawski, et al.

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