Deoxyribonucleoside Incorporation During DNA Repair of Carcinogen-Induced Damage in Human Diploid Fibroblasts

Home , Deoxyribonucleoside

[CANCER RESEARCH 33, 2097-2103, September 1973] Deoxyribonucleoside Incorporation during DNA Repair of Carcinogen-induced Damage in Human Diploid Fibroblasts

Michael W. Lieberman and Miriam C. Poirier

Experimental Pathology Branch, National Cancer Institute, NIH, Bethesda, Maryland 20014

SUMMARY pair plays in the neoplastic process, clearly repair should in volve insertion of purine as well as pyrimidine moieties (13, Incorporation of tritium-labeled deoxyribonucleosides 17, 19, 45). Data from a number oflaboratories suggest that during DNA repair was investigated in confluent human during excision repair of pyrimidine dimers on the order of diploid fibroblasts (WI-38) in conditioned medium contain 30 to 100 bases are removed (5, 10, 36). Thus, assuming that ing hydroxyurea. After damage with either of two proxi repair of dimers does not occur exclusively in long runs of m ate ca rcinogens, N-acetoxy-2-acetylaminofluorene or pyrimidines (isostichs), one would expect that during res 7-bromomethylbenz(a)anthracene, incorporation of radio toration of the damaged segment purine moieties as well as activity into DNA increased 8- to 45-fold when any of the pyrimidine moieties should be inserted. four labeled deoxyribonucleosides was used as a precursor. Initial attempts to show purine deoxyribonucleoside in Similar results were obtained when ultraviolet light-dam corporation directly have been unsuccessful (27, 34, 41, 42). aged cells were allowed to repair in the presence of deoxy However, 2 lines of evidence suggest that purine moieties adenosine-3H. Repair synthesis was also demonstrable when are incorporated during repair: (1) the infectivity of UV deoxyguanosine-3H was used as precursor and bromode damaged virus is restored by cells competent to repair DNA oxyuridine was used as a density label for replicating DNA. damage and functional interferon is produced in cells after Chromatography of nucleosides prepared by enzymatic damage and repair (8, 33), in each case suggesting that ac digestion of repaired DNA's labeled with one of the four curate complementary copies of the genome are made dur tritiated deoxyribonucleosides revealed that virtually all of ing repair; (2) pyrimidine isostich profiles from repaired the radioactivity was recoverable as the expected labeled and replicating cells are indistinguishable, indicating that product. Digestion studies of repaired DNA's with snake â€œrepairâ€•isnot accounted for by simple terminal addition or venom phosphodiesterase suggested that incorporation was insertion of polypyrimidine runs (26). not due to an artifact such as terminal addition. On the basis This paper describes our recent studies on incorporation of these findings we conclude that, during DNA repair syn of purine and pyrimidine deoxyribonucleosides into DNA thesis in human diploid cells, all four deoxyribonucleosides during repair. Because interpretation of â€œrepairphenom are incorporated. These studies are important for assess enaâ€•in relation to chemical mutagenesis and cell transfor ment of the role of repair in chemical carcinogenesis since mation has been difficult in the absence of clear evidence most previous studies have utilized pyrimidine precursors for purine deoxyribonucleoside incorporation, emphasis has for repair, but chemical carcinogens damage primarily been placed on damage by chemical carcinogens. The @ purine moieties. process has been studied in G cells (â€œcontact-inhibitedâ€• human fibroblasts) since they probably represent a more uniform cell population than do the asynchronous cultures INTRODUCTION usually investigated and they allow separation of repair Studies of DNA repair synthesis in human cells have phenomena from replicative phenomena. While our work was in progress we became aware of similar studies in demonstrated incorporation of pyrimidine precursors after another laboratory (7). (Dr. J. E. Cleaver made his manu damage with both UV â€˜(4,5, 10, 11, 15, 35) and proximate script available prior to publication.) chemical carcinogens (6, 18, 24, 38, 40, 43, 44). Little data, however, are available on the incorporation of purine pre cursors during repair. Most of the DNA damage done by MATERIALS AND METHODS alkylating agents including proximate chemical carcinogens results from attack on purine moieties (9, 20â€”22,31). While Treatment of Cells. Wl-38 human diploid fibroblasts were it is not yet certain what role, if any, this damage or its re seeded in 150-mm Petri dishes in diploid growth medium containing neomycin (73.5 mg/liter) and 10% fetal calf 1 The abbreviations used are: NA-AAF, N-acetoxy-2-acetylamino serum. They were allowed to grow to confluence at 37Â°in fluorene; 7-BrMeBA, 7-bromomethylbenz(a)anthracene; TdR, thymidine; CdR, deoxycytidine; AdR, deoxyadenosine; GdR, deoxyguanosine; UV, an incubator gassed with 5% CO2 and maintained without ultraviolet radiation. medium change for 6 to 10 days before use. Assays of cells Received April I I, 1973; accepted May 29, 1973. for Mycoplasma sp. were routinely negative. Confluent

SEPTEMBER 1973 2097

monolayers contain few S-phase cells (12). To suppress (New England Nuclear) in a Beckman liquid scintillation replicative synthesis in any remaining S-phase cells, freshly spectrometer. Efficiency was determined with internal prepared hydroxyurea (Sigma Chemical Co., St. Louis, standards. Calculation of specific activity was as previously (Mo.) was added to 10 mM in Dulbecco's phosphate-buf described (25). fered saline. Fifteen mm later the cells were treated with The protocol for experiments involving bromodeoxyuni either 10 @MNA-AAF (20, 31) (a gift of Dr. E. K. Weis dine was generally similar except as noted below. Bromode burger and Dr. J. H. Weisburger) or 10 zM 7-BrMeBA (9, oxyunidine (10 zg/ml; Schwarz/Mann) was added 10 hr 25) (a gift of Dr. A. Dipple) in redistilled dimethyl sulfoxide before other manipulations were begun. Care was taken to (final concentration, 0.5 to 0.6%). Controls received redis perform operations in reduced light and the cells were in tilled dimethylsulfoxidealone.For the experimentinvolv cubated in the dark. The chromatin solution was made 1 M ing UV, the medium was removed 15 mm after the addition with sodium perchlorate, extracted with chloroform-iso of hydroxyurea, and the monolayers were irradiated with amyl alcohol (30), and dialyzed overnight. This solution was 200 ergs/sq mm from a low-pressure mercury lamp (pri treated with RNase, dialyzed, and centrifuged in an 1l-ml manly 254 nm) at 11 ergs/sq mm/sec. The medium con neutral CsC1 gradient at 45,000 rpm for 65 hr. For removal taming hydroxyurea was then replaced. Unirradiated con of any residual RNA contamination, 0.2-mi aliquots from trols were similarly treated. Five to 15 mm after treatment, each fraction were diluted with 0. 1 ml of a solution of car labeled precursors were added. Both control and damaged ncr DNA (calf thymus DNA, 1 mg/mi). After the addition cells received one of the following: TdR, methyl- 3H 4.1 of 1 ml of 1 MKOH the samples were incubated for 1 hr jsCi/ml (New England Nuclear, Boston, Mass., 50 Ci/ at 37Â°,cooled, precipitated with tnichloroacetic acid, and mmole); CdR, 5-3H 3.3 @tCi/ml (New England Nuclear; collected on Millipore filters. After drying, the samples were 26.5 Ci/mmole); AdR, 8-3H 5 to 10 sCi/ml (New Eng counted for 20 to 50 mm in standard Liquifluor (New Eng land Nuclear, 11 to 17 Ci/mmole); or GdR, 8-3H 5 to 10 land Nuclear)-toluene scintillation fluid. @iCi/ml (Schwarz/Mann, Orangeburg, N. Y.; 5.4 Ci/ Identification of Radioactive Products. After removal of mmole). The cells were incubated for 5 hr at 37Â°. CsCl by dialysis from pooled fractions containing â€œre Preparation and Banding of DNA. The radioactive me pairedâ€• DNA, the DNA was digested enzymatically to the dium was removed, and the monolayers were washed twice nucleoside level as previously described (9, 25) and chro with 0.9% NaCI solution. The cells were then scraped off matographed on paper in either NH3-bonic acid-butyl and pelleted by centrifugation. Nuclei were prepared by alcohol (Method A) (I) or HC1-isopropyl alcohol-water lysing the cells in a 0.32 M sucrose solution containing 0.001 (2, 27). Spots were eluted with 1 ml 0. 1 N HCI, and the M potassium phosphate buffer (pH 7.5), 0.0015 M CaCl2, radioactivity was determined as described above. and 1% Triton X- 100. After collection by centritugation, the Distribution of Label in the DNA. DNA was digested nuclei were washed once in the same solution without Tn with snake venom phosphodiesterase (Worthington) as pre ton X-l00 and once in 0.01 M Tnis-HC1 buffer, pH 7.5. The viously described (24, 29). crude chromatin was dialyzed for 16 hr against 0.01 M Tnis-HC1, pH 7.5, 4Â°(1 volume chromatin to 1000 volumes buffer). DNA was prepared directly from the chromatin by RESULTS isopycnic centnifugation (14, 25). In most cases 8-mi CsCl gradients were centrifuged for 40 hr at 45,000 rpm (50 Ti rotor, Beckman L2-65B centrifuge, 25Â°),and 0.36-mi sam The incorporation of radioactive deoxynibonucleosides ples were collected from the bottom of the tube. For analysis into DNA during repair was investigated by density gradi of pynimidine precursors this technique was sufficient to ent centnifugation. Confluent cells in conditioned medium give good resolution, and additional nebandings produced containing 10 mM hydroxyurea incorporate little radioac no change in specific activity. In the case of punine pre tivity into DNA when incubated for 5 hr with I of the 4 ra cursors, however, a high background was found especially dioactive deoxynibonucleosides (Chart I, A, C, E, and in the initial fractions on the heavy side of the gradient. In G; Table 1). In contrast, treatment with NA-AAF or these cases peak fractions were pooled, and the CsC1 was 7-BrMeBA produces a marked stimulation of incorporation removed by dialysis against 0.01 M Tnis-HCI, pH 7.5. The of radioactivity into DNA in these resting cells (Chart 1, DNA was treated for 1 hr at 37Â°with boiled RNase, 25 B, D, F, and H; Table 1). Similar results were obtained zg/ml (Worthington Biochemical Corp., Freehold, N. J.), in experiments utilizing UV as the damaging agent and after the addition of 0.015 M MgCI2. After RNase treat AdR- 3H as the precursor (Table 1). Specific activities for ment, autodigested Pronase, 20 @tg/m1(Sigma), was added DNA obtained from damaged and undamaged cells were for 30 mm. The solution was then dialyzed against 4 changes calculated from these and similar gradients (Table 1). A of 0.01 M Tnis-HCI, pH 7.5 (1 volume of sample per 1000 large increase in incorporation of radioactivity is seen after volumes of buffer) and rebanded in CsCl. damage. Recovery of DNA from treated and untreated Absorbance at 260 nm was determined in a Gilford 240 cultures was similar, and microscopic examination of spectrophotometer after dilution of the fractions with 0.2 formalin-fixed, Giemsa-stained monolayers revealed no ml H2O. For scintillation counting, a sample (0.05 to 0.2 evidence of karyorrhexis, karyolysis, or sloughing of cells. ml) was removed from each fraction and diluted with water A density-labeling experiment utilizing bromodeoxyuni to a final volume of 1 ml. Samples were counted in Aquasol dine was carried out to confirm that the increased incorpo

2098 CANCER RESEARCH VOL. 33

ration of radioactivity seen with purine deoxyribonucleo I0.0 sides represented incorporation into nonreplicating DNA. Many autoradiographic studies utilizing pyrimidine pre 15 GdR curcors have shown that repair synthesis (â€œunscheduled . CON DNA synthesisâ€•) occurs in non-S-phase cells (4, 6, 11, 24, 35, 41, 43, 44) and that carcinogens and UV inhibit semi 1.0 conservative DNA synthesis (3, 24, 35, 37, 38). Such stud ies provide corroborative evidence that increased incorpo ration of pyrimidine deoxyribonucleosides after damage in 0.5 the system described above represents repair synthesis. While it is unlikely that the increased incorporation of purine deoxyribonucleosides represents anything but the same phenomenon, similar studies with purine deoxyribo 0 nucleosides are difficult to carry out because of their util Â© ization in a variety of metabolic pathways. When confluent CdR cells in conditioned medium are maintained in bromodeoxy 1.0 CON uridine for 10 hr and pulsed with GdR-3H there is little incorporation of label into DNA (Chart 2A). Most of the label is seen in the heavy region of the gradient, and, as 0.5 expected from the small amount of replication present, no

@@ absorbance peak is associated with this radioactivity peak. V.@' â€¢: @ The radioactivity cobanding with the absorbance peak I 0 __ -@-@â€¢-.-a-.-@-..â€”â€”@ .@ probably represents incomplete separation oftrace amounts O 0@ @ of density-labeled DNA from large amounts of normal-den 10 . MR sity DNA (15). If cells maintained for 10 hr in bromode CON oxyuridine are treated with 7-BrMeBA and labeled with GdR-3H either in the presence or absence of hydroxyurea 0.5 (Chart 2, B and C), all of the label is incorporated into the nonreplicating DNA, repreSented by the absorbance peak in the less dense region of the gradient. No label is seen in @==:@â€˜@â€˜@ I the heavy region of the gradient, indicating that the small I.5Â© amount of DNA replication still present in these cells is I0.0 shut off by the carcinogen. TdR TdR We investigated the nature of the radioactivity incorpo CON NA-ME rated during repair by paper chromatography of nucleo 1.0 sides from enzymatic digests of repairing DNA (Table 2; see â€œMaterials and Methodsâ€•). With all 4 radioactive nu cleoside precursors, virtually all the label is recoverable as 0.5 / \ the expected product in both chromatographic systems. @ The small amount of label associated with other nucleosides ; @ and/or bases probably represents incomplete separation 0 I from adj acent ch rom atographic products. 0 5 0 5 20 250 5 0 15 20 The distribution of label in repaired DNA was exam med by digestion with snake venom phosphodiesterase. To Chart I . Incorporation of labeled deoxynibonucleosides into DNA in increase the sensitivity of the assay, the DNA was heat de control (CON) cells and those treated with 10 zMNA-AAF. Control cells were pulsed with GdR-'H (10 @zCi/ml,5.4 Ci/mmole) (A); CdR-'H (3.3 natured and cooled in ice before the addition of enzyme. zCi/ml, 26.5 Ci/mmole) (C); AdR-'H (10 @Ci/ml, 11.1 Ci/mmole (E); DNA from cells damaged with either NA-AAF or 7-Br or TdR-3H (4.1 @.tCi/ml,51.3 Ci/mmole) (G) 30 mm after the addition of MeBA and allowed to repair in the presence of tritiated 10 msi hydroxyurea. Damaged cells (B, D, E, and H) were treated identi GdR, AdR, and CdR were examined. In the case of these cally except that NA-AAF was added 15 mm before the addition of tn precurso rs, absorbance and radioactivity were rendered tium-labeled precursors. CsC1 gradients were run as described in â€œMa acid soluble at the same rate (e.g., Chart 3). These data tenials and Methods.â€•Numbers on the abscissa, fraction number starting suggest that incorporation during repair is not the result with the bottom of the gradient. Values on the right-hand ordinate should of terminal addition since, in the case of terminal addition, be multiplied by 1000 to obtain dpm's. Similar gradients for 7-BrMeBA one would have expected to see a very rapid rate of release are not shown. of label compared to the rate of release of UV-absorbing material (e.g., Ref. 28). It must be borne in mind, however, DISCUSSION that the test is a relatively crude one since one is digesting a heterogeneous, partially-sheared DNA with a partially These results demonstrate that purine deoxyribonucleo purified enzyme. sides as well as pyrimidine deoxyribonucleosides are incor

SEPTEMBER 1973 2099

Table I Specific activities of DNA from control and treated confluent cells

PrecursorControlNA-AAFmm)GdR74'@2643@ (10 zM)7-BrMeBA(10 @zM)UV (200 ergs/sq

(34)CdR (36)25l5V (30) 95 (39) AdR135 68 556 (8.1)3669 222 (3.3)407a (8.1)TdR102 504018 (45) (26) 1704559 4094 (24)2633 aDataarecalculatedfromthegradientsinChartIandsimilargradientsnotshown.Allcultures contained 10 mM hydroxyurea. Specific activity is in dpm/@g DNA. The values in parentheses representthe experimentaltreatment values(Columns 3, 4, and 5) divided by thosein the control column. The amount and specific activity ofthe radioactivity used is: Line I, GdR-3H (10 jzCi/mI, 5.4 Ci/mmole); Line 2, CdR-'H (3.3 zCi/ml, 26.5 Ci/mmole); Line 3, CdR-3H (10 @tCi/m1, 26.5 Ci/mmole); Line 4, AdR-3H (10 MCi/mI, 11.1 Ci/mmole); Line 5, AdR-3H (5 @Ci/ml, 16.8 Ci/mmole). Lines 6 and 7, TdR-'H (4.1 @sCi/ml,51.3 Ci/mmole).

sides occurs during the repair process. One may tentatively 2.5 :: Â® :: Â® :@ Â© conclude that during excision repair, whether the damage .. CON â€¢. 7BrMeBA :@ 7BrMeBA is done to pyrimidine or purine moieties, both pyrimidine @ :: +HU 12.0 and purine deoxyribonucleosides are incorporated. @@ 2.0 â€˜ - Since the proximate carcinogens used in these studies alkylate primarily purine moieties (9, 20, 25, 31), the dem

I.5 onstration that pun ne deoxyribonucleosides a re incorpo rated during repair is additional evidence that a biologically @: Lk, meaningful repair process is taking place in response to chemical damage. It is probable that a â€œcutand patchâ€•

I.0 type of repair is operative for NA-AAF- and 7-BrMeBA induced lesions as well as those induced by UV (13, 45). The failure of previous experiments to demonstrate pu rine incorporation (27, 34, 41, 42) is probably due to the ex @@@ o: 0 (5 10.5 perimental methods used. It is apparent from equilibrium centrifugation studies of DNA prepared directly from cell lysates (see â€œMaterials and Methodsâ€•) that trace contam ination of DNA with RNA (0.1 to 1.0%) can obscure re Chart 2. Incorporation of GdR-3H into DNA from control and dam suits when purine deoxyribonucleosides are used as precur aged cells in the presence of bromodeoxyunidine. Ten hr after the addition sors. This probably results from the reutilization of the of bromodeoxyunidine (10 @g/ml), control cells (A) were pulsed with purine moieties as ribonucleosides. Since the amount of GdR-'H (6@tCi/ml, 5.4 Ci/mmole). Damaged cells were treated identi RNA synthesis greatly exceeds the amount of repair syn cally except that, 15 mm before the addition ofGdR-'H, cells were treated with 10 @iM7-BrMeBA (B) or 10 mM hydroxyurea (HU) 30 mm before thesis, even trace contamination with RNA can obscure re and 10 MM 7-BrMeBA 15 mm before the addition of GdR-'H (C). CsCI pair synthesis. Our previous failure to demonstrate repair gradients were run as described in â€œMaterialsand Methods.â€•Numbers with purine deoxyribonucleosides probably is the result of on the abscissa, fraction numbers starting with the bottom of the gradient. insufficient purification of DNA (i.e., simple KOH diges Values on the right-hand ordinate should be multiplied by 100 to obtain tion verses preparation of nuclei to remove cytoplasmic cpm's. RNA, RNase digestion, and 2 bandings in CsC1). The variation in the amount of stimulation of DNA syn porated into DNA during repair synthesis; furthermore, thesis during repair (Table 1) probably reflects a number of they suggest that the incorporation is probably not ac factors. It is difficult to get good base-line specific activi counted for by an artifact such as terminal addition. We ties because of the low level of counts in the control gradi @ haveutilized2 independentassaysforrepairsynthesis.One ents. Differences in pool size between G and S phase cells measures tritiated nucleoside incorporation in a cell popu for a given nucleotide are known to occur (32). Further @ lation almost devoid of S-phase cells, the latter being sup more, the amount of change in pool size between and pressed with hydroxyurea; the other uses bromodeoxyuri S phase cells may vary from precursor to precursor. In ad dine density labeling to separate DNA replication in dition, levels of the appropriate kinases and competing de S-phase cells from DNA repair synthesis. The data from mandsfor the samepools,especiallyinthe caseof the pu these 2 systems suggest that a meaningful restoration of rine deoxyribonucleosides, probably affect the amount of cellular DNA involving insertion of all 4 deoxyribonucleo incorporation. Obviously, considerable work must be done

2100 CANCER RESEARCH VOL. 33

Table 2 Chromatography of enzymatic digests of DNA from cells damaged with either NA-AA F or 7-BrMeBA and allowed to repair in the presence of the precursor listed in Column I System 1 is the NH3-Bonic acid-butyl alcohol system (1), while System 2 is the HC1-isopropyl alcohol-water system (2, 27). In System 2, depurination occurs from the HCI. In each system the nucleosides or bases are listed in order of increasing RF from left to right.

2GdRCdRAdRTdRGuanineAdenineCdRTdRGdR

PrecursorCarcinogenSystemISystem

7-BrMeBA 99 0 0 0 96.0 1.5 2.0 0.5 CdR NA-AAF 0 99 0 0 0 0 99 0 7-BrMeBA 4.5 93.5 1.5 0.5 0.5 1.2 97.3 1.0 AdR NA-AAF 1.6 3.0 90.3 5.1 6.0 94.0 0 0 7-BrMeBA 9.0 2.0 87.0 0 6.0 93.0 1.0 0 TdRNA-AAF NA-AAF 0 0 4.8 95.2 0 0 0 99 7-BrMeBA99 00 00 9.30 90.798.0 02.0 00 00 99

100 in this area if the real relative rates of incorporation are to be measured. GdR The low level of incorporation of purine deoxyribonucleo 80 7BrMeBA sides into both replicative and repairing DNA in the pres ence of bromodeoxyuridine may reflect some alteration in purine utilization or DNA metabolism by bromodeoxyuri dine or one of its metaboiites. Similar findings are reported from another laboratory (7). Cleaver (7) has reported incorporation of radioactivity during repair synthesis with several purine bases and nude osides as precursors. In general his results and ours are in agreement. However, it is difficult to compare our results directly with his since he found hypoxanthine, a catabolite, to be the most effective precursor in his system and the in corporation products were not identified. In contact-inhibited cells in conditioned medium, the use of hydroxyurea alone is a useful method for studying repair synthesis. Similar results have been obtained with human Iâ€” peripheral blood lymphocytes, another population almost LU C-) completely free of S-phase cells (4, 11, 18, 24, 25). Such a

LU system may not be universally useful, however, since high 0@ concentrations (1 to 10 mM) of some N-hydroxy com pounds may interfere with hydroxyurea and give false positive results in rapidly dividing cells (HeLa) (3). On the other hand, there appears to be little problem in cell popu lations that are largely free of S-phase cells, such as conflu ent WI-38 fibroblasts. The hydroxyurea system has the ad I00 vantage of simplicity and allows the demonstration of Â© incorporation of all 4 naturally occurring deoxyribonucleo 80 sides into DNA. Thus, studies can be carried out in which base composition may be important (e.g., digestion with

60 Chart 3. Release of DNA into acid-soluble material by snake venom phosphodiesterase. After heat denaturation, DNA was digested with en 40 zyme (9, 37). Values on the ordinate, percentage of U V-absorbing material, representing total DNA (0), or radioactivity, representing repaired DNA (C), rendered acid soluble with time. All samples were prepared from 20 hydroxyurea-suppressedcells.DNA from cells damagedwith 7-BrMeBA and allowed to repair in the presence of GdR-'H (A); DNA from cells damaged with 7-BrMeBA and allowed to repair in the presence of CdR 480 3H (B); DNA from cells damaged with NA-AAF and allowed to repair in the presence of AdR-'H (C).

SEPTEMBER 1973 2101

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1973 American Association for Cancer Research. Michael W. Lieberman and Miriam C. Poirier nucleases or preparation of isostich profiles) without con 8. Coppey, J. Repair of Interferon Synthetic Capacity in Ultraviolet cern for the effects of foreign bases. Although it would be irradiated Normal and Hybrid Mammalian Cells. Nature New Biol., 234: 14-15,1971. ultimately desirable to devise a repair system in which no 9. Dipple, A., Brookes, P., Mackentosh, D. S., and Rayman, M. P. Re additions are necessary, the use of the hydroxyurea system action of 7-Bromomethylbenz(a)anthracene with Nucleic Acids, Poly makes it unlikely that an artifact resulting from a number nucleotides and Nucleosides. Biochemistry, 10: 4323â€”4330,1971. of unique properties of bromodeoxyuridine (16, 23, 39) is 10. Edenberg, H., and Hanawalt, P. Size of Repair Patches in the DNA responsible for incorporation of radioactivity during repair of Ultraviolet-irradiated HeLa Cells. Biochim. Biophys. Acta, 272: 361â€”372,1972. synthesis. 11. Evans, R. G., and Norman, A. Unscheduled Incorporation of Thy In conclusion, human diploid fibrobiasts, treated with midine in Ultraviolet-irradiated Human Lymphocytes. Radiation large aryl-alkylating agents or UV, incorporate all 4 bases Res.,36:287â€”298,1968. during repair. Attack of purine bases by agents such as 12. Farber, J., Rovena, G., and Baserga, R. Template Activity of Chro NA-AAF or 7-BrMeBA may be expected to produce dis matin during Stimulation of Cellular Proliferation in Human Diploid Fibroblasts. Biochem. J., 122: 189-195, 1971. tortions in the helix, similar to that induced by UV (13, 17, 13. Fink, L:, Flieg, A., Grunberger, D., and Weinstein, 1. B. Effect of 19, 45). Less bulky agents which may possibly be repaired N-2-Acetylaminofluorene (AAF) Modification of Nucleic Acids on without wide excision (6) have not been examined. Further Their Secondary Structure. Proc. Am. Assoc. Cancer Res., 12: 11, more, it is not certain that these results are applicable to 1971. 14. Flamm, W. G., Birnstiel, M. L., and Walker, P. M. B. Preparation other types of mammalian cells or to rapidly dividing cell and Fractionation and Isolation of Single Strands of DNA by Ultra populations. Nevertheless, these data give further cre centnifugation in Fixed Angle Rotors. In: G. Birnie and S. M. Fox dence to a number of studies in which repair of carcinogen (eds.), Subcellular Components: Preparation and Fractionation, pp. induced DNA damage in human diploid cells was studied 125-155. London: Butterworth and Co., 1969. with pyrimidine precursors (18, 24, 25, 40, 43, 44). It is now 15. Gautschi, J. R., Young, B. R., and Painter, R. B. Evidence for DNA Repair Replication in Unirradiated Mammalian Cellsâ€”Is It An apparent that such cells incorporate all four bases into the Artifact? Biochim. Biophys. Acta, 281: 324-328, 1972. DNA during repair, that this incorporation is unlikely to 16. Gerber, P. Activation of Epstein-Barr Virus by 5-Bromo-deoxyuni result from terminal addition or polypyrimidine runs (26), dine in â€œVirus-freeâ€•HumanCells. Proc. Natl. Acad. Sci. U. S., 69: and that there is removal of bound-carcinogen during such 83â€”85,1972. 17. Grossman, L., Kaplan, J., Kushner, S., and Mahler, I. Enzymatic incorporation (25). All these results suggest that repair syn Mechanisms for the Repair of UV Irradiated DNA. Ann. 1st. Super. thesis represents a faithful restoration of the damaged Sanita, 5: 318â€”333,1969. strand to its original state; however, we are far from being 18. Jacobs, A. J., O'Brien, R. L., Parker, J. W., and Paolilli, P. Abnormal able to determine the accuracy of this process on a nucieo DNA Repair of 4-Nitnoquinoline-l-oxide-induced Damage by Lym tide by nucleotide basis in the living cell. Ultimately, it phocytes in Xenodenma Pigmentosum. Mutation Res., 16: 420â€”424, 1972. would be of interest to examine the error frequency during 19. Kelley, R. B., Atkinson, M. R., Huberman, J. A., and Kornberg, A. repair sincethe introductionof errors might have impor Excision of Thymine Dimers and Other Mismatched Sequences by tant biological consequences if selection plays any role in DNA Polymenase ofEscherichia coli. Nature, 224: 495-501, 1969. the neoplastic process. 20. Kniek, E. On the Mechanism of Action of Carcinogenic Aromatic Amines: I. Binding of 2-Acetylaminofluorene and N-Hydroxy-2- acetylaminofluorene to Rat-Liver Nucleic Acid in Vivo. Chem.-Biol. Interactions, 1: 3â€”17,1969â€”1970. ACKNOWLEDGMENTS 21. Lawley, P. D. Effects of Some Chemical Mutagens and Carcinogens on Nucleic Acids. Progr. Nucleic Acid. Res. Mol. Biol., 5: 89â€”131, We thank Dr. R. R. Bates, Dr. W. G. Flamm, Dr. D. G. Kaufman, 1966. and Dr. M. B. Sporn for their thoughtful comments and Dr. Stuart H. 22. Lawley, P. D., and Thatcher, C. J. Methylation of Deoxyribonucleic Yuspa and David L. Morgan for their advice on cell culture methods. Acid in Cultured Mammalian Cells by N-Methyl-N-nitro-N-nitroso guanidine. Biochem. J., 116: 693-707, 1970. 23. Levitt, D., and Dorfman, A. The Irreversible Inhibition of Differentia REFERENCES tion of Limb-Bud Mesenchyme by Bromodeoxyunidine. Proc. Natl. Acad. Sci. U. S., 69: 1253â€”1257,1972. I. Al-Anif, A., and Sporn, M. B. An Analytical Method for the Separa 24. Liebenman, M. W., Baney, R. N., Lee, R. E., Sell, S., and Farber, E. tion of Sugar-Methylated Ribonucleosides from Base-Methylated and Studies on DNA Repair in Human Lymphocytes Treated with Prox Nonmethylated Ribonucleosides. Anal. Biochem., 48: 386â€”393.1972. imate Carcinogens and Alkylating Agents. Cancer Res., 31: 1297â€” 2. Bendich, A. Methods for the Characterization of Nucleic Acids by 1306,1971. Base Composition. Methods Enzymol., 3: 715-723, 1957. 25. Lieberman, M. W., and Dipple, A. Removal of Bound Carcinogen 3. Brandt, W. N., Flamm, W. G., and Bernheim, N. J. The Value of Hy During DNA Repair in Non-dividing Human Lymphocytes. Cancer droxyurea in Assessing Repair Synthesis of DNA in HeLa Cells. Res.,32:1855â€”1860,1972. Chem.-Biol. Interactions, 5: 327â€”339,1972. 26. Lieberman, M. W., Rutman, J. Z., and Farber, E. Thymidine La 4. Bunk, P. G., Lutznen, M. A., Clarke, D. D., and Robbins, J. H. Ultra beling of Pynimidine Isostichs from Human Lymphocyte DNA dun violet-stimulated Thymidine Incorporation in Xenoderma Pigmento ing Repair after Damage with N-Acetoxy-Acetylaminofluorene or sum Lymphocytes. J. Lab. Clin. Med., 77: 759â€”767,1971. Nitrogen Mustard. Biochim. Biophys. Acta, 247: 497-501, 1971. 5. Cleaver, J. E. Defective Repair Replication of DNA in Xerodenma 27. Lieberman, M. W., Sell, S., and Farber, E. Deoxynibonucleoside In Pigmentosum. Nature, 218: 652â€”656,1968. corporation and the Role of Hydroxyurea in a Model Lymphoxyte 6. Cleaver, J. E. Repair of Alkylation Damage in Ultraviolet-sensitive System for Studying DNA Repair in Carcinogenesis. Cancer Res., (Xenoderma Pigmentosum) Human Cells. Mutation Res., 12: 453- 31: 1307â€”1312,1971. 462, 1971. 28. Lindahl, T., GaIly, J. A., and Edelman, G. M. Properties of Deoxy 7. Cleaver, J. E. DNA Repair with Purines and Pynimidines in Radia nibonuclease III from Mammalian Tissues. J. Biol. Chem., 244: tion- and Carcinogen-damaged Normal and Xerodenma Pigmentosum 5014-5019, 1969. Human Cells. Cancer Res., 33: 362-369, 1973. 29. Loeb, L. A. Purification and Properties of Deoxyribonucleic Acid

2102 CANCER RESEARCH VOL. 33

Polymerase from Nuclei of Sea Urchin Embryos. J. Biol. Chem., 244: Synthesisâ€•)in HeLa and Chinese Hamster Cells following Treatment 1672-1681,1969. with Alkylating Agents. Chem.-Biol. Interactions, 3: 49-68, 1971. 30. Marmun, J. A Procedure for the Isolation of Deoxyribonucleic Acid 39. Schubert, D., and Jacob, F. 5-Bromodeoxyunidine-induced Differenti from Micro-organisms. J. Mol. Biol., 3: 208-218, 1961. tion of a Neunoblastoma. Proc. Nail. Acad. Sci. U. S., 67: 247-254, 31. Miller, J. A. Carcinogenesis by Chemicals: An Overviewâ€”G. H. A. 1970. Clowes Memorial Lecture. Cancer Res., 30: 559-576, 1970. 40. Setlow, R. B., and Regan, J. D. Defective Repair of N-Acetoxy-2- 32. NordenskjÃ¶ld, B. A., Skoog, L., Brown, N. C., and Reichard, P. Dc acetylaminofluorene-induced Lesions in the DNA of Xeroderma Pig oxynibonucleoside Pools and Deoxyribonucleic Acid Synthesis in mentosum Cells. Biochem. Biophys. Res. Commun., 46: 1019-1024, Cultured Mouse Embryo Cells. J. Biol. Chem., 245: 5360-5368, 1970. 1972. 33. Rabson, A. S., Tynell, S. A., and Legallais, F. Y. Growth of Ultra 41. Smets, L. A. Repair Incorporation of Pynimidine Deoxynibonucleo violet-damaged Herpesvirus in Xerodenma Pigmentosum Cells. Proc. sides into DNA of Mammalian Cells Exposed to UV Light. Bio Soc. Exptl. Biol. Med., 132: 802-806, 1969. physik, 6: 85-93, 1969. 34. Rasmussen, R. E., and Painter, R. B. Evidence for Repair of Ultra 42. Smets, L. A. Ultraviolet Light-enhanced Precursor Incorporation and violet Light Damaged Deoxyribonucleic Acids in Cultured Mamma the Repair of DNA Damage. Intern. J. Radiation Biol., 16: 407â€”418, han Cells. Nature, 203: 1360- 1362, 1964. 1969. 35. Rasmussen, R. E., and Painter, R. B. Radiation-stimulated DNA 43. Stich, H. F., and San, R. H. C. DNA Repair and Chnomatin Anom Synthesis in Cultured Mammalian Cells. J. Cell Biol., 29: 11- 19, 1966. alies in Mammalian Cells Exposed to 4-Nitnoquinoline I-Oxide. Mu 36. Regan, J. D., Setlow, R. B., and Ley, R. D. Normal and Defective Re tation Res., 10: 389-404, 1970. pair of Damaged DNA in Human Cells: A Sensitive Assay Utilizing 44. Stich, H. F., San, R. H. C., Miller, J. A., and Miller, E. C. Various the Photolysis of Bromodeoxyunidine. Proc. NatI. Acad. Sci. U. S., Levels of DNA Repair Synthesis in Xenodenma Pigmentosum Cells 68:708-712,1971. Exposed to the Carcinogens N-Hydnoxy- and N-Acetoxy-2-acetyl 37. Roberts, J. J., Brent, T. P., and Crathorn, A. R. Evidence for the In aminofluonene. Nature New Biol., 238: 9- 10, 1972. activation and Repair of the Mammalian DNA Template after Alkyl 45. Weinstein, I. B., and Gnunbergen, D. Structural and Functional ation by Mustard Gas and Half Mustard Gas. European J. Cancer, 7: Changes in Nucleic Acids Modified by Chemical Carcinogens. In: 515â€”524,1971. P. 0. P. Ts'o and J. A. DiPaolo (eds.), World Symposium on Model 38. Roberts, J. J., Pascoe, J. M., Smith, B. A., and Crathonn, A. R. Quan Studies in Chemical Carcinogenesis. New York: Marcel Dekken, in titative Aspects of the Repair of Alkylated DNA in Cultured Mam press. malian Cells: II. Non-Semiconservative DNA Synthesis (â€œRepair

SEPTEMBER 1973 2103

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1973 American Association for Cancer Research. Deoxyribonucleoside Incorporation during DNA Repair of Carcinogen-induced Damage in Human Diploid Fibroblasts

Michael W. Lieberman and Miriam C. Poirier

Cancer Res 1973;33:2097-2103.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/33/9/2097

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/33/9/2097. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.