Excision and Postreplication DNA Repair Capacities, Enhanced Transformation, and Survival of Syrian Hamster Embryo Cells Irradiated by Ultraviolet Light

[CANCER RESEARCH 40, 582-587, March 1980] Excision and Postreplication DNA Repair Capacities, Enhanced Transformation, and Survival of Syrian Hamster Embryo Cells Irradiated by Ultraviolet Light

Jay Doniger1 and Joseph A. DiPaobo

Laboratory of Biology, Carcinogenesis Intramural Program, Division of Cancer Cause and Prevention, Bethesda, Maryland 20205

ABSTRACT prior to a second carcinogen (5â€”7).Maximum enhancement occurs when a 48-hr interval separates the 2 treatments. The frequency of ultraviolet light (UV)-induced neoplastic However, if the interval is 72 hr, the enhancement is negligible. transformation of Syrian hamster embryo cells (HEC) is en The uniqueness of the 48-hr incubation interval is not due to hanced 3- to 10-fold when the cells are first treated with either cytological alterations such as cell synchrony, nate of DNA X-irradiation or with methyl methanesulfonate. Maximum en replication, chromosomal aberrations, changes in mitotic index, hancement occurs when the interval between the two treat or change in length of any cell cycle phase, because none of ments is 48 hr. The relevance of UV-induced transformation to these are observed in the X-irradiated cells (7). The present neoplasia is confirmed because the transformants produce studies have been designed to determine whether an observ tumors when injected into nude mice. Excision and postrepli abbechange in the rate of excision or postreplication repair of cation DNA repair were studied to determine whether the DNA damages is induced by the pretreatment of cells with enhanced transformations were associated with either of these either X-innadiationon MMS and therefore can be responsible repair mechanisms. Independent of X-ray or of methyl for the enhanced transformation. Since repair of DNA damage methanesulfonate pretreatment, approximately 25% of the py caused by UV irradiation (254 nm) is easily quantitated (9) and rimidine dimers are excised within 24 hr in cells irradiated with UV can induce transformation of HEC in vitro (4), this agent is UV with 3 J/sq m. During this period, more than 70% of the used in the combination studies. When the excision of pynimi genome of cells irradiated with UV has been replicated. Post dine dimens,the major UV-induced photoproduct, is measured replication repair is measured by the time required to chase by the sensitivity of the dimers to a specific endonuclease (14), pulse-labeled nascent DNA strands to parental-sized DNA. no differences in excision rates are found in treated and control Regardless of pretreatment, 1 and 3 hr are required for pulse cells. Furthermore, when postreplication repair rates are stud labeled DNA in control and irradiated (10 J/sq m) cells, re ed with standard pulse-chase techniques (2, 13, 17) to meas spectively, to reach parental size. Therefore, no correlation is ure the size of nascent DNA strands, these rates are also found between a change in the rate of excision or postrepli independent of pretreatment. cation repair and enhancement of transformation. Relative to control cloning efficiency, the survival of HEC irradiated with 3 J/sq m is higher than 70% even though HEC contain more MATERIALS AND METHODS than 10@pynimidine dimers/genome. The level of survival is Transformation Assay and Cell Survival. For each transfor similar to the survival of human skin fibnoblasts which excise mation experiment, fresh HEC from fetuses 13 to 14 days in pynimidine dimers four to five times as efficiently. Moreover, gestation (16 to 19 mm crown-rump length) are used. The postreplication repair cannot account for the ability of these tissue culture buffers and medium, the cell culture techniques, cells to survive because it is three times slower than in human and the conditions for the transformation assay have been fibrobbasts. Therefore, other repair mechanisms must be re published (7). Random-bred hamsters are maintained in a room sponsible for HEC survival and transformation. with a 12-hr light cycle, and the fetuses are removed from animals that have been bred at least 3 times and have had 12 INTRODUCTION to 18 viable fetuses. Primary and subsequent passage cells are grown as monobayensin plastic Petnidishes in CM at 37Â°in an The modulation of the frequency of carcinogen-induced 11% CO2humidified atmosphere. In all experiments, 2-day-old transformation with an in vitro model provides a valuable ap secondary or tertiary hamster cultures, obtained by seeding proach for determining the factors important for the conversion 2.5 x 106 cells/i00-mm dish, are used. of normal cells to a neoplastic state (3). Exposure of cells For the quantitative transformation assay, 300 cells in CM sequentially to 2 agents can significantly increase the fre with 20% serum are seeded in a 60-mm plastic Petnidish with quency of transformation. This is accomplished by pretreating or immediately subsequent to the addition of 6 x 10@HEC the cells with the alkybating agent, MMS2, or with X-irnadiation which had been irradiated (100 kVp, 3500 R) as a confluent monolayer culture using a Model T55-433 Picker portable I To whom requests for reprints should be addressed. industrial X-ray apparatus. The batterconstitute the feeder layer 2 The abbreviations used are: MMS, methyl methanesulfonate; HEC, Syrian hamster embryo cells; CM, Dulbecco's modified Eagle's medium supplemented that facilitates the growth of the relatively low number of HEC. with 10% fetal bovine serum unless otherwise specified; CE, cloning efficiency; Transformation and cell survival of HEC are determined in NTE buffer, 100 mM NaCI:50 m@ Tris-HCI, pH 7.5:10 m@ EDTA; BrdUrd, the same dishes. After 7 days of incubation, the colonies are bromodeoxyuridine; saline: EDTA, 0.8% NaCI, 0.1 15% Na2HPO4,and 0.02% each of KH2HPO4,disodium EDTA, and KCI. fixed with methanol and stained with Giemsa. Colony morphob Received July i 2, i 979; accepted November 20, 1979. ogy is determined with a stereoscopic microscope at x 10 to

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1980 American Association for Cancer Research. DNA Repair and Enhanced UV Transformation x40. The coloniesare either bight(semicontiguous)or dense tion at 3000 x g for 5 mm, and the aqueous phase is removed. (contiguous) (4). Nontransfonmed colonies display a regularly To minimize shearing, only cut-off plastic pipet tips are used oriented arrangement of cells while the transformed ones ex for transferring the DNA. The DNA is dialyzed in cobbodionbags hibit a random crisscross piling up of cells not seen in the against 15 ml of NTE buffer for 9 hr, then dialyzed twice against controls. Transformation frequency is calculated either per dish 15 ml buffen(iO mM Tnis-HCI:i mp@iEDTA,pH 7.5) for 10 and or per colony. The enhancement of the transformation is ex 4 hr, and stoned at 4Â°.Pynimidinedimer content is determined pressed as the ratio of the results of the double treatment to by sensitivity of the DNA to MiCroCoCCus!uteus pynimidine those of UV alone. dimer-specific endonuclease (P-i i fraction of Riazuddin and The neoplastic property of morphobogicalbytransformed cob Grossman) (i 5). Extracts of â€˜4C-and3H-labebedDNA are from onies is confirmed by assaying for tumonigenicity. Viable cob irradiated cells that had or had not been incubated, respec nies are identified with a phase-contrast microscope as being tively. The reaction mixture containing excess endonuclease transformed by the same criteria as described for the Giemsa (i 0 @sb)and50 @zbeachof a 3H- and â€˜4C-labeledDNAextract is stained colonies. Colonies are isolated by the ring technique incubated for 30 mm at 37Â°.Thereaction is stopped by cooling and are placed in a 60-mm dish. At confluency, the cells are to 0Â°,and the mixture is layered on top of 0.3 ml of 0.5 M transferred to a 100-mm dish. Approximately 3 weeks from the NaOH:i 0 mM EDTA over a 4.8-mI 5 to 20% (w/v) sucrose time of isolation of the colony, 5 x 106 cells are injected s.c. gradient (0.2 N NaOH:0.5 M NaCl:i 0 mMEDTA), seated on 0.i in nu/nu mice. The animals are then observed for progressively ml of 60% (w/v) sucrose:0.5 N NaOH. The gradients are growingtumors(8). centrifuged at 45,000 rpm in a SW 50.i rotor for about 60 mm. The CE is determined by dividing the average number of Centrifugation is terminated when w2t 8.08 X i 0'Â°radians2/ colonies (greaten than 2 mm) by the number of cells seeded sec. Approximately 20 fractions are collected from each gra per plate, and multiplying by 100. Survival is determined by dient by pumping the gradients through a 20-gauge cannuba comparing the CE of treated to untreated cells. When the inserted to the bottom of the tube. The DNA in each fraction is survival of UV-irnadiated normal human cells (CAL 1220 from precipitated with i N HCI and collected on Whatman GF/C ATC) is measured, no feeder cells are used, and 200 to 500 glass fiber filters. The filters are washed once with 1 N HCI and cells are seeded in 100-mm dishes 7 to 10 hr prior to irnadia twice with 95% ethanol. After the filters are dried, the radio tion. The cultures are incubated for 16 to 18 days; CM is activity is counted in a tobuene-basedscintillation solution. The renewed twice. gradients were precalibrated with the following phage DNA In some experiments, hamster mass cultures are treated with markers: (a) T-2; (b) T-4; (C)T-7; (d) nicked 4Xi 74; and (e) A. either MMS (1 1 @tg/mbofmedium) or X-irradiation (250 A). The data are analyzed and plotted by computer. MMS is added to the cultures in CM for 1 hr. After treatment, The Amount of DNA Replication after UV Irradiation. Cells the cells are washed with Dulbecco's phosphate-buffered sa are plated and prelabebed as for pynimidine dimer excision. line (without calcium or magnesium), trypsinized, and seeded After irradiation (10 J/sq m), the cells are incubated for 6 or for colony formation. 24 hr in fresh CM containing BrdUrd (5 @tg/ml).Subsequently, Cell Treatment Conditions. X-irradiation (126 A/mm) is the medium is removed and the plates are frozen at â€”40Â°. delivered from a distance of 54 cm by a Westinghouse Quan After being thawed, the cells are bysedby the addition of 1% drocondex machine which has the following features: 2 tubes, Sarkosyb in NTE buffer. RNase (50 jig/mI) is added, and the above and below; 235 kVp; and 0.25 mm Cu and 0.55 mm Al dishes are incubated for 30 mm at 37Â°,followed by i hr further filtration. Cells are irradiated with UV 24 cm from a single 15- incubation in the presence of pnoteinase K (50 @g/ml).The watt GeneralElectricgermicidallamp(Gi 5T8) at a fluencerate extracted DNA is sheared by passage through a 20-gauge of 0.6 J/sq rn/sec. The fluence rate is measured by an Inter needle 3 times. 3H-babebedDNA(from cells incubated without national photometer which has been calibrated by the potas BrdUrd), 14C-IabeledDNA (from cells incubated with BrdUrd), sium ferric oxalate procedure. Prior to UV irradiation, the and CsCI [60.8% (w/w) of NTE buffer] are mixed in a i :4:42 medium is removed, and the dishes are covered with a UV ratio by volume. Density equilibrium is established by centnifu transparent Permanox top (Lux Scientific). After irradiation, gation for 48 hr in a SW 50.1 rotor at 33,000 rpm. The fresh CM supplemented with 20% serum is added, and incu gradients are fractionated into about 50 fractions, and the bation continues. For the biochemistry experiments, CM is radioactivity is determined. The density of Fractions i 0, 20, used. and 30 is determined by measuring the refractive index of 2- Pyrlmidlne Dimer Excision. HEC, plated at 5 x 1O@(to ,@bsamples.The data are analyzed and plotted by computer. maintain bog-phasegrowth) in a 60-mm dish, are labeled over PostreplicatlonRepair. HEC are platedand prelabebedover night in CM containing [â€˜4C]thymidmne(0.1@tCi/ml;50 mCi/ night with [â€˜4C]thymidineasabove for pynimidine dimen exci mmol) or[3H]thymidine (0.25 @tCi/ml;0.25 @.tg/ml).Themedium sion. The CM containing radioactivity is removed, the cells are with radioactivityis removed 1 hr prior to UV irradiation,the washed, and 2 ml of fresh CM are added. After a minimum of cells are washed, and 4 ml of fresh CM are added. The cells 0.5 hr, the CM is removed, and the cells are irradiated (10 J/ irradiated with 3 J/sq m are incubated for 2, 6, or 24 hr; the sq m). The same medium is then replaced, the cells incubated medium is removed, and the cells are frozen on the dish at for 25 mm, and pulse-labeled for i 5 mm with [3H]thymidmne(20 _400. After being thawed, the cells are lysed by the addition Ci/mmol, 50 @sCi/mb).Thepulse label is chased for i , 2, or 3 of 1 ml 1% Sankosybin NTE buffer. Protein in the bysate is hr with CM containing 20 @Munlabeledthymidmneand incuba digested by incubation for 1 hr in the presence of proteinase tion is terminated by washing the cells with sabine:EDTA.The K (50 @sg/mI),theDNA solution is extracted with redistibled cells are X-inradiated (1i 00 A, 50 kVp using the Picker source) phenol by very gentle agitation until the liquid phase is com to facilitate the unraveling of the DNA. After a 2-mbsample of pletely emulsified. The 2 phases are separated by centnifuga sabine:EDTAis added to the dishes, the cells are detached with

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1980 American Association for Cancer Research. J. Doniger and J. A. DiPao!o the aid of a rubber policeman, washed once by centrifugation (1000 x g for 4 mm), and resuspended in 0.5 ml sabine:EDTA. Between 5 x 1o@and 1O@cellsare bysedon a 0.3-mbpad of i N NaOH:i 0 mM EDTA on a 5 to 20% (w/v) sucrose gradient (2 MNaCI:0.33 NNaOH:i 0 mi@iEDTA)seated on 0.1 @nlof60% (w/v) sucrose:0.5 N NaOH. The gradients are centrifuged in a SW 50.1 rotor for about i 30 mm at 30,000 rpm and are terminated when w2t 7.7 X i 010radians2/sec. Fractionation, counting of radioactivity and analysis of the data are as de scnibed above.

RESULTS -a > Cell Survival. HEC irradiated 24 hr after seeding on an X > irradiated feeder layer are sensitive to increasing doses of UV In irradiation, as indicated by their colony-forming ability. When the results are normalized by designating the CE of untreated controls as 100%, the dose that results in 37% survival (D37)is 5.5 J/sq m (Chart i ). This lethality is similar to that obtained when normal human fibroblast cells are irradiated with UV. In the latter case, the UV dose corresponding to 37% survival is 6.4 J/sq m. Transformation Enhancement. UV irradiation also causes a dose-dependent increase in transformation (Table i ). The transformed colonies are morphobogicably similar to those which had been described previously, independent of the car cmnogenused (4, 5). The relevance of the morphobogicabby 3 6 9 i2 15 transformed colonies to neoplasia is verified by demonstrating sqm their tumonigenicity. Five discrete colonies have been isolated Chart i . UV survival curves of HEC and normal human fibroblasts. HEC (0), 300 cells/60-mm dish (12 dishes/dose), are seeded with x-irradiated feeder and subcubtured. Homozygous nude mice have been given s.c. cells; human cells (x), 200 to 500 cells/ 100-mm dish (5 dishes/dose), are injections of approximately 5 x i 06 cells. Progressively grow seeded without feeder cells. Within 24 hr incubation, the medium is removed for irradiation. HEC are incubated for 7 days and human cells are incubated for 16 ing tumors result in all cases in approximately 3 to 6 weeks. days before staining. Results are normalized to the CE of nonirradiated HEC Histopathobogical examination of these tumors indicates that (25%) and human cells (30%). they are either undifferentiated sarcomas or fibrosarcomas. The previously published results from this laboratory that Table 1 Enhancement of Uv-induced transformation frequency by MMS or X-irradiation prior treatment with X-irradiation (250 A) enhances the UV pretreatment of HEC 48 hr prior to UV irradiation induced (i .5 or 3.0 J/sq m) transformation frequency in HEC A total of 300 cells were seeded after x-irradiation (250 R) or MMS (11 @g/ are confirmed. When 48 hr separate the 2 treatments, the ml) on a feeder layer. Experiments were terminated after a total of 7 days of enhancement is observed on both an absolute basis, number incubation. of transformed colonies per dish, and on a relative basis, Absolute basisRelativebasisAv. number of transformed colonies per total colonies (Table 1). no.TotalAv. No transformation occurs with X-irradiation alone. When MMS no.of trans (1 i .tg/mI medium for 1 hr) is substituted for X-irradiation, a formedtransno. ofof trans formedcob similar enhancement of UV-induced transformation results. formedcob- En nies/to En Data utilizing MMS pretreatment and 2 UV doses, i .5 and 3 J/ UV doseNo. ofCEcob nies/ hance tal cob hance sq m, shows the effect of increasing doses of UV (Table 1). (%)mentNo(J/sq m)dishes(%)niesdish ment5nies The variability observed between experiments is such that x-Ray012240001 the enhanced transformation with MMS plus i .5 J/sq m at 48 .5242050.210.35x-Ray012130001.51112252.3 hr ranges between 3 and 6 on a per dish basis. In these experiments with UV alone, the number of transformations per ii6.418No dish is more than 2-fold greater when the UV dose is increased MMS012280001.5233060.260.293.01222131.11.7MMS0122410.080.111.52320281.2 from 1.5 to 3 J/sq m. The enhancement ratio for MMS plus 3 J/sq m, however, is approximately one-half (absolute basis) and one-third (relative basis) that obtained with the MMS plus i.5J/sq m. 4.62.17.23.01116242.2 As with X-irradiation plus UV (4), a temporal relationship in 2.04.52.6a the enhancement of transformation is observed when HEC are Ratio of the resuIts ofthe double treatment to thoseof UV alone. treated with MMS prior to UV. The maximum enhancement occurs when UV exposure occurs 48 hr after MMS treatment The very bow transformation frequency observed with MMS (Table 2). At 72 hr, the enhancement obtained with the double treatment alone is not considered in calculating the enhance treatment approaches that which is associated with UV alone. ment.

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Table 2 LOG( MN) MMSenhancementoftransformationinducedby 1.5J/sq m:relationshipof 6 76 8 76 8 76 time interval between treatment 20 SeeTableIdetails.Interval forexperimental ratioPer 15 colony36(hr)Enhancement dishPer 10 48 3.8 4.1 U, 721.6 1.11.6 1.4 C-) Excision of Pyrimidine Dimers under Enhancement Con ct ditions. The rate of pynimidmnedirnerexcision is measured under enhancement and control conditions with the endonucle z @ ase-sensitive site assay. HEC that have been X-irnadiated (250 15 R), or incubatedwith MMS (i i sg/mb)for 1 hr in CM, or not treated are trypsinized and plated. The cells are labeled over 10 night with [14C]thymidmne,irradiatedwith UV (3.0 J/sq m) 48 or 72 hr postpbating, and further incubated for 2, 6, and 24 hr. 5 DNA is extracted and subjected to pyrimidmnedimen-specific endonuclease digestion. The size of the DNA digestion prod 0 ucts is analyzed by alkaline sucrose sedimentation. The rate of 1.0 OS 1.0 0.5 .@ os o loss of UV-induced endonuclease-sensitive sites in pretreated DISTANCE SEDIMENTED and control cells is the same. Independent of pretreatment Chart 2. Loss of pyrimidine dimer-specific endonuclease-sensitive sites. HEC are treated with MMS (11 @g/mlof medium), plated at 1O@cells/60-mm dish, conditions, approximately 25% of the pynimidmnedimensare and labeled overnight with [â€˜4C]thymidine.The cells are irradiated with UV (3.0 removed within 24 hr post-UV (Chart 2; Table 3). By 72 hr J/sq m) 48 hr post-MMS. DNA is extracted, subjected to pyrimidine dimer post-UV, no more than 35% of the pynimidine dimers are specific endonuclease, and sedimented through alkaline sucrose. A minimum of 2000 cpm are applied to each gradient, and fractions are counted for 2 or 5 mm. excised in any of the above cases (data not shown). Therefore, A to C,cellspretreatedwithMMSbeforeplatin9;Dto F, noMMS;0, 0 J/sq m the enhancement of transformation is not the result of a change UV; A, 3.0 J/sq m, no incubation; x , 3.0 J/sq m, incubated for 2 (A and D), 6 in the rate of pyrimidine dimer excision. (B andE), or 24 (CandF) hr. Postreplication Repair under Enhancement Conditions. In Table 3 this study, the rate of postreplication repair is functionally Excision of pyrimidine dimers from UV-irradiated HEC (3 J/sq m) defined as the time required for small pulse-labeled DNA to be See Chart 2 for protocol. chased into parental-sized DNA in cells irradiated with UV. This dimers excised (%)a at PretreatmentPyrimidine rate is measured in HEC, pretreated 48 or 72 hr with 250 A of incubationsTime following post-UV X-ray (conditionswhich enhance transformation),and com UVTreatment(hr)2 Before pared to the rate in HEC treated with UV (i 0 J/sq m) alone. hrNonei hr 6 hr 24 When the size of pulse-labeledDNA nascentstrandsis deter 23MMS485 8 mined by alkaline sucrose sedimentation, their nate of chase 26X-Ray487 6 into parental-length DNA is the same whether or not the cells 25X-Ray724 7 27a 6 had been treated with X-ray (48 or 72 hn)prior to UV (Chart 3). The size of pulse-labeled nascent strands is smaller in UV @ ESNI ES, irradiated than in non-UV-inradiated cells under all pretreatment % of pyrimidine dimer excised = x 100 conditions (compare Chart 3, A and B). Furthermore, for nas ESNI cent strandsto be chased into parental size in cells irradiated 5(2x108\ I_2xi08 @@ with or without UV, 1 and more than 3 hr. respectively,are E â€” ______required (Chart 3, B and F). Results similar to those in Chart 3 in which ES is (endonuclease-sensitive sites/ 108 daltons); NI is not incubated; I are obtained with cells X-irradiated 24 hr prior to UV. Therefore, is incubated; the enhancement of transformation by X-ray does not result >:@@ from a change in the rate of postreplication repair. Mw = The Amount of DNA Replication In HEC Irradiated with UV. The percentage of DNA replicated in a given period can be in which fi is the amount of radioactivity in the ?thfraction and mi is the molecular measured by determining the fraction of DNA that has a hybrid weight of DNA which sediments to the ith fraction. density (heavy-light) in cells grown with BndUrd in the medium. By 24 hr post-UV(3.0 J/sq m), more than 70% of the dimens irradiated cells is heavy-bightby 24 hr (Chart 4B). Evidence for still remain in the DNA (Table 3). The BndUrd density shift further rounds of replication cannot be observed because fully technique demonstrates that UV (i 0 J/sq m) slows the rate of BrdUnd-substituted DNA contains no label. At this UV dose, DNA replication(Chart 4) relativeto the controls.By 6 hr after there are approximately 15 dimens/replicon and 8 x i O@ UV, only 10% of the DNA in irradiated cells is replicated, dimens/genome, assuming that the replicon size and the av compared to 50% of the DNA in nonirnadiated cells (Chart 4A); erage amount of DNA per chromosome is the same as in the while by 24 hr post-UV, 70% of the DNA has replicated in Chinese hamster (i 0, i 1) whose dipboid number is one-half irradiated cells and virtually all of the prelabebed DNA in non that of the Syrian hamster. Therefore, DNA replication

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J. Doniger and J. A. DiPaolo LOG( MN)

8 76 8 76 8 76 20 HL LL HL LL U, ++ ++ 15 Iâ€” 20 A -B z I II 10 â€˜I U) L) z :4 :@ -J . II C-) a: Iâ€” ct II Is DO Iâ€”

@ z I- 10 . A 5 @ 15 Li 5- C.) 10 LU : :@ 0@ 5. 5 I I I 5

0 I S 5 I 5 _@_L@!@II I 5 1.0 O.S 1.0 0.5 1.0 0.5 a i.e o.e o.e 0.4 0.2 1.0 0.1 o.e 0.4 0.2 0 DISTANCE SEDIMENTED Chart 3. Postreplication repair. HEC secondaries are X-irradiated (250 R), DISTRNCE FROM TOP plated, and labeled overnight with [â€˜4Cjthymidine.Thecells are irradiated with Chart 4. Inhibition of DNA replication by IJV. HEC secondaries are plated and UV (10 J/sq m) 48 or 72 hr after plating and pulse-labeled for 15 mm with [3H]- labeledovernightwith[â€˜4C]thymidlne.AfterUVirradiation,thecellsareincubated thymidmne25 mm after UV. The pulse label is chased in the presence of cold In the presence of BrdUrd for 6 (A) or 24 (B) hr. DNA is extracted and subjected thymidine, the cells are lysed in 1 NNaOH on alkaline sucrose gradients, and the to isopyknic banding in CsCI. A minimumof 5000 cpm Is added in each gradient. released DNA is sedimented. There is a minimum of 2000 cpm of each label per Fractions are counted for at least 2 mm. LL, light-light DNA, density of 1.71; HL, gradient. Fractions are counted for 2 or 5 mm. A, no UV, no chase; B, no UV, 1- heavy-light DNA, density of i .76; 0, non-Uv-irradiated cells; A, cells irradiated hrchase;C, 10 J/sq m, 2-hrchase;D, 10 J/sq m, no chase;E, 10 J/sq m, 1-hr with 10 J/sq m. chase; F, 10 J/sq m, 3-hr chase. 0, parental [â€˜4C]DNAfromcells receiving no X ray; A, 3H pulse label, cells receiving no X-ray; x , 3H pulse label, cells X irradiated 48 hr prior to UV; K@,3Hpulse label, cells X-lrradiated 72 hr prior to UV. i and 2) affects neither the ability of cells to excise pynimidine dimers (Chart 2; Table 3) nonthe rate of postreplication repair procedes in the presence of bargenumbers of damages in the (Chart 3). The endonuclease-sensitive site assay used to meas template strands. Similar results have been observed with ure the removal of pynirnidinedimers from DNA is independent Chinese hamster cells (i 2). of the type of excision mechanism. The rate of excision has been measured up to 3 days post-UV which is 5 days post DISCUSSION plating. Any changes in excision repair occurring after this time would not be expected to affect the observed transformation MMS pretreatment of HEC, as does X-irradiation, results in frequency because those experiments are terminated on Day the enhancement of transformation induced by UV irradiation. 7. The rate of postreplication repair can be measured only MMS alone produces rare transformation while X-inradiation during the first several hr post-UV because, by 6 hr post-UV, does not (5). However, the bowbevelof MMS-induced transfor the size of pulse-labeled nascent strands increases to that of mation cannot account for the enhancement ratio obtained with those in noninradiated cells and postreplication repair is no the 2 agents. The interval between MMS and UV which gives longer needed. Neither excision of pyrimidine dimensnonpost maximum enhancement is 48 hr (Table 2), the same as for X replication repair can be responsible for the enhancement of irradiation plus UV. Moreover, the enhancement resulting from transformation frequency. X-irradiation is greaten than that attributable to MMS. This is Numerous studies have shown that excision and postrepli consistent with a previous study of X-ray and MMS enhance cation repair are the 2 most important mechanisms used by ment in which a variety of carcinogens belonging to diverse prokaryotic organisms to protect themselves from potentially chemical classes had been used (5). Finally, the transformation lethal damages in their DNA. Postreplication repair in prokar frequency is dependent on the UV dose in both enhanced and yotic organisms involves the filling of gaps in daughter strands nonenhanced conditions (Table i ). Similar results occur when which have been replicated on damaged templates (16). Most HEC are transformed by benzo(a)pyrene (7). Thus, transfor investigations of DNA repair in mammalian cells have, there mation is a random event induced by the carcinogen treatment. fore, been predicated on the assumption that these are also In the past, this laboratory has referred to UV-induced trans the 2 most important mechanisms for mammalian cell survival. formations of HEC as morphological transformations because These results, however, suggest that these 2 repair mecha animal studies had not been performed to confirm their tumor nisms have a minimal effect on HEC survival when the cells are igenicity. UV transformants are morphobogicabbysimilarto those irradiated with 3 J/sq m, a â€˜â€˜physiologicaldose'â€˜thatresults in induced by chemical carcinogen treatment and produce the only about 30% lethality. With respect to excision repair, at same types of tumors when injected s.c. into nude mice. beast70% of the DNA is replicated within 24 hr even though Pretreatment of HEC with agents which increase their sus only 25% of the pynimidine dimers are removed (Chart 4; Table ceptibibity to UV light-induced transformation (Chart i ; Tables 3). A dose of 3 J/sq m yields 2.4 x i O@pynimidinedimers/

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1980 American Association for Cancer Research. DNA Repair and Enhanced UV Transformation genome and 4.5 pynimidine dimers/neplicon. Since the exci Biochem. Biophys. Res. Commun., 36: 203-208, 1969. 3. DiPaolo, J. A., and Casto, B. C. Chemical carcinogenesis. In: R. C. Galbo sion and replication measurements are from cells irradiated (ed), Recent Advances in Cancer Research: Cell Biology, Molecular Biology, with 3 and 10 J/sq m, respectively, it is likely that even more and Tumor Virology, Vol. 1, pp. 17â€”47.Cleveland:CRC Press, 1977. than 70% of the DNA would have been replicated within the 4. DiPaolo, J. A., and Donovan, P. J. In vitro morphologic transformation of Syrian hamster cells by UV-irradiation is enhanced by X-irradiation and first 24 hr if HEC had received the lower dose. With respect to unaffected by chemical carcinogens. Int. J. Radiat. Biol. Relat. Stud. Phys. postreplication repair, the size of pulse-labeled nascent strands Chem. Med., 30: 41-53, 1976. in cells irradiated with 10 J/sq m recovers to that of nonirra 5. DiPaobo,J. A., Donovan, P. J., and Casto, B. C. Enhancement by alkylating agents of chemical carcinogen transformation of hamster cells in culture. diated cells by 6 hr post-UV, as has been observed in other Chem.-Biol. Interact., 9: 351â€”364,1974. mammalian cells , i 3). Moreover, by 6 hr post-UV, only i 0% 6. DiPaolo, J. A., Donovan, P. J., and Nelson, A. L. X-irradiation enhancement of the DNA has been replicated (Chart 4). Therefore, only a of transformation by benzo(a)pyrene in hamster embryo cells. Proc. NatI. Acad. Scm.U.S. A., 68: i734-i737, i97i. small percentage of the genome is subject to postreplication 7. DiPaobo,J.A., Donovan, P. J., and Popescu, N. C. Kinetics of Syrian hamster repair. HEC excise pynimidine dimers at one-fourth to one-fifth cells during X-irradiation enhancement of transformation in vitro by chemical carcinogens. Radiat. Res., 66: 310â€”325,1976. the rate of human cells (Table 3; Ref. 7) and require 3 times 8. Evans, C. H., and DiPaobo,J. A. Comparison of nude mice with the host longer to chase pulse-labeled nascent strands to parental size species for evaluation of the tumorigenicity of guinea pig and hamster cells (Chart 2). Although the repair rates for excision and postrepli transformed in vitro by chemical carcinogens. Cancer Res., 36: 128â€”i31, 1976. cation differ significantly,the survival curves for UV-tneated 9, Hanawalt, P. C., Freidberg, E. C., and Fox, C. F. (eds.). In: DNA repair human fibrobbasts and HEC are similar (Chart i ). Therefore, mechanisms. ICN-UCLA symposia on molecular and cellular biology, Vol. repair mechanismsother than excision of pynimidinedimers IX, pp. 1-800. New York: Academic Press, Inc., 1978. 10. Huberman, J. A., and Riggs, A. D. On the mechanism of DNA replication in and postreplication repair must be responsible for the survival mammalianchromosomes. J. Mob.Biol., 32: 327-341 , 1968. of HEC irradiated with UV. 11. Kornberg, A. In: DNA synthesis, p. 17. San Francisco: W. H. Freeman and Other repair systems, such as the recovery of nascent Co.,1974. 12. Meyn, R. E., Hewitt, R. R., Thomson, L. F., and Humphrey, R. M. Effects of strands to normal size, may be important to cell survival and to ultraviolet irradiation on the rate and sequence of DNA replication in syn the mechanism of transformation and its enhancement. chronized Chinese hamster cells. Biophys. J., 16: 517â€”525,1976. 13. Meyn, R. E., and Humphrey, R. M. Deoxyribonucleic acid synthesis in ultraviolet-light-irradiated Chinese hamster cells. Biophys. J., 11: 295â€”301, ACKNOWLEDGMENTS 1971. 14. Paterson, M. C. Use of purified lesion-recognizing enzymes in vivo. Adv. The authors wish to express their gratitude to Anthony DeMarinis for excellent Radiat. Biol., 7: 1â€”53,1978. technical assistance and to Dr. Richard J. Reynolds for his advice on endonucle 15. Riazuddin, S., and Grossman, L. Micrococcus luteus correndonucleases: I. ase-sensitive site assays. Resolution and purification of two endonucleases specific for DNA contain ing pyrimidmnedimers.J. Biol. Chem., 252: 6280â€”6286,1977. 16. Rupp, W. D., and Howard-Flanders, P. Discontinuities in the DNA synthe REFERENCES sized in an excision defective strain of Escherichia coli following ultraviolet irradiation. J. Mob.Biol., 31: 291â€”304,1968. 1. BuhI, 5. N., Setbow, R. B., and Regan, J. D. Recovery of the ability to 17. Rupp, W. D., Zipser, E., von Essen, C., Reno, D., Prosnitz, L., and Howard synthesize DNA in segments of normal size at long times after ultraviolet Flanders, P. Repair and reconstruction of chromosomal DNA after replica irradiationofhumancells.Biophys.J.,13: 1265â€”1275,1973. tion. In: Time and Dose Relationships in Radiation Biology as Applied to 2. Cleaver, J. E., and Thomas, G. H. Single strand interruptions in DNA and the Radiotherapy, Publication BN1 50203(C-57), pp. 1â€”13.Brookhaven,N. V.: effects of caffeine in Chinese hamster cells irradiated with ultraviolet light. Brookhaven National Laboratory, 1970.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1980 American Association for Cancer Research. Excision and Postreplication DNA Repair Capacities, Enhanced Transformation, and Survival of Syrian Hamster Embryo Cells Irradiated by Ultraviolet Light

Jay Doniger and Joseph A. DiPaolo

Cancer Res 1980;40:582-587.

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