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

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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 (5—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 37°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 582 CANCER RESEARCH VOL. 40 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 4°.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 ‘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 ‘4C-labeledDNAextract is stained colonies. Colonies are isolated by the ring technique incubated for 30 mm at 37°.Thereaction is stopped by cooling and are placed in a 60-mm dish. At confluency, the cells are to 0°,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'°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).
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