Analyses of differential sensitivities of synchronized HeLa S3 cells to radiations and chemical carcinogens during the cell cycle

219

Mutation Research, 71 (1980) 219--231

© Elsevier/North-Holland Biomedical Press

ANALYSES OF D I F F E R E N T I A L SENSITIVITIES OF S Y N C H R O N I Z E D HeLa $3 CELLS TO RADIATIONS AND CHEMICAL CARCINOGENS D U R I N G THE CELL CYCLE PART V. RADIATION- AND CHEMICAL CARCINOGEN-INDUCED MUTAGENESIS

MASAMI WATANABE and MASAKATSU HORIKAWA Division of Radiation Biology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa 920 (Japan)

(Received 26 June 1979) (Revision received 16 January 1980) (Accepted 28 January 1980)

Summary 8-Azaguanine(8AG)-resistant mutations induced b y X-rays, ultraviolet radiation (UV) and a chemical carcinogen, 4-hydroxyaminoquinoline 1-oxide (4-HAQO) were examined during the cell cycle of synchronized HeLa $3 cells. Mutants induced by 400 R of X-rays occurred in a higher frequency in the X-ray-sensitive G1--S b o u n d a r y phase than in the X-ray-resistant GI, S and early G2 phases. 8AG-resistant mutants induced by treatment with 10 -s M 4-HAQO for 20 rain appeared in a higher frequency in the early to middle S phases than in the other phases. In the case of UV, however, we found no significant difference in the induced mutation frequencies during the cell cycle, because the mutation frequencies induced b y the UV doses (0--20 J / m 2) used were t o o low for detection of the difference. These results suggest that there is a close correlation between the critical damage induced in DNA molecule(s) at the DNAsynthetic phase in the cell cycle and mutagenesis, because mitotic cells have a low mutability in spite of their high radio~sensitivity.

In cells that have sustained severe damage in their DNA, their abilities to repair the damage, their survival rate and the mutagenesis cannot be considered separately. The fate of these cells m a y depend partly upon the nature and extent of the primary damage induced in their DNA and partly upon their ability to repair the damage. And some of the survivors m a y have mutational changes because either an irreparable DNA lesion has generated a replication error or an error-prone repair system has changed the base sequence in the

220 course of restoration to a viable DNA structure [6,9--11,14]. The repair mechanisms involved in error-free, error-prone, and/or lethal functions are well documented for microbial systems but are not fully understood for mammalian cells. Differential sensitivities to radiations and chemical carcinogens during the cell cycle of cultured mammalian cells provide a potentially useful system for studies on the relationships among DNA damage and its repair, survival of cells and mutagenesis. In previous studies [21--23], by using a HeLa $3 population synchronized by the combined method of colcemid and harvesting techniques [20], we examined DNA damage induced by X-rays, UV, 4-nitroquinoline 1-oxide (4-NQO) and its derivative 4-HAQO, and their repair during the cell cycle, and found that the cyclic fluctuations of survivals to UV and chemical carcinogens (4-NQO and 4-HAQO) may be due to differences in the amount of UV-induced thymine dimers in DNA and of 4-NQO and 4-HAQO adducts to DNA, resp. For X-rays, however, we could not find any significant differences in the extent of single-strand breaks induced in cellular DNA and in the extent of their rejoining throughout the cell cycle. The cyclic-variation curve of X-ray survivals was rather similar to t h a t o f the content of non-protein sulphydryls during the cell cycle except the mitotic phase. In addition, we examined the correlation between sensitivity of the cells to X-rays and X-ray-induced mutation frequency through the cell cycle [23]. X-Ray-induced mutants resistant to 8AG occurred in a higher frequency in the X-ray-sensitive GI--S boundary phase than in the X-ray-resistant G1, S and early G2 phases. These results indicate a close correlation between the extent of lethal radiation damage to the cells and their mutability. In these experiments, however, the conditions for the selection of 8AG-resistant mutants were not stringent enough, so that unstable mutants were isolated. In the investigation reported here we have extended the earlier study by using X-rays, UV and a chemical carcinogen 4-HAQO as the agents, and a modified selection procedure for 8AG-resistant mutants, to find out whether cultured mammalian cells exhibit differential sensitivities to mutation induction by various physico-chemical agents as a function of position in the cell cycle. The results obtained suggest that there is a close correlation between critical DNA damage sustained in the DNA synthetic phase of HeLa $3 cells and their mutagenesis. Materials and methods Cells and cell synchronization HeLa $3 cells were grown in a medium containing 90% Eagle's MEM and 10% bovine serum. For mutagenesis experiments, the same batch of serum was used to normalize the influence of serum components on mutant recovery among different experiments. For obtaining a large highly purified synchronized population from the HeLa $3 cells, mitotic cells were collected by the combined method of colcemid and harvesting techniques described previously [20]. Determination o f sensitivity o f the cells to radiations and a chemical carcinogen The sensitivity to X-rays, UV and 4-HAQO (Tosin Chemical Industry,

221 T o k y o ) of HeLa $3 cells at different phases of t h e cell cycle was determined by the colony-forming method. Mitotic cells were collected, inoculated onto 90-mm glass petri dishes containing 10 ml of the culture medium and incubated at 37°C in a humidified 5% CO2 incubator. After various periods of incubation, aliquots of 6 × l 0 s cells were suspended in a short pyrex test-tube (inside diameter 10 mm, length 100 mm) containing 4 ml of fresh medium and irradiated with various doses of 200 kV X-rays, at a dose rate in air of 75 R/min at room temperature. For UV irradiation, 6 × l 0 s cells were suspended in 4 ml of Eagle's MEM free of phenol red, spread over the surface of a 90-mm glass petri dish, and then irradiated with various doses of UV, at 1 j/m2/sec at the surface of a dish. For 4-HAQO treatment, 6 X l 0 s cells were suspended in 4 ml of the culture medium containing various concentrations of 4-HAQO, and incubated at 37°C for 20 min. The cells were rinsed and suspended in fresh medium. Aliquots of 250 unirradiated (untreated) or irradiated (treated) cells were plated on 60-mm glass petri dishes containing 5 ml of the culture medium, and the dishes were incubated in a CO2 incubator for 14 days. The colonies in each dish were fixed and stained, and the colonies containing more than 50 cells were counted.

Mutagenesis experiments Before the mutagenesis experiments, we determined a concentration of 8AG for selecting 8AG-resistant mutants from HeLa S3 cells in the following way. Samples of 10 s HeLa $3 cells were inoculated into 90-mm glass petri dishes containing 10 ml of the medium with various concentrations of 8AG (Sigma Chemical Co., St. Louis). The dishes were placed in a CO2 incubator for 16 days, with changes of newly prepared SAG medium every 4 days. The colonies in each dish were fixed and stained, and the colonies containing more than 50 cells were counted. Fig. 1 shows the results. Concentrations of 8AG higher than 0.4 pg/ml were evidently suitable for the selection of mutants. Therefore, mutants were selected with 8AG at 0.5 gg/ml in the following experiments. Mutagenesis experiments were performed in the following manner: 6 × l 0 s cells at different phases of their cycle were irradiated (treated) with X-rays, UV and 4-HAQO as described above. Then from each unirradiated (untreated) or irradiated (treated) sample, 5 × l 0 s cells were transferred to a 100-ml square culture bottle containing 5 ml of fresh medium, and the bottles were incubated at 37°C for the mutation expression for various times. During the expression periods the cells were subcultured every 3 days to maintain exponential growth. After incubation, l 0 s cells from each bottle were distributed into each of 10 (90-mm) glass petri dishes containing 10 ml of a selection medium with 8AG at 0.5 pg/ml, and the dishes were placed in a CO2 incubator for 16 days with changes of newly prepared 8AG medium every 4 days. The colonies in each dish were fixed and stained, and those containing more than 50 cells were counted. The number of 8AG-resistant m u t a n t colonies induced by X-rays, UV and 4-HAQO was corrected for the decrease in survival (determined by the colonyforming m e t h o d described above) of cells incubated for various times after irradiation (treatment) with these agents; i.e. the mutation frequencies were expressed as the number of 8AG-resistant colonies per l 0 s surviving colonies.

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Concentration of BAG (pg I ml ) Fig. 1. C o l o n y - f o r m i n g ability o f 10 5 HeLa S3 ceils euJ.tured in each dish containing 10 ml o f the m e d i u m with various concentrations of 8AG for 16 days. The dishes were replenished with newly prepared 8AG m e d i u m e v e r y 4 days. The bars indicate s t a n d a r d e r r o r s o f t h e m e a n s o f 5 i n d e p e n d e n t d e t e r m i n a t i o n s .

For determining the recovery efficiency of 8AG-resistant m u t a n t cells under these selective conditions, a mixture of 250 8AG-resistant mutant cells and l 0 s parental HeLa $3 cells was inoculated into 90-mm petri dishes c o n t a i n i n g 1 0 ml of the medium with 8AG at 0.5 pg/ml, and the dishes were placed in a CO2 incubator for 16 days with changes of newly prepared 8AG medium every 4 days. The colonies in each dish were fixed and stained, and those containing more than 50 cells were counted.

Analysis of the properties of 8A G-resistant mutant clones Mutant colonies that appeared in selection plates containing 8AG were separately isolated by micro-pipettes, grown and their properties were analyzed. For determining the sensitivity to SAG of cells, aliquots of 250 cells were inoculated into 60-mm glass petri dishes containing 5 ml of medium with 8AG at 5 or 30 pg/ml, and the dishes were placed in a CO2 incubator for 14 days without medium change. The colonies in each dish were fixed, stained, and those containing more than 50 cells were counted. For testing the incorporation of radioactive hypoxanthine into the cells, a b o u t 3 × l 0 s cells were inoculated onto cover-slips in each 60-ram glass petri dish. After 48 h of incubation, [3H]-8-hypoxanthine (1.8 mCi/mmole; The

223 Radiochemical Centre, Amersham) was added to a final concentration of 1 pCi/ ml. After another 24 h of incubation, the cover-slips were removed, washed with ice-cold phosphate-buffered saline, and treated twice with 5% cold perchloric acid for 10 min each time. The cover-slips were fixed, dipped in Sakura NR-M2 emulsion, exposed for 14 Clays, and stained with Giemsa solution. Hypoxanthine--guaninephosphoribosyl-transferase (HGPRT, EC 2.4.2.8) activity of the cells was measured by the conversion of [~4C]-8-hypoxanthine (41.6 mCi/mmole; New England Nuclear, Boston, MA) into [14C]-IMP in cell extracts, according to the m e t h o d described in the previous paper [7]. Protein content was determined by the m e t h o d of Lowry et al. [13]. Results

Mutation expression time Because the length of the mutation expression time markedly affects the maximal expression of m u t a n t phenotypes induced by radiations and chemicals, the optimal expression times for mutations induced by X-rays, UV and 4-HAQO were studied. Figs. 2a and 2b show the expression--time curves for the mutations to SAG resistance of asynchronous HeLa $3 cells after X-irradiation and 4-HAQO treatment. With X-rays (Fig. 2a), the mutation frequency increased with time and reached a maximum at 72 h with all X-ray doses. The expression time was independent of X-ray dose. Beyond this time the mutation frequency detected tended to decrease except at lower doses, 100 and 200 R. In contrast with this, the mutation frequency induced by 20-min treatment with 4-HAQO reached a maximum at 48--72 h of expression with all 4-HAQO concentrations (Fig. 2b). The expression time was again independent of

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224 4-HAQO concentration, and the mutation frequency decreased after 72 h of expression. The possibility that mutations induced at different phases during the cell cycle required different lengths of time for optimal expression was tested by comparing the X-ray- and 4-HAQO-induced mutation frequencies at various expression times. Figs. 3a and 3b show the expression time curves for SAGresistant mutations induced in HeLa $3 cells at different phases of the cell cycle after 400 R of X-irradiation and 20-min treatment with 10 -s M 4-HAQO. Similarly, the mutation frequency induced by X-rays of 400 R reached a maximum at 72 h of expression time at all phases of the cell cycle, although there was a cell-cycle-dependent variability in mutation induction by X-rays (Fig. 3a). The mutation frequency decreased after 72 h. The 4-HAQO-induced mutation frequency at all phases of the cell cycle also reached a maximum at 48-72 h of expression time, although there was a cell-cycle-dependent variability in the mutation frequency induced by 20-min treatment with 10 -s M 4-HAQO (Fig. 3b). Here, too, the mutation frequency decreased after 72 h of expression. We could not determine the optimal expression time for the mutations induced by UV, because the mutation frequencies induced in either asynchronous and synchronous HeLa $3 cells by UV irradiation with 0--20 J/m 2 were too low even when up to 360 h was allowed as the expression time. Based on these results, 72 h as the mutation expression time following treatment with radiation and chemical carcinogen was applied for the following mutagenesis experiments.

Cell-cycle response in radiation-and chemical carcinogen-induced mutagenesis The cyclic fluctuation curves of sensitivity to X-irradiation with 400 R through the cell cycle of HeLa $3 cells and of the induced mutation frequency

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Fig. 3. T h e e f f e c t o f t h e m u t a t i o n e x p r e s s i o n t i m e o n t h e f r e q u e n c i e s o f S A G - r e s i s t a n t m u t a t i o n s i n d u c e d in s y n c h r o n o u s H e L a SS cells at d i f f e r e n t p h a s e s o f t h e cell c y c l e a f t e r i r r a d i a t i o n w i t h 4 0 0 R o f X - r a y s (a) a n d t r e a t m e n t w i t h 1 0 -5 M 4 - H A Q O f o r 2 0 r a i n (b). E a c h p o i n t s h o w s t h e m e a n o f 3 i n d e p e n d e n t e x p e r i m e n t s , o, M (0 h ) ; e, G 1 (4 h ) ; ~, G I - - S b o u n d a r y (8 h ) ; m, e a r l y S ( 1 0 h ) ; z~ m i d d l e S ( 1 2 h ) ; A l a t e S--G2 (16 h).

225

after X-irradiation are shown in Fig. 4. 8AG-resistant mutants were induced in a higher frequency in the X.ray-sensitive G1--S boundary phase than in the X-ray-resistant GI, S and early G2 phases, as already seen in Fig. 3a. However, the cells in the mitotic phase did not show this correlation, as already reported [23], because they have a lower mutability in spite of their high radiosensitivity. Fig. 5 shows the changes in 4-HAQO (10 -s M for 20 min) survivals and the mutation frequencies of 8AG-resistant cells induced by 20-min treatment with 10 -s M 4-HAQO during the cell cycle of synchronous HeLa $3 cells. As shown, 8AG-resistant mutations were induced in a higher frequency in the most 4-HAQO-sensitive early to middle S phases than in the other phases. (See also Fig. 3b.) The mutation frequency induced in the mitotic phase was again very low in spite of the high 4-HAQO sensitivity. For UV, we found no significant difference in the induced mutation frequencies during the cell cycle though the survival rate showed a marked fluctuation, because the mutation frequencies of SAG-resistant cells induced by UV at 10 J/m 2 were too low throughout the cell cycle (Fig. 6). No significant increase in the induced mutation frequency was obtained even after expression periods up to 360 h after irradiation with 0--20 J/m 2. Properties o f 8 A G-resistan t m u tan ts

15 mutant colonies (3 spontaneous and 8 X-ray- and 4 4-HAQO-induced) that appeared in selection plates containing 8AG were isolated at random and

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TABLE 1

32 33 35 36

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4-HAQO 4-HAQO 4-HAQO 4-HAQO

rain rain rain min

(G 1) (S) (S) (S)

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PROPERTIES OF THE ORIGINAL HeLa $3 CELLS AND FIFTEEN 8AG-RESISTANT MUTANT CLONES

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228 TABLE 2 EFFICIENCY OF RECOVERY OF SAG-RESISTANT MUTANTS FROM ARTIFICIAL MIXTURES WITH PARENTAL HeLa $3 CELLS AT CELL PLATING DENSITIES OF 10 s CELLS PER 90-rnm GLASS PETRI DISH Clone

Cloning e f f i c i e n c y (%) in n o r m a l m e d i u m a

C l o n i n g e f f i c i e n c y (%) i n selective 8 A G medium b

Recovery of mutants in selective 8 A G m e d i u m (%)

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grown in 8AG-free medium, and their properties were analyzed 2 months after their isolation. As shown in Table 1, the colony-forming abilities of the 15 SAG-resistant clones in medium with 8AG at 5 or 30 pg/ml were very high compared with that of the original HeLa $3 cells. On the other hand, H G P R T activities of the 9 8AG-resistant clones tested were very low, that is, 1.5--3.9% of that of the original HeLa $3 cells. This was confirmed by the autoradiographic assay of cellular uptake of [ 3H]-8-hypoxanthine. Extensive incorporation of [ 3H]-8-hypoxanthine into nucleic acid was observed in HeLa $3 cells, whereas the 11 mutant clones examined showed no significant incorporation. These data suggest that only 8AG-resistant mutants deficient in H G P R T were recovered in the selection procedure. Their properties were stable even after cultivation during 2 months in the absence of the selective agent. In addition, there were no differences in p h e n o t y p e of 8AG-resistant mutants induced by different agents at different phases in the cell cycle. On the other hand, reconstruction experiments with artificial mixtures of parental HeLa $3 cells and mutant cells showed that, at a total cell plating density of 105 cells per 90-mm glass petri dish, the cloning efficiency of 8AGresistant mutants in 8AG medium was ~ 1 0 0 % of that observed for the same mutants cloned in normal medium (Table 2}. Discussion We wanted to know whether the capacity of various physico-chemical agents to induce mutations varies with position in the cell cycle upon treatment. In the experiments with asynchronous HeLa $3 populations, as well as synchronous populations obtained b y colcemid and harvesting techniques [20], we found that the optimal phenotypic expression times for SAG resistance induced by doses (or concentrations) less than 800 R of X-rays and 2 × 10 -s M 4-HAQO were 72 and 48--72 h, resp., and were essentially independent of the position in the cell cycle. There were no differences in p h e n o t y p e of SAGresistant mutants induced by these agents at different phases in the cell cycle. All isolated mutants were resistant to 8AG and had less than 4% of the H G P R T activity of the wild-type cells even 2 months after their isolation. In additior~,

229 reconstruction experiments with artificial mixtures of parental HeLa $3 cells and m u t a n t cells revealed that our mutagenesis experiments are quantitatively accurate. The mutation of resistance to 8AG is based on a recessive mutation of the H G P R T gene or genes located in the X-chromosome, leading to a loss or reduction of the H G P R T enzymic activity [4,5,16]. Therefore, we analyzed the k a r y o t y p e of HeLa $3 cells by the Giemsa banding method [17], and found that HeLa 83 cells are aneuploid, extend from 58 to 72 chromosomes, but cann o t be thoroughly characterized in terms of chromosomes. On the other hand, the mutation frequencies induced in either asynchronous or synchronous HeLa $3 cells by doses of UV less than 20 J / m 2 were t o o low even when up to 360 h was allowed as expression time. We do n o t yet have an explanation of this although it is known that UV and a chemical carcinogen 4-nitroquinoline 1-oxide (or its derivative 4-HAQO) have similar biological and mutagenic activities [12,18,19]. Some papers suggest the presence of differences in their activities [8,22]. If there are differences in the biological and mutagenic activities between UV and 4-HAQO, the reduction in the number of UV-induced mutants might be explained by the additional cytotoxicity of 8AG to survivors after UV irradiation. We shall discuss this later. As regards radiation-induced mutation during the cell cycle, Arlett and Potter [1] first observed that G2 cells were more mutable by 7-irradiation than were G1 or S cells, as determined by the appearance of mutants resistant to 8AG in synchronized cells of Chinese hamster V79-4. Carver et al. [2] reported that both G1 and S of synchronous Chinese hamster ovary cells were practically identical in sensitivity with X-ray-induced 8AG-resistant mutations. However, in our experiment, we found that the mutation frequencies induced by 400 R of X-rays were higher in the X-ray-sensitive G1--S boundary phase than in the X-ray-resistant G1, S and G2 phases. This result agrees with that obtained by the selection procedure without change of 8AG medium, as we reported previously [23]. Similarly, 8AG-resistant mutations induced b y treatment with 10 -s M 4-HAQO for 20 min were high in the most 4-HAQO-sensitive early to middle S phases than in the other phases. These results suggest that there is a close correlation between the critical damage induced in DNA molecule(s) at the DNA synthetic phase in the cell cycle and mutagenesis, because mitotic cells have a low mutability in spite of their high radio-sensitivity. On the other hand, we found no significant difference in the mutation frequencies induced by UV during the cell cycle, because the frequency of mutations in 8AG-resistant cells induced by UV at 10 J / m 2 was very low throughout the cell cycle, as mentioned above. However, in a preliminary experiment using the selection procedure w i t h o u t change of 8AG medium, we found that unstable quasi-8AG-resistant mutants, which had a b o u t 20--40% of the H G P R T activity of the wild-type cells, appeared in a higher frequency in the S phase than in the G~ and G2 phases after irradiation at 10 J / m 2. Recently, Riddle and Hsie [15] showed that mutation induction of 6-thioguanine resistance by UV occurred maximally in early S phase of Chinese hamster ovary cells. All these findings suggest that there is a close correlation between cellular DNA damage induced by UV at the DNA synthetic phase and mutagenesis. The findings in our preliminary experiment also suggest that the low mutability in UV-irradi-

230 ated HeLa $3 cells may be due to the additional cyto-toxicity of 8AG to survivors after UV irradiation. There are several possible explanations for higher mutability at the S phase, if DNA is the cellular target for mutations induced by radiations and chemicals. For instance, it is possible that, during the replication time, DNA is susceptible to the initiation of mutational change. Cerd~-Olmedo et al. [3] reported that the majority of the mutations induced by nitrosoguanidine in E s c h e r i c h i a coli are located in the replication region of the chromosome. In the present experiments, however, we found that maximal mutation induction to SAG resistance by X-rays and 4-HAQO occurred in different periods of the S phase, that is, the G,--S boundary phase was sensitive to X-rays and 4-HAQO in the early to middle S phases. Our preliminary experiment showed that unstable quasi-8AGresistant mutants appeared in a higher frequency in the most UV-sensitive middle S phase than in the other periods of the S phase after UV irradiation. These findings indicate that the extent of DNA damage (or the extent of lethal damage of the cells) induced by each mutagen during different periods of the S phase is at least partly related to their mutability. Further, it is conceivable that potentially mutational changes occurring in DNA in S phase, or to a lesser extent in G1 and G: phases, are more likely to become fixed in the genome by replication before they can be repaired. We need more detailed study to obtain a definite conclusion about this. Acknowledgement We thank Professor T. Sugahara for his warm encouragement and reading of the manuscript. This study was supported in part by research funds from the Ministry of Education, Science and Culture in Japan. References 1 A r l e t t , C . F . , a n d J . P o t t e r , M u t a t i o n t o 8 - a z a g u a n i n e r e s i s t a n c e i n d u c e d b y 7 - r a d i a t i o n in a C h i n e s e h a m s t e r cell line, M u t a t i o n R e s . , 1 3 ( 1 9 7 1 ) 5 9 - - 6 5 . 2 C a r v e r , J . H . , W.C. D e w e y a n d L . E . H o p w o o d , X - R a y - i n d u c e d m u t a n t s r e s i s t a n t t o 8 - a z a g u a n i n e , II. Cell c y c l e d o s e r e s p o n s e , M u t a t i o n Res., 3 4 ( 1 9 7 6 ) 4 6 5 - - 4 8 0 . 3 C e r d ~ - O l m e d o , E., P.C. H a n a w a l t a n d N. G u e r o l a , M u t a g e n e s i s o f t h e r e p l i c a t i o n p o i n t b y n i t r o s o g u a n i d i n e : m a p a n d p a t t e r n o f r e p l i c a t i o n o f t h e Escherichia coli c h r o m o s o m e , J. Mol. Biol., 3 3 (1968) 705--719. 4 F u j i m o t o , W . Y . , J . H . S u b a k - S h a r p e a n d J . H . S e e g m i l l e r , H G - P R T d e f i c i e n c y ; C l i n i c a l a g e n t s selective f o r m u t a n t o r n o r m a l c u l t u r e d f i b r o b l a s t s in m i x e d a n d h e t e r o z y g o u s c u l t u r e s , P r o c . N a t l . A c a d . Sci. (U.S.A.), 68 (1971) 1516--1519. 5 G o l d s t e i n , J . L . , J . F . M a r k s a n d S.M. G a r t l c r , E x p r e s s i o n o f t w o X - l i n k e d g e n e s in h u m a n h a i r follicles o f d o u b l e h e t e r o z y g o t e s , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 6 8 ( 1 9 7 1 ) 1 4 2 5 - - - 1 4 2 7 . 6 Higgins, N.P., K. K a t o a n d B. S t r a u s s , A m o d e l f o r r e p l i c a t i o n r e p a i r in m a m m a l i a n cells, J. M o l . Biol., 101 (1976) 417--425. 7 H o r i k a w a , M., F. S u z u k i a n d S. B a n , S t u d i e s o n s o m a t i c cell m u t a t i o n s , II. R a d i a t i o n - i n d u c t i o n o f 8 - a z a g u a n i n e - r e s i s t a n t a n d -sensitive m u t a n t s i n C h i n e s e h a m s t e r Hal cells i n v i t r o , J p n . J . G e n c t . , 51 (1976) 253--264. 8 I k e n a g a , M., Y. Ishii, M. T a d a , T. K a k u n a g a , H. T a k e b e a n d S. K o n d o , E x c i s i o n o r e p a i r o f 4 - n i t r o q u l n o l i n e - l - o x i d e d a m a g e r e s p o n s i b l e f o r killing, m u t a t i o n , a n d c a n c e r , in: P.C. H a n a w a l t a n d R . B . S e t i o w ( E d s . ) , M o l e c u l a r M e c h a n i s m s f o r R e p a i r o f D N A , P a r t B, P l e n u m , N e w Y o r k , 1 9 7 5 , p p . 7 6 3 - 771. 9 I k e n a g a , M., H . T a k e b e a n d Y. Ishii, E x c i s i o n r e p a i r o f D N A b a s e d a m a g e in h u m a n cells t r e a t e d w i t h t h e c h e m i c a l c a r c i n o g e n 4 - n i t r n q u l n o l i n e 1 - o x i d e , M u t a t i o n Res., 4 3 ( 1 9 7 7 ) 4 1 5 - - 4 2 7 . 1 0 K a k u n a g a , T., C a f f e i n e i n h i b i t s cell t r a n s f o r m a t i o n b y 4 - n i t x o q u i n o l i n e 1 - o x i d e , N a t u r e ( L o n d o n ) , 258 (1975) 248--250.

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