JAPAN. J. GENETICS Vol. 54, No. 6: 415-426 (1979) X-RAY-INDUCED CELL KILLING AND MUTATIONS IN CULTURED HUMAN CELL LINES (XERODERMA PIGMENTOSUM CELLS AND HELA 53 CELLS) YOSHIHIRO MURAII~, SACHIKO TATSUKAWAZ~ AND MASAKATSU HORIKAWA Division of Radiation Biology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa 920 Received August 3, 1979 This paper deals with the development of assay systems for radiation-induced mutations to 8-azaguanine (8AG) resistance, using XP2OS cells which belong to complementation group A of xeroderma pigmentosum (XP) and the familiar human cancer HeLa S3 cells. The results indicated that these mutation assay systems to purine analogue resistance using XP2OS and HeLa S3 cells are useful for determining the mutation frequency induced by X-rays and that the data obtained by using these assay systems can be used for predicting the mutation frequency expected after exposure of man to low doses of ionizing radiation. In addition, a plot of induced mutation frequency against log surviving fraction yielded an approximately linear relationship for five cell types including human diploid cells, Chinese hamster cells and mouse lymphoma cells, which have already been studied by other workers. This relationship seems to suggest that mammalian cells generally have the same fixed probability of mutation induction relative to the extent of damage caused by ionizing radiation. INTRODUCTION During the last decade, several mammalian cell systems suitable for the detection and quantitative evaluation of radiation-induced mutational events have been described by many workers (Kao and Puck 1969; Bridges and Huckle 1970; Arlett and Potter 1971; Chu 1971; Albertini and DeMars 1973; Knaap and Simons 1975; Cox and Masson 1976; Zeeland and Simons 1976; Thacker et al. 1977). Among these Chinese hamster cell systems hold a special place because of a number of characteristics, such as relatively short generation time, high plating efficiency and small number of chromosomes 1) Present address: Department of Pathology, Toyama Medical and Pharmaceutical University, School of Medicine, 2630 Sugitani, Toyama 930-01. 2) Present address: Research and Development Division, Olympus Optical Co. Ltd., Hachioji 192.
416 Y. MURAI, S. TATSUKAWA AND M. HORIKAWA of the cells and stable maintenance of their properties in culture. In the previous studies (Ban et al. 1976; Horikawa et al. 1976), we have also established a sensitive forward mutation system, for detecting the mutations induced by radiations and chemical carcinogens or mutagens, using prototrophic CH-hai Cl 23 cells which were isolated from an original Chinese hamster hai cell line. However, it was found in our recent experiments (Suzuki et al. 1977, 1979) that this forward mutation system using prototrophic CH-hai Cl 23 cells is not sensitive enough to detect the mutations induced by low doses (especially, less than 200R) of X-rays, because Chinese hamster hai cells have the high repair ability for cell killing and mutational damage induced by low doses of X-rays. These results seem to indicate that it is necessary to use cell lines which have a low repair ability for the sublethal damage (or have an unshouldered exponential survival curve) such as human diploid fibroblasts (Cox and Masson 1976) for maximal detection of the mutations induced by low doses of ionizing radiation. In the present communication we report the development of the assay systems which permit quantitation of forward mutations to 8AG-resistance induced by low doses (which were corresponding to shoulder region on the dose-survival curve) of X- rays, using a human cell line derived from an XP patient and a familiar human cell line, HeLa S3 cells, and the comparison of the results obtained by these assay systems with those by the other mutation systems (Knaap and Simons 1975; Cox and Masson 1976; Thacker et al. 1977). These approaches may also be valuable for the further understanding of molecular mechanisms in mammalian cells to repair the damage which are related to cell killing and mutational change induced by ionizing radiation and nonionizing radiation. MATERIALS AND METHODS Cells and media XP2OS fibroblast cells which were kindly supplied by Dr. H. Takebe and HeLa S3 cells which were routinely maintained in our laboratory were used in the present study. XP2OS cells were originally derived from a 6-year-old female XP patient and transformed with SV 40 virus in culture (Takebe et al. 1974). This XP cell line belongs to complementation group A (Ikenaga et al. 1977; Takebe et al. 1977). After arrival in our laboratory, these cells were grown in a medium composed of 75% Eagle's MEM plus 10% TC-199 medium supplemented with 15% calf serum. The average doubling time and plating efficiency of the cells in this medium at 37 C were 30 h and 23%. HeLa S3 cells used as a control were routinely grown in Eagle's MEM supplemented with 10% bovine serum in our laboratory. The average doubling time and plating efficiency of the cells in this medium were 20 h and 70%. Mutagenesis experiments One ml suspensions containing 106 XP2OS or HeLa S3 cells were suspended in short test tubes, and irradiated with various doses of X-rays, at a dose rate in air of 75R/ min. Then, each of unirradiated or irradiated cell suspension was transferred to a 200 ml square culture bottle containing 9 ml of normal culture medium, and the bottles
X-RAY-INDUCED MUTATIONS IN CULTURED HUMAN CELL LINES 417 were incubated at 37 C for the mutation expression for various lengths of time. In this case, 10 ml of each fresh culture medium were added to each culture bottle at 72 h of incubation, to prevent the selection of the cells by subculture. After the incubation, 105 cells from each bottle were distributed into 90-mm petri dishes which contained 10 ml of each selection medium containing 8AG (5,ug/ml for XP20S cells and 0.5 ig/ml for HeLa S3 cells), and incubated at 37 C in a C02 incubator for 17 days for XP20S cells and 16 days for HeLa S3 cells. During these incubation periods, each selection medium was changed every fourth day. Then, the colonies in each dish were fixed, stained, and the colonies containing more than 50 cells were counted. Cell survival experiments Immediately after irradiation of the cells with various doses of X-rays and after incubation for various lengths of time following irradiation as described above, each 1 ml cell suspension containing 1,200 of XP20S cells or 200 of HeLa S3 cells was distributed in a 60-mm glass petri dish with an additional 4 ml of each normal medium. The dishes were incubated for 17 days for XP20S cells and 14 days for HeLa S3 cells, and the colonies were counted. Determination of induced mutation frequency The number of 8AG-resistant mutant colonies induced by X-irradiation was corrected for the decrease in survival of cells incubated for various lengths of time following irradiation with X-rays, i.e., the mutation frequencies were expressed as the number of 8AG-resistant colonies per 105 surviving colonies. The induced mutation frequencies were obtained by subtracting the control mutation frequency from the frequency in the irradiated population. The control mutation frequencies varied from 0.31.8 x 10-5 for both XP20S and HeLa S3 cells. Analysis of the properties of 8AG-resistant mutants induced in HeLa S3 cells For determining the sensitivity to 8AG of the cells, aliquots of 250 8AG-resistant mutant and original HeLa S3 cells were inoculated into 60-mm glass petri dishes which contained 5 ml of a medium containing 0.5 tcg/ml of 8AG and incubated in a C02 incubator for 16 days. During this incubation period, medium was changed every fourth day. Then, the colonies in each dish were fixed, stained, and counted. For testing the incorporation of radioactive hypoxanthine into the cells, about 3 X 105 mutant and original HeLa S3 cells were inoculated onto coverslips in each of 60-mm glass petri dish. After 48 h of incubation, [3H] 8-hypoxanthine (1 mci/mmole; The Radiochemical Centre, Amersham) was added to a final concentration of 1 pci/ml. After another 24 h of incubation, coverslips were removed, washed with cold phosphate buffered saline, and treated three times with 1 % cold perchloric acid for 10 min each. Then, the coverslips were fixed, dipped in Sakura NR-M2 emulsion, exposed for 15 days, and stained with Giemsa solution. Hypoxanthine-guanine-phosphoribosyl-transf erase (HGPRT, EC 2.4.2.8) activity of both mutant and original HeLa S3 cells was measured by the conversion of [14C] 8- hypoxanthine (41.6 mci/mmole; New England Nuclear, Boston, Mass.) into [14C] IMP
418 Y. MURAI, S. TATSUKAWA AND M. HORIKAW A in cell extracts, according to the method described in the previous paper (Horikawa et al. 1976). Protein content was determined by the method of Lowry et al. (1951). RESULTS Sensitivity to X-rays of XPZOS and HeLa S3 cells Differences in sensitivity to X-rays of XP2OS and HeLa S3 cells as determined by the colony-forming ability are shown in Fig. 1. As seen in this figure, XP2OS cells were more sensitive to X-rays than HeLa S3 cells. The Do values were 95 and 130R, and the extrapolation numbers (ns) were 5.9 and 5.4 for XP2OS and HeLa S3 cells, respectively. On the other hand, XP2OS cells were extremely sensitive to ultraviolet light (UV) compared with HeLa S3 cells, Do values being 0.5 and 9.5 J/m2, and ns were 1.0 and 1.2 for XP2OS and HeLa S3 cells, respectively. Mutation expression time Since it is known that the length of the mutation expression time markedly affects the number of mutant colonies that arise, the optimal expression time for X-rays was Fig. 1. Differences in sensitivity to X-rays of XP20S and HeLa S3 cells determined by colony-forming method. The bars indicate standard deviations of the mean for eight independent experiments.
X-RAY-INDUCED MUTATIONS IN CULTURED HUMAN CELL LINES 419 Fig. 2. The effect of the mutation expression time on the mutation frequencies of XP2OS (a) and HeLa S3 cells (b) following irradiation with various doses of X-rays. studied. Figs. 2a and 2b show the expression time curves for mutants resistant to 8AG appeared after irradiation of XP2OS and HeLa S3 cells with various doses of X- rays. In the both cell lines, the mutation frequency for any dose seems to increase with time up to 72 h and then decrease again after 72 h. From these findings, 72 h as an expression time for the both cell lines after X-irradiation was used for the following experiments. Properties of 8AG-resistant mutants Five 8AG-resistant mutant colonies (2 spontaneous and 3 X-ray-induced) appeared in HeLa S3 cells incubated for the mutation expression of 72 h after X-irradiation were separately isolated and grown in 8AG-free medium and their properties were examined 2 months after their isolation. As shown in Table 1, the colony-forming abilities of 5 8AG-resistant clones in medium with 0.5 ig/ml of 8AG were very high compared with that of the original HeLa S3 cells. On the other hand, HGPRT activities of the 4 8AG-resistant clones tested were very low, that is, 3.8 to 6.4% of that of the original HeLa S3 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 S3 cells, while the 5 mutant clones examined showed no significant incorporation. These data suggest that only 8AG-resistant mutants deficient in HGPRT were reco-
420 Y. MURAI, S. TATSUKAWA AND M. HORIKAWA Table 1. Properties of the original HeLa S3 cells and five 8AG-resistant mutant clones Fig. 3. a) Dose-survival curves of XP2OS and HeLa S3 cells plotted against doses up to 400R of X-rays. Each point shows the mean of four to five independent experiments. b) Induced mutation frequency of 8AG-resistant mutants versus doses of X-rays for XP2OS and HeLa S3 cells. Each point shows the mean of three to four independent experiments.
X-RAY-INDUCED MUTATIONS IN CULTURED HUMAN CELL LINES 421 Fig. 4. a) Dose-survival curves of XP2OS and HeLa S3 cells plotted against doses less than 160R of X-rays. Each point shows the mean of four to five independent experiments. b) Induced mutation frequency of 8AG-resistant mutants versus doses of X-rays for XP2OS and HeLa S3 cells. Each point shows the mean of seven to eight independent experiments. vered in the selection procedure and their properties were stable even after cultivation for 2 months in the absence of the selective agent 8AG. Dose-response of the mutation induction for X-rays Fig. 3a shows the dose-survival curves of XP2OS and HeLa S3 cells irradiated with doses up to 400R of X-rays. The induced mutation frequencies for these cell lines incubated for the mutation expression of 72 h after irradiation are also shown in Fig. 3b. As seen in these figures, the induced mutation frequencies for both XP2OS and HeLa S3 cells have an inverse relation to their survivals, that is, the induced mutation frequency for XP2OS cells, which were more sensitive to X-rays, increased more highly than HeLa S3 cells with increasing exposure to X-rays. Fig. 4b shows the induced mutation frequencies of XP2OS and HeLa S3 cells for doses less than 160R of X-rays, with comparable data on the survival of these cells immediately after irradiation (Fig. 4a). As seen in Fig. 4b, the dose-response curves for the both cell lines have approximately linear relationships for doses in the range used, when the dose and the induced mutation frequency plotted on a linear scale. Moreover, it can also be seen in these figures that the induced mutation frequencies
422 Y. MURAI, S. TATSUKAWA AND M. HORIKAWA for both XP2OS and HeLa S3 cells have an inverse relation to their survivals. DISCUSSION This paper deals with the development of mutant selection systems, using 8AGresistance as a marker in XP2OS cells which belong to complementation group A and the familiar human cancer HeLa S3 cells. It has been shown by Takebe et al. (1974) that XP2OS cells are extremely sensitive to UV and defective in excision repair of UV-induced DNA lesions. However, so far as examined in our laboratory, XP2OS cell line found to be an aneuploid line with the wide range of 65 to 95 chromosomes in number. This may be produced by the transformation with SV 40 virus. For this reason, we used an aneuploid HeLa S3 cell line instead of using primary diploid human cells, as a control. In this experiment, we found that the acquisition of 8AG-resistance as a marker in mutation studies can be used in XP2OS cells as well as in HeLa S3 cells, and that the optimum phenotypic expression times for 8AG-resistance induced by doses less than 400R of X-rays are 72 h in the both cell lines. All mutants isolated from the original HeLa S3 cells were resistant to 8AG, possessed less than 6.4% of the HGPRT activity of the wild-type cells even 2 months after their isolation. It was found in the mutation induction experiments that the assay systems using these both XP2OS and HeLa S3 cells are able to detect a significant increase in the frequency of mutations induced by a dose as low as 40R of X-rays, corresponding to shoulder regions on the dose-survival curves. In addition, an inverse relation was observed between the induced mutation frequencies and the survivals of both XP2OS and HeLa S3 cells, that is, the induced mutation frequency of XP2OS cells, which were more sensitive to X-rays, increased more highly than HeLa S3 cells with increasing exposure to X-rays. Such an inverse relation between the radiation-induced mutation frequencies and cell survivals is also observed for various cell types including human diploid cells (Cox and Masson 1976), Chinese hamster cells (Thacker et al. 1977) and mouse lymphoma cells (Knaap and Simons 1975), as have already been studied by other workers. These results indicate that there is a close correlation between the degree of the radiationinduced damage of the cells and their mutability, similarly to the previous results obtained from the dose rate, split-dose and potentially lethal dose experiments using Chinese hamster hai cells (Suzuki et al. 1979). On the other hand, the relationship between surviving fraction and induced mutation frequency in these various cell types irradiated with X- or r-rays is summarized in Fig. 5. In this figure, the induced mutation frequencies in human diploid cells, Chinese hamster V79-4 cells and mouse lymphoma L5178Y cells have been determined by the appearance of 6-thioguanine (6TG) resistant colonies after X- or r-irradiations. As seen in this figure, a plot of induced mutation frequency against log surviving fraction yields an approximately linear relationship for these five cell types. This relationship seems to suggest that mammalian cells generally have the same fixed probability of mutation induction relative to the extent of damage caused by ionizing
X-RAY-INDUCED MUTATIONS IN CULTURED HUMAN CELL LINES 423 Fig. 5. Relationship between surviving fraction and induced mutation frequency in various cell types irradiated with X- or r-rays. In this figure, data obtained after irradiation with various doses of X- or y-rays which were measured in R or rad unit were plotted in a figure, for comparison., HeLa S3 cells (This report) ; 0, XP2OS cells (This report) ; o, Human diploid HF 19 cells (Cox and Masson 1976) ; A, Chinese hamster V79-4 cells (Thacker et al. 1977) ; x, Mouse lymphoma L5178Y cells (Knaap and Simons 1975). radiation. Table 2 also shows the rate of forward mutations at the HGPRT locus induced in various cell types by ionizing radiation, as recently investigated by several workers. The induced mutation rates to purine analogue resistance in cultures of human, Chinese hamster and mouse cells are now in good agreement at the order of magnitude with the average rate (1.7X 10-'/locus/R) obtained for several specific loci of the mouse after irradiation (UNSCEAR 1972). This suggest that the mutation assay system to purine analogue resistance using various mammalian cell types including XP2OS cells are useful for determining the mutation frequency induced by ionizing radiation and that the data obtained by using these mammalian cell systems can be used for predicting the mutation frequency expected after exposure of man to ionizing radiation. In addition, mutation-survival plot as shown in Fig. 5 may facilitate comparison of mutagenicities of various kinds of mutagen and also comparison of mutation response to a mutagen of various cell types including hereditary sensitive cells. In this sense, it remains to be clarified how XP2OS cells which are extremely sensitive to UV respond to mutagenicity of UV and its mimetic substance, 4-nitroquinoline 1-oxide (4-NQO), etc.
424 Y. MURAI, S. TATSUKAWA AND M. HORIKAWA
X-RAY-INDUCED MUTATIONS IN CULTURED HUMAN CELL LINES 425 ACKNOWLEDGMENTS The authors wish to thank Dr. H. Takebe, Radiation Biology Center, Kyoto University, for his kind gift of XP2OS cells, and Dr. T. Sugahara, Kyoto University, for his many helpful discussions and reading of the manuscript. This study was supported in part by research funds from the Ministry of Education, Science and Culture, Japan. LITERATURE CITED Albertini, R. J., and R. DeMars, 1973 Somatic cell mutation, detection and quantification of X- ray-induced mutation in cultured, diploid human fibroblasts. Mutation Res. 18: 199-224. Arlett, C. F., and J. Potter, 1971 Mutation to 8-azaguanine resistance induced by r-radiation in a Chinese hamster cell line. Mutation Res. 13: 59-65. Ban, S., F. Suzuki, and M. Horikawa, 1976 Studies on somatic cell mutation, I. Radiation- and chemical-induction of nutritionally deficient and sufficient mutants in Chinese hamster hai cells in vitro. Japan. J. Genetics 51: 237-251. Bridges, B. A., and J. Huckle, 1970 Mutagenesis of cultured mammalian cells by X-radiation and ultraviolet light. Mutation Res. 10: 141-151. Chu, E. H. Y., 1971 Mammalian cell genetics, III. Characterization of X-ray-induced forward mutations in Chinese hamster cell cultures. Mutation Res. 11: 23-34. Cox, R., and W. K. Masson, 1976 X-ray-induced mutation to 6-thioguanine resistance in cultured human diploid fibroblasts. Mutation Res. 37: 125-136. Horikawa, M., F. Suzuki, and S. Ban, 1976 Studies on somatic cell mutations, II. Radiationinduction of 8-azaguanine-resistant and -sensitive mutants in Chinese hamster hai cells in vitro. Japan. J. Genetics 51: 253-264. Ikenaga, M., H. Takebe, and Y. Ishii, 1977 Excision repair of DNA base damage in human cells treated with the chemical carcinogen 4-nitroquinoline 1-oxide. Mutation Res. 43: 415-427. Kao, F. T., and T. T. Puck, 1969 Genetics of somatic mammalian cells, IX. Quantitation of mutagenesis by physical and chemical agents. J. Cell. Physiol. 74: 245-258. Knaap, A. G. A. C., and J. W. I. M. Simons, 1975 A mutational assay system for L5178Y mouse lymphoma cells, using hypoxanthine-guanine-phosphoribosyl-transferase (HGPRT)-deficiency as marker. The occurrence of a long expression time for mutations induced by X-rays and EMS. Mutation Res. 30: 97-110. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, 1951 Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 265-275. Suzuki, F., H. Hoshi, and M. Horikawa, 1977 Repair of the sublethal and mutational damage induced by X-rays in Chinese hamster hai cells in vitro. Cell Biol. Int. Reports 1: 463-467. Suzuki, F., H. Hoshi, and M. Horikawa, 1979 Repair of radiation-induced lethal and mutational damage in Chinese hamster cells in vitro. Japan. J. Genetics 54: 109-119. Takebe, H., S. Nii, M. I. Ishii, and H. Utsumi,1974 Comparative studies of host-cell reactivation, colony forming ability and excision repair after UV irradiation of xeroderma pigmentosum, normal human and some other mammalian cells. Mutation Res. 25: 383-390. Takebe, H., Y. Miki, T. Kozuka, J. Furuyama, K. Tanaka, M. S. Sasaki, Y. Fujiwara, and H. Akiba, 1977 DNA repair characteristics and skin cancers of xeroderma pigmentosum patients in Japan. Cancer Res. 37: 490-495. Thacker, J., and R. Cox, 1975 Matation induction and inactivation in mammalian cells exposed to ionizing radiation. Nature 258: 429-431. Thacker, J., A. Stretch, and M. A. Stephens,1977 The induction of thioguanine-resistant mutants of Chinese hamster cells by y-rays. Mutation Res. 42: 313-326.
426 Y. MURAI, S. TATSUKAWA AND M. HORIKAWA United Nations Scientific Committee on the Effects of Atomic Radiation, Ionizing Radiation: Levels and Effects, Vol. II, Effects. A/8725: General Assembly Official Records, 27th Session Supplement No. 25, United Nations, New York, 1972. Zeeland, A. A. van, and J. W. I. M. Simons,1976 Linear dose-response relationships after prolonged expression times in V-79 Chinese hamster cells. Mutation Res. 35: 129-138.