Mutatwn Research, 230 (1990) 159-166

159

Elsevier MUT 04860

Molecular changes in UV-induced and "t-ray-induced mutations in human lymphoblastoid cells Akira Tachibana, Tadaaki Ohbayashi, Hiraku Takebe and Kouichi Tatsumi Departments of Molecular Oncology and Experimental Radwlogy, Faculty of Medzcme, Kyoto Unwerszty, Kyoto 606 (Japan) (Recewed 7 September 1989) (Revasion recewed 5 January 1990) (Accepted 9 January 1990)

Keywords UV-induced mutataon; ~,-Ray-mduced mutatmn; Spontaneous mutatmn; Human lymphoblastold cells; HPRT gene, molecular analysis

Summary We have characterized the structural changes in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene of 14 UV-induced, 15 "t-ray-induced and 17 spontaneous mutants of human lymphoblastoid cells selected for 6-thioguanine (6TG) resistance. Southern blot analysis using the full-length HPRT eDNA as a probe revealed that 29% (5/17) of the spontaneous mutants contained detectable alterations in their restriction fragment patterns. Among the 15 mutants induced by ,/ rays, 7 (47%) had such alterations indicative of large deletions in the HPRT gene. In contrast, all 14 UV-induced mutants exhibited hybridization patterns indistinguishable from those of the wild-type cells. These results suggest that UV is likely to induce point mutations at the HPRT locus on the human chromosome and that the molecular mechanism of UV-induced mutation is quite different from that of ionizing radiation-induced mutation or spontaneous mutation in human cells.

Somatic mutations apparently play an important role in the emergence of human cancer (Setlow, 1978; Bishop, 1987). Clarifying the mechanism of mutation induction in human somatic cells must, therefore, benefit the understanding of carcinogenesis. The hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene locus has been

Abbremations: HPRT, hypoxanthme-guanine phosphoribosyltransferase; APRT, ademne phosphonbosyltransferase; 6TG, 6-thioguamne. Correspondence: Dr. K. Tatsumi, Department of Molecular Ontology, Faculty of Medicine, Kyoto University, Sakyo, Kyoto 606 (Japan).

one of the most extensively studied loci for mutation analysis in cultured mammalian cells (A1bertini et al., 1989). In culture, cells bearing mutations at the hprt gene can be selected out by growing the cells in 6-thioguanine (6TG)-containing medium. We have examined the mutagenesis by various agents in human lymphoblastoid cell lines, and have isolated a great number of 6TG-resistant mutant clones (Tatsumi and Takebe, 1984; Tatsumi et al., 1987). To understand the process(es) of mutagenesis in mammalian cells, it is imperative to elucidate the molecular nature of spontaneous and induced mutations. The cloning of HPRT eDNA (Brennand et al., 1982; Konecki et al., 1982; Brennand

0027-5107/90/$03.50 © 1990 Elsewer Science Publishers B V. (Biomedical Davislon)

160 et al., 1983; Jolly et al., 1983) has made it possible to investigate the relationship between the imtial DNA damage by mutagens and the ultimate changes in the mutated hprt gene. More than half of the X-ray-induced 6TG-resistant mutants had major alterations of DNA in human cells (Skulimowski et al., 1986; Liber et al., 1987) and also in hamster cells (Vneling et al., 1985; Stankowski and Hsie, 1986; Thacker, 1986, Fuscoe et al., 1986; Gibbs et al., 1987), whereas the majority of UV-induced mutations at the HPRT locus were suggested to be point mutations in hamster cells (Fuscoe et al., 1983; Stankowski and Hsie, 1986; Vrieling et al., 1989). Since there is as yet no description of the molecular basis of UV-induced mutations leading to HPRT deficiency in human somatic cells, we have analyzed UV-induced mutants for 6TG resistance using Southern blotting and have compared them with y-ray-induced and spontaneous mutants. While large deletions or rearrangements of DNA seem to be causative for a substantial proportion of spontaneous and y-ray-induced 6TG-resistant mutants, no change in restriction fragment pattern is detectable with UV-induced mutants of human lymphoblastoid cells. Materials and methods

Cell culture The lymphoblastoid cell line TK6 (Skopek et al., 1978), derived from a male spherocytosls patient, was provided by Dr. W.G. Thilly, Massachusetts Institute of Technology, Cambridge, MA. The cells were grown in RPMI 1640 medium supplemented with 17% fetal calf serum, and maintained by daily dilution to 4 × 105 cells/ml. Prior to each experiment, TK6 cells were incubated in complete medium containing 10 -5 M cytidine, 2 x 10 -4 M hypoxanthine, 2 x 10 -7 M aminopterin and 1.75 x 10 -5 M thymidine (CHAT medium) for 2 days to reduce the HPRT- mutant fraction (Furth et al., 1981). Following CHAT treatment, cells were centrifuged and resuspended in THC medium (the same formulation as CHAT medium but without aminopterin) to maintain the exponential growth, and diluted with the complete medium without THC for 3-5 days prior to irradiation.

Mutagenests experiments Methods to determine cytotoxicity and mutagenesis following UV or ,/ irradiation have been described previously (Thilly et al., 1980; Furth et al., 1981; DeLuca et al., 1983; Tatsurm and Takebe, 1984; Tatsurm et al., 1987). Briefly, 5 × 107-108 cells per point were centrifuged and resuspended at 5 x 10 6 cells/ml in 'RPMI saline' (0.1 M NaC1, 11 mM glucose, 5.6 mM Na2HPO 4, 5.4 mM KC1, 0.4 mM Ca(NO3)2, 0.4 mM MgSO4, pH 7.0) (DeLuca et al., 1983). For UV irradiation the cells were subjected to UV light emitted from a 15-W germicidal lamp (GL-15, Toshiba, Tokyo) at a fluence rate of - 3 J/m2/man. The UV fluence was measured with a UV photometer (Topkon UVR-254, Tokyo Kogaku, Tokyo). For y irradiation the cells were exposed to a 137Cs source at a dose rate of - 4 G y / m i n at room temperature. Following irradiation, cells were resuspended in a 10 × volume of complete medium. Cytotozaclty was determined by back-extrapolating the exponential portion of growth curves of the cultures (DeLuca et al., 1983; Tatsumi and Takebe, 1984). For mutation assay, cells were incubated for 8 days to allow maximum expression of 6TG resistance. The cells were dispensed into mlcrowells of 96-well microtiter plates at a density of 1-4 × 104 cells/well in the presence of 6TG (5 t~g/ml). Positive wells were scored at 10-12 days after plating. Mutant colonies were recloned from independent cultures, expanded to approximately 107 cells, and frozen for subsequent DNA isolation.

Molecular analysts High-molecular-weight nuclear DNA was isolated by proteinase K digestion and organic solvent extraction (Maniatls et al., 1982) and was digested with restriction enzymes according to the methods recommended by the supplier (Takara, Kyoto). Digested DNA samples (5 or 10/xg) were electrophoresed in 0.7% agarose gel in Tris-acetate buffer at 2.3 V/cm. DNAs were transferred from agarose gel to Gene Screen PlusT M nylon membrane according to the protocols recommended by the supplier (New England Nuclear, Boston, MA). After fixation by UV irradiation (1.5 kJ/m2), the blotted filters were prehybridized in 0.2% polyvinyl-pyrrolidone (MW 40,000), 0.2% FicoU (MW 400,000), 0.2% bovine serum albumin, 0.05 M

161 Tris-HC1, p H 7.5, 0.1% sodium pyrophosphate, 1% SDS, 10% dextran sulfate (MW 500,000) and 100 g g / m l denatured salmon sperm D N A for 20-24 h at 65 o C. The human H P R T c D N A probe was obtained from a plasmid, pPR-1, constructed by Dr. D. Jolly and provided for us by Dr. R.J. Monnat, University of Washington, Seattle, WA. The 0.9-kb c D N A insert was excised with the restriction enzyme PstI. The probe was labeled with [32P]dCTP (Amersham) using a multi-prime D N A labeling system according to the supplier's instructions (Amersham). Hybridization with radiolabeled probe was carried out for 15-20 h at 65 ° C in the same buffer as used for prehybridization. The filters were washed twice for 5 min in 2 × SSC at room temperature, twice for 30 n'fin in 2 × SSC, 1% SDS at 65 ° C and once for 30 min in 0.5 × SSC, 1% SDS at 65°C. After washing with 0.1 x SSC, the filter was exposed to Fuji X-ray

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Results Spontaneous mutant clones were collected by first seeding 200 cells into 24-well microplates. Twenty-four such sib-cultures were expanded to - 1 × 107 cells in T-75 flasks and then exposed to 6 T G selection in 96-well microplates. We examined the structural changes in 17 spontaneous T K 6 mutant clones thus independently obtained from the positive sib-cultures. Genomic D N A of these mutant clones was digested with EcoRI and subjected to Southern blot analysis using the fulllength h u m a n H P R T c D N A as a probe. Fig. 1A shows the result of a blot hybridization experiment. The EcoRI-generated H P R T banding pattern for the wild-type T K 6 DNA, shown in the

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F~g. 1. Southern blot analysis of DNA isolated from spontaneous 6TG-resistant mutants. (A) Genorrac DNA was digested with EcoRI and probed with a full-length HPRT cDNA probe. The left ordinate shows s~zes of X-linked HPRT fragments and pseudogene (1/,) assignments. Lane 1: $13; lane 2: $14; lane 3: $15; lane 4: $16; lane 5: $60; lane 6: $62; lane 7: $63; WT: wild-type TK6 cells. (B) PstI-digestedgenomic DNA was hybridized with an HPRT cDNA probe. The left ordinate shows X-lmked HPRT exon assignments and autosome-linkedpseudogene fragments (~). Lane 1. S07; lane 2: $16; lane 3: $17; WT: wild-type TK6 cells.

162 ' W T ' lane m Fig. 1A, revealed 4 autosome-linked pseudogene bands and 4 X-linked bands. This pattern was m close agreement with the previous reports (Patel et al., 1984, 1986; K i m et al., 1986). The proposed exon assignments of the 4 EcoRIgenerated X-linked H P R T fragments were as follows: the l l . 0 - k b band contains exon 1, the 8.3-kb band contains exons 2 and 3, the 10.5-kb band contains exons 4 and 5, and the 8.0-kb band contains exons 6, 7 and 8 (Patel et al., 1986; Kam et al., 1986). We observed fragment sizes very similar to these, and yet could not always detect the 11- and 10.5-kb fragments. Those fragments sometimes appeared but gave at best a very weak signal on most blots. It was, therefore, conceivable that we failed to detect some mutants with major alterations involving exon 1 or exons 4 and 5. Five out of 17 spontaneous mutants had discernible alterations in the restriction fragment patterns. These structural alterations in the spontaneous deletion mutants were confirmed by analyzing PstI-generated H P R T banding patterns (Fig. 1B). With this enzyme, we detected 6 X-linked H P R T bands so that we could map the deleted regions in more detail. Those data are diagrammed in Fig. 4. For instance, the 10.5-, 8.3-, 8.0-kb EcoRI-generated bands were lost in the S16 mutant clone, which suggested deletion from exon 2 to exon 8. In PstI-generated banding patterns, the bands which correspond to exons 2, 3, 4 - 6 were lost, but the band which contains exons 7 - 9 remained (Fig. 1B). Therefore, we concluded that the deletion in the S16 mutant clone encompassed exons 2-6, and that the remaining EcoRl fragment containing exons 7 and 8 was too small to be detected on our Southern blot with EcoRI. The remaimng 12 mutants with no change in the restriction pattern for EcoRI were regarded to have resulted from point mutations including base-pair substitutions and very small deletions or additions ( < 200 base pairs). Exposure to 1 and 2 G y of 3' rays resulted in an approximately 9-fold and a 12-fold increase m the 6TG-resistant mutant fraction, respectively, with a background fraction of 1.2 × 1 0 - 6 for unirradiated cultures. Of 15 mutants isolated, 7 had alterations when D N A was digested with EcoRI (Fig. 2). The structural alterations in the deletion mutants induced by 1 G y of 3' rays were analyzed

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2.3 2.0 Fig. 2 Analysis of T-ray-reduced mutants for 6TG resistance by Southern blot hybndtzauon with the restncuon enzyme EcoRI Lane 1. G22, lane 2. G24, lane 3: G26, lane 4 G27, lane 5 G28, lane 6. G52.

using digestion with PstI, and are summarized in Fig. 4. In the PstI banding patterns of the G27 mutant, the upper band of the doublet seen at 5.3-5.5 kb was lost, but the lower band of the doublet remained. Yang et al. (1984) did not assign exon 5 to either the upper band of the doublet which contains exon 4 or the lower one which contains exon 6. On drawing Fig. 4, we tentatively assigned exon 5 to the lower band following the restricUon m a p reported by Patel et al. (1986), although Gennett and Thilly (1988) reported that their exon 4 - 5 probe could not detect a doublet on the PstI-generated banding pattern, which indicated that exon 5 could be assigned to the upper b a n d containing exon 4. F r o m U V (4.4 j/m2)-irradiated T K 6 cells, 6TG-resistant mutant clones were isolated at a

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frequency of 1.7 × 10 -5. EcoRI digestion of genomic DNA from 14 mutants exhibited hybridization patterns indistinguishable from those of the wild-type cells. Fig. 3 illustrates the banding patterns of 6 of the 14 UV-induced mutant clones. Discussion

We examined the molecular nature of spontaneous, y-ray-induced and UV-induced mutants at the HPRT locus in a human lymphoblastoid cell line, TK6, and compared among these 3 classes of mutations the degree of involvement of large molecular changes in DNA. Table 1 summarizes the proportion of mutants with alterations in banding pattern. Among the spontaneous mutants, 5/17 (29%) had visible changes in the restriction fragment patterns, in

general agreement with the following previous reports. Liber et al. (1987) reported that 36% (5/14) of spontaneous mutants at the HPRT locus in TK6 cells had altered restriction fragment patterns. Gennett and Thilly (1988) reported that 39% (33/85) of spontaneous HPRT- mutants from the same cell line contained alterations of banding patterns. Similar results have been obtained by Monnat (1989) with a human promyelocytic leukemia cell line, HL-60: 38% (17/45) of spontaneous 6TG-resistant mutants had restriction fragment alterations. Among the v-ray-induced mutants, 7/15 (47%) had alterations in restriction fragment patterns (Table 1). Because the v-ray-induced mutant fractions were approximately 10 times as high as the baseline fraction, it was unlikely that the proportion of mutants with restriction fragment alterations in v-ray-induced mutants was appreciably affected by the possible coexistence of spontaneous mutants in the analyzed samples. Our data on y-ray-induced mutants can be compared with earlier molecular studies on ionizing radiation-induced mutations at the HPRT locus in human cells. Skulimowski et al. (1986) cloned X-ray-induced mutants for 6TG resistance from human peripheral T lymphocytes. Southern blot analysis on these mutants revealed that 17 of 33 (52%) mutant clones contained major molecular changes in the gene. Liber et al. (1987) reported that 54% (15/28) of X-ray (1.5 Gy)-induced HPRTmutants in TK6 cells had structural abnormalities. However, there are several differences between these previous reports and our present study. Skulimowski et al. (1986) reported that 11 of 33 mutants contained novel bands, whereas neither Liber et al. (1987) nor the present study detected such new bands. Our data summarized in Fig. 4 indicated that none of the deletions observed for TABLE 1 FREQUENCY OF DELETION MUTATIONS AT THE HPRT LOCUS IN TK6 CELLS Treatment

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SO7 $16 $17 G22 G27 G52 F~g. 4 The deleted regtons m the hprt gene of spontaneous (S07, S16, S17) and y-ray-mduced (G22, G27, G52) m u t a n t s are diagrammed based on both EcoRI- and PstI-generated Southern blot patterns. The H P R T exon a h g n m e n t s are gwen m the top part of the figure following the pubhshed results (Patel et al., 1986, Kam et a l , 1986). The regions where deletions were obvaous are shown w~th ttuck hnes and those regtons where deletions were uncertain are shown w~th than hnes

mutants induced by 1 Gy -/-irradiation encompassed the entire hprt gene. Liber et al. (1987) reported, however, that all deletxon mutants except one lost the total fragments. These differences in the extent of deletions may be due to the different type of cells employed (B lymphoblastotd cells for both Liber et al. and this work vs. peripheral T lymphocytes for Skulimowski et al.), the quality of iomzing radiation (X rays for Liber et al. and Skulimowskl et al. vs. V rays for this study), a n d / o r the different stringency in the selection for 6TG resistance, i.e., 0.5 /xg/ml for Liber et al., 2.5/xg/ml for Skulimowskl et al., and 5.0/~g/ml for this study. None of the 14 UV-induced 6TG-reslstant mutants were found to have an evident alteration in the size of the X-linked H P R T fragments (Table 1), indicating that those human cell mutants did not result from either large intragenic rearrangements or major deletions. For rodent cells, Fuscoe et al. (1983) reported that 1 of 9 UV-induced mutants of V79 cells had major deletions affecting the H P R T locus, but other studies analyzing 23 H P R T - CHO mutants (Stankowski and Hsie, 1986), 34 adenine phosphoribosyltransferase-deficient ( A P R T - ) C H O mutants (Drobetsky et al., 1987), 20 H P R T - V79 mutants and 22 H P R T - V-H1 (a UV-sensitive cell line isolated

from V79) mutants (Vneling et al., 1989) disclosed that blotting patterns for UV-mduced mutants were all indistinguishable from those for their corresponding wild-type ceils. Rodent cells have been shown to be deficient in removing UV-induced pyrlmidine dimers from their genomes overall, despite the essentially similar sensitivity to cell kilhng by UV as human cells, which efficiently repair the UV-mduced lesions. Rodent cells only remove a relatively small fraction of UV-induced pyrimidine dimers from their total D N A (Zelle et al., 1980) as compared to human cells (van Zeeland et al., 1981). In spite of such difference m the kinetics of repairing UV-induced damages, the molecular change of the gene in UV mutagenesis as well as UV mutability does not seem to be much different between human cells and rodent cells. This situation could be understood in view of the recent discovery that rodent cells preferentially repair transcriptionally actwe genes while the repair of inactive genes and bulk D N A is very poor (Bohr et al., 1985; Madhani et al., 1986). Drobetsky et al. (1987) cloned and determined the sequences of UV-induced mutants at the A P R T locus of C H O cells. Vrieling et al. (1989) investigated the sequence alterations of the UV-induced H P R T mutations in both normal (V79) and UVsensitive (V-H1) Chinese hamster cells. Both of these studies have indicated that UV reduces mainly single-base substitutions together with tandem double mutations and frameshift mutations. Our data are consistent w~th the expectation that the majority of UV-induced mutations in human cells may be point mutations as in hamster cells. In conclusion, D N A rearrangements and deleUons account for a large proportion of ionizing radlauon-induced mutations, while UV induces exclusively point mutations in human cells. The difference in mutation spectrum indicates that the mechanism of UV-induced mutations should be different from that of iomzing radiation-induced mutations or spontaneous mutations in human cells.

Acknowledgements The authors thank Dr. R.J. Monnat for pPR-1, Dr. I.M. Jones and Dr. R.J. Monnat for useful

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comments on molecular analysis of the hprt gene, and Dr. M. Meuth and Dr. T. Yagi for critically reading the manuscript. The technical assistance by Ms M. Toyoda and the secretarial help by Ms M. Nishihara are also acknowledged. This work was supported in part by grants for cancer research from the Ministry of Education, Science and Culture and a grant from the Science and Technology Agency of Japan. References Alberum, R.J, I N. Gennett, B Lambert, W G. Thilly and H. Vnehng (1989) Mutation at the hprt locus. Workshop on mutation at the HPRT locus, Stockholm, May 26-28, 1988, Mutauon Res., 216, 65-88. Bishop, J.M (1987) The molecular genetics of cancer, Science, 235, 305-311. Bohr, V.A., C A. Srmth, D.S. Okumoto and P.C. Hanawalt (1985) DNA repair m an active gene: removal of pyrinudine dimers from the DHFR gene of CHO cells is much more efficient than in the genome overall, Cell, 40, 359-369. Brennand, J., A.C. Chinanlt, D.S. Konecki, D.W Melton and C.T. Caskey (1982) Cloned eDNA sequences of the hypoxanthine/guanine phosphonbosyltransferase gene from a mouse neuroblastoma cell hne found to have amphfied genormc sequences, Proc. Nati Acad. Sci. (U.S.A.), 79, 1950-1954. Brermand, J., D.S. Konecki and C.T. Caskey (1983) Expression of human and Chinese hamster hypoxanthme-guamne phosphonbosyltransferase eDNA recombmants in cultured Lesch-Nyhan and Chinese hamster fibroblasts, J. Biol. Chem., 258, 9593-9596. DeLuca, J G., L. Weinstean and W.G Thilly (1983) Ultraviolet light-reduced mutauon of diploid human lymphoblasts, Mutation Res., 107, 347-370 Drobetsky, E.A., A.J. Grosovsky and B.W. Giickman (1987) The specificity of UV-induced mutations at an endogenous locus in mammalian cells, Proc. Natl. Acad. SCL (U S.A.), 84, 9103-9107 Furth, E.E, W.G. Thflly, B.W. Penman, H.L. Lther and W M. Rand (1981) Quantitative assay for mutation m &plmd human lymphoblasts using rmcrotiter plates, Anal. Blochem., 110, 1-8. Fuscoe, J.C, R.(3. Fenwmk, D.H. Ledbetter and C.T. Caskey (1983) Deletion and amphfication of the HGPRT locus in Clunese hamster cells, Mol. Cell. Biol., 3, 1086-1096. Fuscoe, J.C, C.H. Ockey and M. Fox (1986) Molecular analysis of X-ray-reduced mutants at the HPRT locus in V79 Clunese hamster cells, Int. J. Radiat. Biol., 49, 1011-1020. Gennett, I.N., and W.G Tlully (1988) Mappmg large spontaneous delenon endpolnts in the human HPRT gene, Mutation Res., 201, 149-160. Gibbs, R.A., J. Camakaris, G.S. Hodgson and R.F. Martin (1987) Molecular characterization of 125I decay and X-ray induced HPRT mutants in CHO cells, Int. J Radmt. Biol., 51, 193-199

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the HPRT locus m cultured Chinese hamster ceils are mostly large deletions, Mutation Res, 144, 281-286. Vrieling, H., M.L Van Rooijen, N.A Groen, M Z Zdraemcka, J.W.I.M. Slmons, P H M Lohrnan and A A van Zeeland (1989) DNA strand specificity for UV-mduced mutahons in mammahan cells, Mol. Cell Blol, 9, 1277-1283 Yang, T P , P.I Patel, A.C Chinault, J T Stout, L.G. Jackson, B M. Hddebrand and C T. Caskey (1984) Molecular evidence for new mutauon at the hprt locus m Lesch-Nyhan patients, Nature (London), 310, 412-414 Zelle, B, R J Reynolds, M.J. Kottenhagen, A Schmte and P H M. Lohman (1980) The influence of the wavelength of ultraviolet radlaUon on survwal, mutation induction and DNA repair m irradiated Chinese hamster cells, Mutation Res, 72, 491-509