Mutation Research, 249 (1991) 161-167 © 1991 Elsevier Science Publishers B.V. 0027-5107/91/$03.50 ADONIS 002751079100137Q
161
MUT 04989
Molecular characterization of mutations at the hprt locus in V79 Chinese hamster cells induced by bleomycin in the presence of inhibitors of DNA repair Beate KOberle and Giinter Speit Abteilung Klinische Genetik, Universitiit Ulm, D- 7900 Ulm (F. R. G.)
(Received 25 September 1990) (Revision received 10 December 1990) (Accepted 7 January 1991)
Keywords: Hprt locus; Mutation; DNA-repair inhibitors; Bleomycin; Southern analysis; V79 cells
Summary The molecular basis of bleomycin (BLM)-induced mutations in the absence and presence of inhibitors of D N A repair was investigated in V79 cells with Southern hybridization techniques. 43% of the BLM-induced thioguanine-resistant mutants suffer from large alterations of hprt DNA sequences. To understand the role of DNA repair in the process of mutagenesis, the effect of inhibitors of D N A repair on the frequency and types of BLM-induced mutations was tested. The inhibitors used were arabinofuranosyl cytosine (araC), didesoxythymidine (ddThd) and 3-aminobenzamide (3AB), which inhibit different steps of excision repair. Only 3AB caused a comutagenic effect. The increased mutation frequency was mainly due to additionally induced gene deletions. In the presence of 3AB, 70% of the HPRT-deficient mutants revealed partial or total deletions of the hprt coding sequences. Thus, it could be shown that BLM induces a broad range of types of mutation and that inhibited repair of BLM-induced D N A damage leads to specific types of mutations.
In order to understand the mechanisms of mutagenesis it is important to analyze the nature of spontaneous and induced mutations at the molecular level. The hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene locus has been used extensively for mutagenesis studies in mammalian cells (for review see Albertini et al., 1989). The hprt gene is an excellent choice for studying the process of mutagenesis, owing to its large size
Correspondence: Dr. G. Speit, Abteilung Klinische Genetik, Universifiit Ulm, Postfach 4066, D-7900 Ulm (F.R.G.).
( > 30 kb), X-hnked location and homology in mammals, ease of mutant selection and availability of probes and sequence data. H P R T is an enzyme that catalyzes the reutilization of preformed purine bases. In man, complete H P R T deficiency results in the L e s c h - N y h a n syndrome and it has been shown that this phenotype is the result of a heterogenous group of mutations (for review see Stout and Caskey, 1988). In a number of studies, mutation induction by ionizing radiation has been investigated at the D N A level. Using a cloned e D N A and Southern blot analysis, it could be demonstrated that the
162 majority of these mutations are caused by large deletions (Vrieling et al., 1985; Thacker, 1986; Fuscoe et al., 1986; Whaley and Little, 1990). In contrast, mutants induced by UV irradiation or alkylating agents almost all seem to arise from point mutations (Vrieling et al., 1988; Albertini et al., 1989; Thacker and Ganesh, 1989). Thus the hprt gene mutation system is able to detect different types of mutation and is a sensitive method for distinguishing the mutagenic action of different DNA-damaging agents. We therefore tried to characterize the mutagenic action of the antitumor drug bleomycin (BLM) in V79 Chinese hamster cells. BLM is widely used as part of a combination therapy of advanced testicular cancer in young men. As a cure is possible for over 90% of the patients (Einhorn, 1981), the mutagenic action of BLM is relevant with respect to the development of secondary malignancies and the genetic risk for offspring of surviving cancer patients. BLM has been shown to produce chromosome aberrations comparable to those induced by X-irradiation and is also known to induce DNA breaks through the production of free radicals (Takeshita et al., 1978) similar to the indirect action of radiation. In a recent molecular study (Wood and Moses, 1989) it was found that bleomycin generated deletion mutations at the hprt locus in xeroderma pigmentosum cells. Not only with regard to the types of damage it produces does bleomycin appear to be similar to X-rays, the enzymatic mechanisms for the repair of BLM and X-ray damage also seem to be essentially the same (Utsumi and Elkind, 1989). Our understanding of the biochemical basis of D N A repair in mammalian cells has been greatly enhanced over the past several years through the use of specific enzyme inhibitors. A role in D N A repair could be established for DNA polymerase a, D N A polymerase fl and poly(ADP-ribose)/ D N A ligase (for review see Collins et al., 1984). We used specific inhibitors for each of these 3 processes to analyze the relative importance of these steps for the repair of BLM-induced D N A damage. We investigated the consequences of inhibited repair on the frequencies and types of mutation at the cellular and molecular levels. The results contribute to a better understanding of the mechanisms of mutation and the influence of D N A repair.
Materials and methods
Cell culture and mutation experiments The experiments were performed with a clonal derivative of the V79 Chinese hamster cell line (provided by Dr. D. Wild, Freiburg). Cells were cultivated in minimal essential medium (MEM) with Earle's salts supplemented with 10% fetal calf serum and antibiotics in a humidified incubator at 37°C with 5% CO 2 at a p H of 7.2. Mutagenicity experiments were performed in 175-cm2 flasks (Nunc). About 5 x 10 6 cells were treated in each experiment according to one of the following protocols: (i) treatment for 21 h with one of the repair inhibitors; (ii) treatment for 21 h with bleomycin alone; (iii) treatment with bleomycin in combination with one of the repair inhibitors. The inhibitors used were arabinofuranosyl cytosine (araC), didesoxythymidine (ddThd) and 3-aminobenzamide (3AB). Bleomycin (BLM) and the inhibitors were purchased from Sigma. Survival (relative plating efficiency.) was determined by plating 200 cells into 4 replica petri dishes (60 x 15 mm) at the end of mutagen treatment. Colonies were fixed, Giemsa-stained and counted 7 days later. The treated cultures were transferred as needed during the expression period. After 7 days, 103 cells were replated into 5 replica petri dishes (100 x 15 mm) with selective medium (10/~g/ml 6-thioguanine). At the time of replating into selective medium, the plating efficiency was determined in non-selective medium (four 60-mm replica petri dishes with 200 cells each). After 1 week, thioguanine-resistant colonies were isolated from different petri dishes and cultivated in normal medium for the molecular analysis. For the determination of the mutation frequency, the colonies were fixed with methanol, stained with Giemsa and counted. Mutation frequencies were corrected for cell survival. DNA isolation DNA was isolated from wild-type V79 cells and H P R T mutants (6-TG-resistant colonies) by treatment with protease K (100 # g / m l ) , 1% SDS and homogenization buffer (100 mM NaC1, 10 mM Tris, p H 7.6, 10 mM EDTA, p H 7.6) for 1 h at 37°C. After phenol extraction (3 times to purify
163
DNA) the D N A solution was dialyzed for 3 x 8 h to STE buffer (100 mM NaC1, 10 mM Tris, pH 7.6, 1 mM EDTA, pH 7.6), followed by treatment with RNase (100 ~ g / m l ) for 1 h at 37°C and protease K (100 /~g/ml) for 1 h at 37°C. D N A solution was again purified by phenol extraction, followed by chloroform/isoamylalcohol (4%) extraction and precipitated by 1 / 1 0 volume 1 M K-acetate and 2 volumes 100% cold ethanol. D N A was stored at 4°C in 10 mM Tris (pH 7.6)-1 mM EDTA (pH 7.6).
Restriction endonuclease digestion and Southern blot High-molecular weight DNA (15 ~g) was digested with the restriction endonucleases for 5 h under conditions recommended by the manufacturer (Boehringer Mannheim). The fragments were separated on 1% agarose gel at 40 V for 16 h. The gel was washed in 0.25 M HC1 for 30 min and blotted for 4 h with N a O H (0.4 M) onto nylon filter (Hybond N ÷, Amersham). The filter was washed in 2 x SSC for 10 rain.
Nick translation and filter hybridization 270 Fg of a full-length hamster hprt cDNA cloned into pBR322 (pHPT12, kindly supplied by Dr. J. Thacker) was nick-translated using 30 /xCi [32p]dCTP at 15°C with nick-translation kit (Boehringer Mannheim). The filter was prehybridized for 1 h at 65°C in 5 x SSPE, 5 x Denhardt's solution, 0.5% SDS, then the nick-translated probe (preboiled for 10 min) was added and the filter was hybridized for about 16 h at 65°C. The filter was then washed in 2 changes of 2 x SSC, 0.1 x SDS for 5 min, 0.1 × SSC, 0.1% SDS for 15 rain at 55°C, 15 min at 60°C and 10 min at 65°C. The filter was then exposed to X-ray film for several days at - 7 0 ° C .
Molecular weight markers The sizes of hprt gene fragments were estimated with reference to fragments of known size from molecular-weight markers (Boehringer).
Cytogenetic analysis Cytogenetic analysis was done by ing. Cells were cultivated with BrdU for 5 h, chromosome preparations according to standard procedures and
RBG-band(10 /~g/ml) were made stained with
a fluorescence plus Giemsa (FPG) technique (Speit and Haupter, 1985). Results
Treatment of V79 cells with BLM for 21 h resulted in an increased frequency of mutations at the hprt locus. 10 /~g/ml BLM caused 26-42 mutants/106 surviving cells (Table 1). This treatment reduced initial cell survival to values between 13% and 22%. Lower BLM concentrations influenced mutation frequency only marginally and a higher concentration (20 /xg/ml) caused more mutations but at the same time showed high toxicity (relative plating efficiency below 10%). Therefore we chose the BLM concentration of 10 /~g/ml for our experiments, which are summarized in Table 1. The DNA-repair inhibitors 3AB, araC and ddThd were not mutagenic under the present conditions. Only araC reduced survival of V79 cells (to 40% PE at 2 x 1 0 - 7 M ) . After combined treatment, 3AB slightly increased the toxicity of BLM and showed a comutagenic effect. Mutation frequencies were nearly doubled with 10 -3 M
TABLE I EFFECTS O F I N H I B I T O R S OF D N A - R E P A I R O N BLMI N D U C E D M U T A T I O N S IN V79 CELLS Mutagen treatment
Relative plating efficiency (%)
None
100
3
3AB (10 -3 M) 3AB ( 2 × 10 -3 M) BLM (10 ~ g / m l ) BLM + 3AB (10 -3 M) B L M + 3 A B ( 2 x 10 -3 M)
100 100 19 14 13
0 0 26 46 81
araC (10 - 7 M) araC ( 2 x 10 - 7 M) BLM (10/xg/ml) BLM + araC (10 -7 M) B L M + araC ( 2 x 1 0 -7 M)
85 40 22 20 8
2 5 36 42 40
100 90 22 17 11
3 6 31 40 42
ddThd (5 × 10 - 4 M) ddThd (10- 3 M) BLM (10 # g / m l ) BLM + ddThd ( 5 × 1 0 - 4 M) BLM + ddThd (10- 3 M)
Frequency of 6-TG-resistant cells/10 6
164
kb
M C I! 12 13 14 15 1617 18 19
kb
23.19.46.5-
51 C II 12 13 14 15 16 17 18 19
21.2-
4.3-
5.1~ 4.94.23.5m n
2.01.91.51.3-
Fig. 1. Southern blot hybridization of a hprt gene probe to genomic D N A of 9 HPRT-deficient m u t a n t s induced by bleomycin in the presence of 3-aminobenzamide. D N A was digested with the restriction endonuclease EcoRl. M, molecular-weight markers; C, control (V79 parent cell).
Fig. 2. Southern blot hybridization analysis as in Fig. 1, but genomic D N A was digested with Bglll.
with EcoRI and hybridized to p H P T 12, 3 bands located at 17.5 kb, 11.3 kb and 1.2 kb were visualized by autoradiography. The 2 main bands at 17.5 kb and 11.3 kb are part of the functional hprt gene whereas the small fragment at 1.2 kb represents a pseudogene (Fuscoe et al., 1983; Thacker, 1986). BglII digestion revealed 6 fragments. The 4 bands located at 4.3, 2.1, 1.5 and 1.2 kb represent the functional hprt gene, while the other 2 bands (at 3.4 and 1.9 kb) indicate pseudogene sequences. Each mutant D N A was digested with these 2 endonucleases (separately) and the resulting pat-
3AB and tripled with 2 x 10 _3 M 3AB. Simultaneous treatment with BLM and araC or BLM and ddThd caused enhanced toxicity but had no clear effect on the mutation frequency. F r o m the experiments with 3AB, mutant clones were isolated from controls, BLM-treated cultures and from cultures treated with BLM plus 3AB (10-3 M). The overall results of the Southern blot analysis are shown in Table 2 and examples of the data are illustrated in Figs. 1 and 2. When genomic D N A from wild-type V79 cells was cleaved
TABLE 2 A N A L Y S I S O F D N A F R O M H P R T - D E F I C I E N T M U T A N T S O F V79 CELLS F O R C H A N G E S IN hprt G E N E S T R U C T U R E Inducing
N u m b e r of
N u m b e r of m u t a n t s with large changes
agent
m u t a n t s examined
total deletions
partial deletions/ rearrangements
%
N u m b e r of m u t a n t s with no detectable changes
Control BLM 3AB + BLM
20 30 30
0 9 14
0 4 8
0 43 70
20 17 8
165 terns allowed the differentiation of 3 types of mutation: (i) apparently total gene deletions which lacked all functional hprt sequences; (ii) partial deletions a n d / o r rearrangements of the hprt gene; (iii) mutants without detectable loss of gene sequences. The restriction pattern of all 20 spontaneous H P R T mutants analyzed could not be distinguished from the V79 wild-type pattern, indicating that point mutations or small deletions ( > 300 bp) are responsible for the H P R T phenotype. Among the BLM-induced mutants, 57% showed the wild-type pattern, while 43% revealed changes in the fragment pattern: 9 out of 30 mutants apparently were total gene deletions and 4 were partial deletions. After combined treatment with 3AB the portion of mutants with large changes was increased from 43% to 70%. 14 out of 30 mutants lacked all functional gene fragments, 8 out of 30 lacked parts of the hprt coding sequences and only 8 out of 30 showed the wild-type pattern. Figs. 1 and 2 show examples of this type of result from EcoRI and BgllI digests of mutants induced by BLM in combination with 3AB. The EcoRI digest (Fig. 1) reveals that mutants 11, 14, 15 and 18 lack all functional gene fragments, only the pseudogene fragment at 1.2 kb is seen. Mutants 12, 17 and 19 have restriction fragment patterns identical to that of V79 D N A while the patterns of mutants 13 and 16 indicate a reduction in size of the 2 fragments. Hybridization analysis with the same set of mutants digested with BgllI confirmed the total deletion of hprt coding sequences in mutants 11, 14, 15 and 18 and the wild-type pattern in mutants 12, 17 and 19. Mutants 13 and 16 both lack the fragment at 1.5 kb but have additional bands at 3.2 kb (mutant 13) and 3.3 kb (mutant 16). Thus these 2 H P R T - clones suffer from a partial deletion or rearrangement of hprt gene fragments. A limited sample of 10 of these induced mutant clones was selected for karyotypic analysis. The X chromosome of the Chinese hamster has a late replicating heterochromatic long arm. BrdU incorporation followed by F P G staining renders this arm faintly stained with 2 distinct dots (nucleolus organizers), therefore allowing an easy identification of the X chromosome. The short arm of the X
chromosome which contains the hprt gene is darkly stained and banded. We compared the X chromosome of 10 H P R T mutants to that of the wild-type V79 cells and did not find any difference in length or banding pattern. Discussion
Our results show that BLM induces mutations at the hprt locus in V79 Chinese hamster cells. Because of the selection conditions (10 # g / m l 6-thioguanine) it can be expected that the H P R T mutants are completely devoid of H P R T activity (Fuscoe et al., 1986). The mutagenic action of BLM is accompanied by a strong toxicity which possibly is a consequence of a clastogenic effect. In the range of concentrations used in our study, BLM induces many chromosome aberrations in V79 cells (Speit et al., 1984). It could be demonstrated here that BLM generates a broad spectrum of types of mutation and not only large chromosomal aberrations. The alteration of D N A sequences which can be detected with molecular methods may even be more relevant for genetic effects in offspring of BLM-treated cancer patients. The molecular analysis of the BLM-induced mutations revealed a high percentage of large changes in the hprt gene structure. 43% of the mutants analyzed showed partial or total deletion of the hprt coding sequences. In contrast, no changes in Southern blot hybridization pattern compared to the wild-type pattern were found in spontaneous mutations. In earlier reports on molecular characterization of hprt mutations in Chinese hamster cells, it was also found that large changes seldom occur in spontaneous mutations but are frequently induced by ionizing radiation (Vrieling et al., 1985; Fuscoe et al., 1986; Thacker 1986; Gibbs et al., 1987; Thacker and Ganesh, 1989). The frequency and distribution of partial and total deletions are similar in X-ray- and BLM-induced mutations. Thus, even at the D N A level it can be demonstrated that the DNAdamaging and mutagenic action of BLM is radiomimetic. In all cases, the actual incidence of deletions may be higher than indicated since small deletions are not detectable by Southern analysis. Although the use of 2 endonucleases (EcoRI and BgllI) is suitable to get both the coverage of the
166 whole functional gene and the potential to detect small alterations (Thacker, 1986), the limit of resolution of this approach is probably between 50 and 500 bases. The molecular mechanism by which large deletions as observed in our experiments occur is still unclear. It has been suggested that unsuccessful recombination-type repair may result in large deletions (Gibbs et al., 1987). However, lesions induced by radiation and BLM are mainly shortlived and do not stimulate recombination between replicated D N A molecules as measured by sisterchromatid exchange (Perry and Evans, 1975; Speit et al., 1984). BLM-induced D N A damage seems to be repaired by 2 processes which can be distinguished by their kinetics (Utsumi and Elkind, 1989) and there is evidence that polymerase fl is primarily responsible for repair synthesis induced by BLM (Miller and Chinault, 1982). Agents capable of inhibiting the resynthesis step of D N A excision repair induce aborted repair which is manifested as single-strand breaks at each initiated site. ddThd appears to inhibit specifically the fl polymerase in mammalian cells. However, we have been unable to demonstrate a ddThd effect on BLM-induced mutations. One possible explanation is that ddThd is not phosphorylated in m a m m a l i a n cells and thus inhibition of repair does not occur. We therefore did the same experiments with d d T T P but the mutation frequency was not influenced at all (data not shown). It may be that inhibition of polymerase fl increases the amount of lethal but non-mutagenic lesions because after treatment with ddThd or d d T T P reduced survival was observed. Both inhibitors increased the frequency of sister chromatid exchanges under the same experimental conditions indicating that they are taken up by the cells and interact with the D N A metabolism. We therefore conclude that the resynthesis step of BLM-induced DNA-repair is not critical for mutagenesis. Besides other biological effects, araC inhibits polymerase et. In the presence of araC, repairing sites are held open, resulting in DNA-strand breaks. D N A break accumulation with araC is strongest after treatment with UV or UV-like damaging agents, i.e., it correlates with the occurrence of the 'long-patch' repair mode. BLM-induced D N A damage is mainly repaired by the
'short-patch' pathway and araC did not influence the BLM-induced mutation frequency in our experiments. These findings may indicate that araC does not interact with BLM-induced repair. But it could be shown that treatment with araC following X-irradiation of human lymphocytes in the G 0 stage of the cell cycle increased the yield of induced chromosome aberrations (Natarajan et al., 1986). This effect was found with high araC concentrations (5 x 10 4 M) which were toxic for V79 cells. The inefficiency of araC treatment in our experiments with BLM can also be explained by differences in the repair capacity of lymphocytes and V79 cells, differences between resting and proliferating cells or differences between the origin of gene and chromosome mutations. As araC enhanced ethyl methanesulfonate (EMS)-induced hprt mutations in V79 cells (Tachi-Shinkawa et al., 1987) the type of mutagenic lesion seems to be of special importance. 3AB inhibits poly(ADP-ribose) synthesis and the ligation step of D N A repair (Creissen and Shall, 1982). D N A ligase II is involved in both the 'short-patch' and the 'long-patch' repair. 3AB potentiated the cytotoxic and clastogenic effects of certain D N A - d a m a g i n g agents and increased EMS-induced mutations at the hprt locus (Schwartz et al., 1985). As 3AB only had an effect on 6-thioguanine-resistant mutations which are point mutations or deletions and did not increase the frequency of ouabain-resistant cells, which only arise through point mutations, it was suggested that 3AB increases the incidence of deletion mutations (Schwartz et al., 1985). Our results with BLM confirm the comutagenic action of 3AB and provide direct evidence for the occurrence of deletions. After combined treatment with 3AB (10 -3 M) the mutation frequency is nearly doubled and the amount of deletions increased from 43% to 70%. Thus most of the additionally found mutations arose through deletions. These findings indicate that increasing the frequency and duration of DNA-strand breaks at the site of ligation can be manifested as deletion mutations. Obviously, delayed rejoining of D N A - s t r a n d breaks allows more time for the formation of abnormal rearrangements between broken D N A molecules. Possibly the same mechanism causes the increased yield of mutagen-induced chromosome aberrations ob-
167 s e r v e d a f t e r 3 A B t r e a t m e n t . H o w e v e r , t h e 10 mutant clones analyzed here did not show aberrat i o n s o f the X c h r o m o s o m e . B e s i d e s t h e l i m i t e d s a m p l e size this m a y b e d u e to t h e d i f f e r e n t resol u t i o n o f the 2 m e t h o d s . W h i l e a d e l e t i o n o f a c h r o m o s o m a l b a n d s p a n s m o r e t h a n 1000 kb, t h e d e l e t i o n s r e p o r t e d h e r e m a y b e in the r a n g e o f 30 k b ( W h a l e y a n d Little, 1990). T a k e n t o g e t h e r , o u r results i n d i c a t e t h a t t h e c o m u t a g e n i c e f f e c t o f i n h i b i t o r s o f D N A - r e p a i r is d u e to a n i n c r e a s e in specific types of mutation. Thus, investigations of interactions between mutagens and repair inhibitors c o n t r i b u t e to a b e t t e r u n d e r s t a n d i n g o f t h e r o l e o f D N A - r e p a i r in t h e p r o c e s s o f m u t a g e n e s i s .
Acknowledgement T h e skillful t e c h n i c a l a s s i s t a n c e o f M r s . S a b i n e H a u p t e r is h i g h l y a p p r e c i a t e d .
References Albertini, R.J., I.N. Gennett, B. Lambert, W.G. Thilly and H. Vrieling (1989) Mutation at the hprt locus, Mutation Res., 216, 65-88. Creissen, D., and S. Shall (1982) Regulation of DNA ligase activity by poly (ADP-ribose), Nature (London), 296, 271272. Collins, A., C.S. Dowries and R.T. Johnson (1984) DNA Repair and its Inhibition, IRL Press, Oxford, Washington, DC. Einhorn, L.H. (1981) Testicular cancer as a model for curable neoplasm, Cancer Res., 41, 3275-3280. Fuscoe, J.C., C.H. Ockey and M. Fox (1986) Molecular analysis of X-ray-induced mutants at the HPRT locus in V79 Chinese hamster cells, Int. J. Radiat. Biol., 49, 1011-1020. Gibbs, R.A., J. Camakarist, G.S. Hodgson and R.F. Martin (1987) Molecular characterization of 125I decay and X-rayinduced HPRT mutants in CHO cells, Int. J. Radiat. Biol., 51, 193-199. Miller, M.R., and D.N. Chinault (1982) Evidence that DNA polymerase a and fl participate differentially in DNA repair synthesis induced by different agents, J. Biol. Chem., 257, 46-49. Natarajan, A.T., L.F.H. Mullenders and T.S.B. Zwanenburg (1986) Modulation of mutagen-induced biological effects by inhibitors of DNA repair, in: Genetic Toxicology of Environmental Chemicals, Part A: Basic Principles and Mechanisms of Action, pp. 373-384.
Perry, P., and H.J. Evans (1975) Cytological detection of mutagen-carcinogen exposure by sister chromatid exchange, Nature (London), 258, 121-125. Schwartz, J.L., W.F. Morgan, P. Brown-Lindquist, V. Afzal, R.R. Weichselbaum and S. Wolff (1985) Comutagenic effects of 3-aminobenzamide in Chinese hamster ovary cells, Cancer Res., 45, 1556-1559. Speit, G., and S. Haupter (1985) On the mechanism of differential Giemsa staining of bromodeoxyuridine-substituted chromosomes. II. Differences between the demonstration of sister chromatid differentiation and replication patterns, Hum. Genet., 70, 126-129. Speit, G., R. Hochsattel and W. Vogel (1984) The contribution of DNA single-strand breaks to the formation of chromosome aberrations and SCEs, in: R. Tice and A. Hollaender (Eds), Sister Chromatid Exchanges, Plenum, New York, pp. 229-243. Stout, J.T., and C.T. Caskey (1988) The Lesch-Nyhan syndrome: clinical, molecular and genetic aspects, Trends Genet., 4, 175-178. Tachi-Shinkawa, K., Y. Kuroda, K. Morimoto and A. Koizumi (1987) Enhancing effects of cytosine arabinoside on ethyl methanesulfonate induced 6-thioguanine resistance mutations in Chinese hamster V79 cells, Mutation Res., 191, 37-40. Takeshita, M., A.P. Groilman, E. Ohtusbo and H. Ohtusbo (1978) Interaction of bleomycin with DNA, Proc. Natl. Acad. Sci. (U.S.A.), 75, 5983-5987. Thacker, J. (1986) The nature of mutants induced by ionizing radiation in cultured hamster cells. III. Molecular characterization of HPRT deficient mutants induced by "t-rays or a-particles showing that the majority have deletions of all or part of the hprt gene, Mutation Res., 160, 267-275. Thacker, J., and A.N. Ganesh (1989) Molecular analysis of spontaneous and ethylmethanesulphonate-induced mutations of the hprt gene in hamster cells, Mutation Res., 210, 103-112. Utsumi, H., and M.M. Elkind (1989) Bleomycin-induced potentially lethal damage and its repair, Radiat. Res., 119, 534-541. Vrieling, H., J.W.I.M. Simons, F. Arwert, A.T. Natarajan and A.A van Zeeland (1985) Mutations induced by X-rays at the HPRT locus in cultured Chinese hamster cells are mostly large deletions, Mutation Res., 144, 281-286. Whaley, J.M., and J.B. Little (1990) Molecular characterization of hprt mutants induced by low- and high-LET radiations in human cells, Mutation Res., 243, 35-45. Wood C.M., and R.E. Moses (1989) Ethyl methane sulfonateand bleomycin-generated deletion mutations at HPRT locus in xeroderma pigmentosum complementation group D fibroblasts, Somat. Cell Mol. Genet., 15, 345-357.
161
MUT 04989
Molecular characterization of mutations at the hprt locus in V79 Chinese hamster cells induced by bleomycin in the presence of inhibitors of DNA repair Beate KOberle and Giinter Speit Abteilung Klinische Genetik, Universitiit Ulm, D- 7900 Ulm (F. R. G.)
(Received 25 September 1990) (Revision received 10 December 1990) (Accepted 7 January 1991)
Keywords: Hprt locus; Mutation; DNA-repair inhibitors; Bleomycin; Southern analysis; V79 cells
Summary The molecular basis of bleomycin (BLM)-induced mutations in the absence and presence of inhibitors of D N A repair was investigated in V79 cells with Southern hybridization techniques. 43% of the BLM-induced thioguanine-resistant mutants suffer from large alterations of hprt DNA sequences. To understand the role of DNA repair in the process of mutagenesis, the effect of inhibitors of D N A repair on the frequency and types of BLM-induced mutations was tested. The inhibitors used were arabinofuranosyl cytosine (araC), didesoxythymidine (ddThd) and 3-aminobenzamide (3AB), which inhibit different steps of excision repair. Only 3AB caused a comutagenic effect. The increased mutation frequency was mainly due to additionally induced gene deletions. In the presence of 3AB, 70% of the HPRT-deficient mutants revealed partial or total deletions of the hprt coding sequences. Thus, it could be shown that BLM induces a broad range of types of mutation and that inhibited repair of BLM-induced D N A damage leads to specific types of mutations.
In order to understand the mechanisms of mutagenesis it is important to analyze the nature of spontaneous and induced mutations at the molecular level. The hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene locus has been used extensively for mutagenesis studies in mammalian cells (for review see Albertini et al., 1989). The hprt gene is an excellent choice for studying the process of mutagenesis, owing to its large size
Correspondence: Dr. G. Speit, Abteilung Klinische Genetik, Universifiit Ulm, Postfach 4066, D-7900 Ulm (F.R.G.).
( > 30 kb), X-hnked location and homology in mammals, ease of mutant selection and availability of probes and sequence data. H P R T is an enzyme that catalyzes the reutilization of preformed purine bases. In man, complete H P R T deficiency results in the L e s c h - N y h a n syndrome and it has been shown that this phenotype is the result of a heterogenous group of mutations (for review see Stout and Caskey, 1988). In a number of studies, mutation induction by ionizing radiation has been investigated at the D N A level. Using a cloned e D N A and Southern blot analysis, it could be demonstrated that the
162 majority of these mutations are caused by large deletions (Vrieling et al., 1985; Thacker, 1986; Fuscoe et al., 1986; Whaley and Little, 1990). In contrast, mutants induced by UV irradiation or alkylating agents almost all seem to arise from point mutations (Vrieling et al., 1988; Albertini et al., 1989; Thacker and Ganesh, 1989). Thus the hprt gene mutation system is able to detect different types of mutation and is a sensitive method for distinguishing the mutagenic action of different DNA-damaging agents. We therefore tried to characterize the mutagenic action of the antitumor drug bleomycin (BLM) in V79 Chinese hamster cells. BLM is widely used as part of a combination therapy of advanced testicular cancer in young men. As a cure is possible for over 90% of the patients (Einhorn, 1981), the mutagenic action of BLM is relevant with respect to the development of secondary malignancies and the genetic risk for offspring of surviving cancer patients. BLM has been shown to produce chromosome aberrations comparable to those induced by X-irradiation and is also known to induce DNA breaks through the production of free radicals (Takeshita et al., 1978) similar to the indirect action of radiation. In a recent molecular study (Wood and Moses, 1989) it was found that bleomycin generated deletion mutations at the hprt locus in xeroderma pigmentosum cells. Not only with regard to the types of damage it produces does bleomycin appear to be similar to X-rays, the enzymatic mechanisms for the repair of BLM and X-ray damage also seem to be essentially the same (Utsumi and Elkind, 1989). Our understanding of the biochemical basis of D N A repair in mammalian cells has been greatly enhanced over the past several years through the use of specific enzyme inhibitors. A role in D N A repair could be established for DNA polymerase a, D N A polymerase fl and poly(ADP-ribose)/ D N A ligase (for review see Collins et al., 1984). We used specific inhibitors for each of these 3 processes to analyze the relative importance of these steps for the repair of BLM-induced D N A damage. We investigated the consequences of inhibited repair on the frequencies and types of mutation at the cellular and molecular levels. The results contribute to a better understanding of the mechanisms of mutation and the influence of D N A repair.
Materials and methods
Cell culture and mutation experiments The experiments were performed with a clonal derivative of the V79 Chinese hamster cell line (provided by Dr. D. Wild, Freiburg). Cells were cultivated in minimal essential medium (MEM) with Earle's salts supplemented with 10% fetal calf serum and antibiotics in a humidified incubator at 37°C with 5% CO 2 at a p H of 7.2. Mutagenicity experiments were performed in 175-cm2 flasks (Nunc). About 5 x 10 6 cells were treated in each experiment according to one of the following protocols: (i) treatment for 21 h with one of the repair inhibitors; (ii) treatment for 21 h with bleomycin alone; (iii) treatment with bleomycin in combination with one of the repair inhibitors. The inhibitors used were arabinofuranosyl cytosine (araC), didesoxythymidine (ddThd) and 3-aminobenzamide (3AB). Bleomycin (BLM) and the inhibitors were purchased from Sigma. Survival (relative plating efficiency.) was determined by plating 200 cells into 4 replica petri dishes (60 x 15 mm) at the end of mutagen treatment. Colonies were fixed, Giemsa-stained and counted 7 days later. The treated cultures were transferred as needed during the expression period. After 7 days, 103 cells were replated into 5 replica petri dishes (100 x 15 mm) with selective medium (10/~g/ml 6-thioguanine). At the time of replating into selective medium, the plating efficiency was determined in non-selective medium (four 60-mm replica petri dishes with 200 cells each). After 1 week, thioguanine-resistant colonies were isolated from different petri dishes and cultivated in normal medium for the molecular analysis. For the determination of the mutation frequency, the colonies were fixed with methanol, stained with Giemsa and counted. Mutation frequencies were corrected for cell survival. DNA isolation DNA was isolated from wild-type V79 cells and H P R T mutants (6-TG-resistant colonies) by treatment with protease K (100 # g / m l ) , 1% SDS and homogenization buffer (100 mM NaC1, 10 mM Tris, p H 7.6, 10 mM EDTA, p H 7.6) for 1 h at 37°C. After phenol extraction (3 times to purify
163
DNA) the D N A solution was dialyzed for 3 x 8 h to STE buffer (100 mM NaC1, 10 mM Tris, pH 7.6, 1 mM EDTA, pH 7.6), followed by treatment with RNase (100 ~ g / m l ) for 1 h at 37°C and protease K (100 /~g/ml) for 1 h at 37°C. D N A solution was again purified by phenol extraction, followed by chloroform/isoamylalcohol (4%) extraction and precipitated by 1 / 1 0 volume 1 M K-acetate and 2 volumes 100% cold ethanol. D N A was stored at 4°C in 10 mM Tris (pH 7.6)-1 mM EDTA (pH 7.6).
Restriction endonuclease digestion and Southern blot High-molecular weight DNA (15 ~g) was digested with the restriction endonucleases for 5 h under conditions recommended by the manufacturer (Boehringer Mannheim). The fragments were separated on 1% agarose gel at 40 V for 16 h. The gel was washed in 0.25 M HC1 for 30 min and blotted for 4 h with N a O H (0.4 M) onto nylon filter (Hybond N ÷, Amersham). The filter was washed in 2 x SSC for 10 rain.
Nick translation and filter hybridization 270 Fg of a full-length hamster hprt cDNA cloned into pBR322 (pHPT12, kindly supplied by Dr. J. Thacker) was nick-translated using 30 /xCi [32p]dCTP at 15°C with nick-translation kit (Boehringer Mannheim). The filter was prehybridized for 1 h at 65°C in 5 x SSPE, 5 x Denhardt's solution, 0.5% SDS, then the nick-translated probe (preboiled for 10 min) was added and the filter was hybridized for about 16 h at 65°C. The filter was then washed in 2 changes of 2 x SSC, 0.1 x SDS for 5 min, 0.1 × SSC, 0.1% SDS for 15 rain at 55°C, 15 min at 60°C and 10 min at 65°C. The filter was then exposed to X-ray film for several days at - 7 0 ° C .
Molecular weight markers The sizes of hprt gene fragments were estimated with reference to fragments of known size from molecular-weight markers (Boehringer).
Cytogenetic analysis Cytogenetic analysis was done by ing. Cells were cultivated with BrdU for 5 h, chromosome preparations according to standard procedures and
RBG-band(10 /~g/ml) were made stained with
a fluorescence plus Giemsa (FPG) technique (Speit and Haupter, 1985). Results
Treatment of V79 cells with BLM for 21 h resulted in an increased frequency of mutations at the hprt locus. 10 /~g/ml BLM caused 26-42 mutants/106 surviving cells (Table 1). This treatment reduced initial cell survival to values between 13% and 22%. Lower BLM concentrations influenced mutation frequency only marginally and a higher concentration (20 /xg/ml) caused more mutations but at the same time showed high toxicity (relative plating efficiency below 10%). Therefore we chose the BLM concentration of 10 /~g/ml for our experiments, which are summarized in Table 1. The DNA-repair inhibitors 3AB, araC and ddThd were not mutagenic under the present conditions. Only araC reduced survival of V79 cells (to 40% PE at 2 x 1 0 - 7 M ) . After combined treatment, 3AB slightly increased the toxicity of BLM and showed a comutagenic effect. Mutation frequencies were nearly doubled with 10 -3 M
TABLE I EFFECTS O F I N H I B I T O R S OF D N A - R E P A I R O N BLMI N D U C E D M U T A T I O N S IN V79 CELLS Mutagen treatment
Relative plating efficiency (%)
None
100
3
3AB (10 -3 M) 3AB ( 2 × 10 -3 M) BLM (10 ~ g / m l ) BLM + 3AB (10 -3 M) B L M + 3 A B ( 2 x 10 -3 M)
100 100 19 14 13
0 0 26 46 81
araC (10 - 7 M) araC ( 2 x 10 - 7 M) BLM (10/xg/ml) BLM + araC (10 -7 M) B L M + araC ( 2 x 1 0 -7 M)
85 40 22 20 8
2 5 36 42 40
100 90 22 17 11
3 6 31 40 42
ddThd (5 × 10 - 4 M) ddThd (10- 3 M) BLM (10 # g / m l ) BLM + ddThd ( 5 × 1 0 - 4 M) BLM + ddThd (10- 3 M)
Frequency of 6-TG-resistant cells/10 6
164
kb
M C I! 12 13 14 15 1617 18 19
kb
23.19.46.5-
51 C II 12 13 14 15 16 17 18 19
21.2-
4.3-
5.1~ 4.94.23.5m n
2.01.91.51.3-
Fig. 1. Southern blot hybridization of a hprt gene probe to genomic D N A of 9 HPRT-deficient m u t a n t s induced by bleomycin in the presence of 3-aminobenzamide. D N A was digested with the restriction endonuclease EcoRl. M, molecular-weight markers; C, control (V79 parent cell).
Fig. 2. Southern blot hybridization analysis as in Fig. 1, but genomic D N A was digested with Bglll.
with EcoRI and hybridized to p H P T 12, 3 bands located at 17.5 kb, 11.3 kb and 1.2 kb were visualized by autoradiography. The 2 main bands at 17.5 kb and 11.3 kb are part of the functional hprt gene whereas the small fragment at 1.2 kb represents a pseudogene (Fuscoe et al., 1983; Thacker, 1986). BglII digestion revealed 6 fragments. The 4 bands located at 4.3, 2.1, 1.5 and 1.2 kb represent the functional hprt gene, while the other 2 bands (at 3.4 and 1.9 kb) indicate pseudogene sequences. Each mutant D N A was digested with these 2 endonucleases (separately) and the resulting pat-
3AB and tripled with 2 x 10 _3 M 3AB. Simultaneous treatment with BLM and araC or BLM and ddThd caused enhanced toxicity but had no clear effect on the mutation frequency. F r o m the experiments with 3AB, mutant clones were isolated from controls, BLM-treated cultures and from cultures treated with BLM plus 3AB (10-3 M). The overall results of the Southern blot analysis are shown in Table 2 and examples of the data are illustrated in Figs. 1 and 2. When genomic D N A from wild-type V79 cells was cleaved
TABLE 2 A N A L Y S I S O F D N A F R O M H P R T - D E F I C I E N T M U T A N T S O F V79 CELLS F O R C H A N G E S IN hprt G E N E S T R U C T U R E Inducing
N u m b e r of
N u m b e r of m u t a n t s with large changes
agent
m u t a n t s examined
total deletions
partial deletions/ rearrangements
%
N u m b e r of m u t a n t s with no detectable changes
Control BLM 3AB + BLM
20 30 30
0 9 14
0 4 8
0 43 70
20 17 8
165 terns allowed the differentiation of 3 types of mutation: (i) apparently total gene deletions which lacked all functional hprt sequences; (ii) partial deletions a n d / o r rearrangements of the hprt gene; (iii) mutants without detectable loss of gene sequences. The restriction pattern of all 20 spontaneous H P R T mutants analyzed could not be distinguished from the V79 wild-type pattern, indicating that point mutations or small deletions ( > 300 bp) are responsible for the H P R T phenotype. Among the BLM-induced mutants, 57% showed the wild-type pattern, while 43% revealed changes in the fragment pattern: 9 out of 30 mutants apparently were total gene deletions and 4 were partial deletions. After combined treatment with 3AB the portion of mutants with large changes was increased from 43% to 70%. 14 out of 30 mutants lacked all functional gene fragments, 8 out of 30 lacked parts of the hprt coding sequences and only 8 out of 30 showed the wild-type pattern. Figs. 1 and 2 show examples of this type of result from EcoRI and BgllI digests of mutants induced by BLM in combination with 3AB. The EcoRI digest (Fig. 1) reveals that mutants 11, 14, 15 and 18 lack all functional gene fragments, only the pseudogene fragment at 1.2 kb is seen. Mutants 12, 17 and 19 have restriction fragment patterns identical to that of V79 D N A while the patterns of mutants 13 and 16 indicate a reduction in size of the 2 fragments. Hybridization analysis with the same set of mutants digested with BgllI confirmed the total deletion of hprt coding sequences in mutants 11, 14, 15 and 18 and the wild-type pattern in mutants 12, 17 and 19. Mutants 13 and 16 both lack the fragment at 1.5 kb but have additional bands at 3.2 kb (mutant 13) and 3.3 kb (mutant 16). Thus these 2 H P R T - clones suffer from a partial deletion or rearrangement of hprt gene fragments. A limited sample of 10 of these induced mutant clones was selected for karyotypic analysis. The X chromosome of the Chinese hamster has a late replicating heterochromatic long arm. BrdU incorporation followed by F P G staining renders this arm faintly stained with 2 distinct dots (nucleolus organizers), therefore allowing an easy identification of the X chromosome. The short arm of the X
chromosome which contains the hprt gene is darkly stained and banded. We compared the X chromosome of 10 H P R T mutants to that of the wild-type V79 cells and did not find any difference in length or banding pattern. Discussion
Our results show that BLM induces mutations at the hprt locus in V79 Chinese hamster cells. Because of the selection conditions (10 # g / m l 6-thioguanine) it can be expected that the H P R T mutants are completely devoid of H P R T activity (Fuscoe et al., 1986). The mutagenic action of BLM is accompanied by a strong toxicity which possibly is a consequence of a clastogenic effect. In the range of concentrations used in our study, BLM induces many chromosome aberrations in V79 cells (Speit et al., 1984). It could be demonstrated here that BLM generates a broad spectrum of types of mutation and not only large chromosomal aberrations. The alteration of D N A sequences which can be detected with molecular methods may even be more relevant for genetic effects in offspring of BLM-treated cancer patients. The molecular analysis of the BLM-induced mutations revealed a high percentage of large changes in the hprt gene structure. 43% of the mutants analyzed showed partial or total deletion of the hprt coding sequences. In contrast, no changes in Southern blot hybridization pattern compared to the wild-type pattern were found in spontaneous mutations. In earlier reports on molecular characterization of hprt mutations in Chinese hamster cells, it was also found that large changes seldom occur in spontaneous mutations but are frequently induced by ionizing radiation (Vrieling et al., 1985; Fuscoe et al., 1986; Thacker 1986; Gibbs et al., 1987; Thacker and Ganesh, 1989). The frequency and distribution of partial and total deletions are similar in X-ray- and BLM-induced mutations. Thus, even at the D N A level it can be demonstrated that the DNAdamaging and mutagenic action of BLM is radiomimetic. In all cases, the actual incidence of deletions may be higher than indicated since small deletions are not detectable by Southern analysis. Although the use of 2 endonucleases (EcoRI and BgllI) is suitable to get both the coverage of the
166 whole functional gene and the potential to detect small alterations (Thacker, 1986), the limit of resolution of this approach is probably between 50 and 500 bases. The molecular mechanism by which large deletions as observed in our experiments occur is still unclear. It has been suggested that unsuccessful recombination-type repair may result in large deletions (Gibbs et al., 1987). However, lesions induced by radiation and BLM are mainly shortlived and do not stimulate recombination between replicated D N A molecules as measured by sisterchromatid exchange (Perry and Evans, 1975; Speit et al., 1984). BLM-induced D N A damage seems to be repaired by 2 processes which can be distinguished by their kinetics (Utsumi and Elkind, 1989) and there is evidence that polymerase fl is primarily responsible for repair synthesis induced by BLM (Miller and Chinault, 1982). Agents capable of inhibiting the resynthesis step of D N A excision repair induce aborted repair which is manifested as single-strand breaks at each initiated site. ddThd appears to inhibit specifically the fl polymerase in mammalian cells. However, we have been unable to demonstrate a ddThd effect on BLM-induced mutations. One possible explanation is that ddThd is not phosphorylated in m a m m a l i a n cells and thus inhibition of repair does not occur. We therefore did the same experiments with d d T T P but the mutation frequency was not influenced at all (data not shown). It may be that inhibition of polymerase fl increases the amount of lethal but non-mutagenic lesions because after treatment with ddThd or d d T T P reduced survival was observed. Both inhibitors increased the frequency of sister chromatid exchanges under the same experimental conditions indicating that they are taken up by the cells and interact with the D N A metabolism. We therefore conclude that the resynthesis step of BLM-induced DNA-repair is not critical for mutagenesis. Besides other biological effects, araC inhibits polymerase et. In the presence of araC, repairing sites are held open, resulting in DNA-strand breaks. D N A break accumulation with araC is strongest after treatment with UV or UV-like damaging agents, i.e., it correlates with the occurrence of the 'long-patch' repair mode. BLM-induced D N A damage is mainly repaired by the
'short-patch' pathway and araC did not influence the BLM-induced mutation frequency in our experiments. These findings may indicate that araC does not interact with BLM-induced repair. But it could be shown that treatment with araC following X-irradiation of human lymphocytes in the G 0 stage of the cell cycle increased the yield of induced chromosome aberrations (Natarajan et al., 1986). This effect was found with high araC concentrations (5 x 10 4 M) which were toxic for V79 cells. The inefficiency of araC treatment in our experiments with BLM can also be explained by differences in the repair capacity of lymphocytes and V79 cells, differences between resting and proliferating cells or differences between the origin of gene and chromosome mutations. As araC enhanced ethyl methanesulfonate (EMS)-induced hprt mutations in V79 cells (Tachi-Shinkawa et al., 1987) the type of mutagenic lesion seems to be of special importance. 3AB inhibits poly(ADP-ribose) synthesis and the ligation step of D N A repair (Creissen and Shall, 1982). D N A ligase II is involved in both the 'short-patch' and the 'long-patch' repair. 3AB potentiated the cytotoxic and clastogenic effects of certain D N A - d a m a g i n g agents and increased EMS-induced mutations at the hprt locus (Schwartz et al., 1985). As 3AB only had an effect on 6-thioguanine-resistant mutations which are point mutations or deletions and did not increase the frequency of ouabain-resistant cells, which only arise through point mutations, it was suggested that 3AB increases the incidence of deletion mutations (Schwartz et al., 1985). Our results with BLM confirm the comutagenic action of 3AB and provide direct evidence for the occurrence of deletions. After combined treatment with 3AB (10 -3 M) the mutation frequency is nearly doubled and the amount of deletions increased from 43% to 70%. Thus most of the additionally found mutations arose through deletions. These findings indicate that increasing the frequency and duration of DNA-strand breaks at the site of ligation can be manifested as deletion mutations. Obviously, delayed rejoining of D N A - s t r a n d breaks allows more time for the formation of abnormal rearrangements between broken D N A molecules. Possibly the same mechanism causes the increased yield of mutagen-induced chromosome aberrations ob-
167 s e r v e d a f t e r 3 A B t r e a t m e n t . H o w e v e r , t h e 10 mutant clones analyzed here did not show aberrat i o n s o f the X c h r o m o s o m e . B e s i d e s t h e l i m i t e d s a m p l e size this m a y b e d u e to t h e d i f f e r e n t resol u t i o n o f the 2 m e t h o d s . W h i l e a d e l e t i o n o f a c h r o m o s o m a l b a n d s p a n s m o r e t h a n 1000 kb, t h e d e l e t i o n s r e p o r t e d h e r e m a y b e in the r a n g e o f 30 k b ( W h a l e y a n d Little, 1990). T a k e n t o g e t h e r , o u r results i n d i c a t e t h a t t h e c o m u t a g e n i c e f f e c t o f i n h i b i t o r s o f D N A - r e p a i r is d u e to a n i n c r e a s e in specific types of mutation. Thus, investigations of interactions between mutagens and repair inhibitors c o n t r i b u t e to a b e t t e r u n d e r s t a n d i n g o f t h e r o l e o f D N A - r e p a i r in t h e p r o c e s s o f m u t a g e n e s i s .
Acknowledgement T h e skillful t e c h n i c a l a s s i s t a n c e o f M r s . S a b i n e H a u p t e r is h i g h l y a p p r e c i a t e d .
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