Inhibition of DNA synthesis is not sufficient to cause mutagenesis in Chinese Hamster cells

CNRS symposium, May 1982 - TOULOUSE

BIOCHIMIE, 1982, 64, 809-810.

Inducible responses to D N A damages

Inhibition of DNA synthesis is not sufficient to cause mutagenesis in Chinese Hamster cells. Toby G. ROSSMAN o and Donna S. STONE-WOLFF.

Departement o[ Environmental Medicine, New York University School of Medicine, 550 First Avenue, New Y o r k - New Y o r k 10016 U.S.A.

R~sum~.

Summary.

Des experiences ont ~td mises au point a[in de dOterminer si l'inhibition de la synthkse du D N A dans des cellules de hamsters chinois V79 provoquerait une augmentation de mutagenkse par un mgcanisme similaire ~ celui de la <~r~paration SOS ~ chez E. coli. Le traitement des cellules pendant 16 heures avec un excOs des d£soxynucl~osides TdR, UdR, A d R et GdR se r~vkle mutagkne, alors qu' aucun effet mutagkne ne peut Otre d£montr~ h la suite d'un traitement par l'hydroxyur~e. La mutag~nicitd de TdR ne peut pas Otre revers~e par l'addition de CdR. Chez E. coli l'inhibition de la synthkse du D N A par une courte exposition gz l'hydroxyur~e a pour r~sultat l'induction du prophage lambda et une mutagenkse augment~e.

Experiments were designed to test whether the inhibition o[ D N A synthesis in Chinese hamster V79 cells would result in increased mutagenesis by a mechanism similar to <> in E. coli. Treatment o] cells [or 16 hours with excess of the deoxynucleosides TdR, UdR, A d R and GdR was mutagenic, whereas treatment with hydroxyurea demonstrated no mutagenic effect The mutagenicity o[ T d R could be reversed by the addition of CdR. In E. coli, inhibition o[ D N A synthesis by a short exposure to hydroxyurea resulted in the induction o[ ), prophage and increased mutagenesis.

Ces rdsultats montrent qu'alors que chez E coli la presence d'une [ourche de r£plication bloqude peut provoquer une mutagenkse par l'induction du <>, ce ph~nomkne ne semble pas se produire dans les celhdes de hamster chinois V79. Le m~canisme mutagdnique de fortes concentrations de d~soxynucIdosides dans les cellules de hamster chinois V79 est vraisemblablement dft ?l des erreurs de r@lication r~sultant d'alt&ations dans les < de d~soxynucl~osides.

These results show that whereas the presence o[ a stalled replication fork in E. coli can result in mutagenesis via induction of the <~SOS system >>, the same phenomenon does not seem to occur in Chinese hamster V79 cells, The mutagenic mechanism of high concentrations of deoxynucleosides in Chinese hamster V79 cells is likely to be due to replication errors which result from alterations in deoxynuclotide pools,

Mots-cl~s : mutagen~se / ceUules de hamsters chinois / syst~me SOS / d~soxynucl~otides / hydroxyur~e.

Key-words : mutagenesis / Chinese hamster cells / SOS system / deoxynueleotides / hydroxyurea.

In E. coli, an error-prone DNA repair or replication system leading to mutagenesis is one of a number of induced functions known as the <> [1]. SOS functions are triggered concurrently by most treatments which damage DNA or halt its replication [1, 2]. The SOS system can be

induced by treatments which interrupt DNA synthesis without necessarily damaging the DNA molecule. It was of interest to determine whether a similar mechanism for mutagenesis exists in mammalian cells. D N A synthesis in animal cells can be blocked by high concentrations of thymidine (TdR), 2' deoxyadenosine (AdR), or 2' deoxyguanosine (GdR) in the medium [3-5]. The inhibition of DNA syn-

To whom all correspondence should be

sent.

810

T. G. R o s s m a n and D. S. Stone-Woll[.

thesis occurs via f e e d b a c k inhibition of r i b o n u c l e o tide reductase, causing a d e p l e t i o n in the d C T P p o o l [6]. H y d r o x y u r e a ( H U ) is also an inhibitor of ribon u c l e o t i d e reductase, but a p p e a r s to w o r k b y destroying the tyrosine r a d i c a l n e e d e d for enzyme a c t i o n [7]. A d d i t i o n of H U to c u l t u r e d ceils causes d e p l e t i o n of d G T P a n d d A T P p o o l s a n d the inhibition of D N A synthesis [8]. R e p o r t e d here are results of e x p e r i m e n t s designed to test w h e t h e r any or all of these inhibitors of D N A synthesis cause m u t a t i o n s in Chinese h a m s t e r V 7 9 cells. Effects of these agents on some SOS functions in E. coli are p r e s e n t e d for c o m p a rison.

Chemicals. Hydroxyurea and all 2' deoxynucleosides were purchased from Sigma Chemical Company, St. Lot~is, Missouri.

Results. E l f e c t s of h y d r o x y u r e a and deoxynucleosides on survival and D N A synthesis in V 7 9 cells. I n o r d e r to a v o i d excess toxicity, d o s e - r e s p o n s e curves representing 16 h o u r exposures to all d e o x y n u c l e o t i d e s and to H U were d e t e r m i n e d

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Materials and Methods. Animal cell mutagenesis. The origin and culture of Chinese hamster cell line V79 has been described previously [9]. Mutagenesis experiments using the ouabain resistance marker were performed as described by Chang et al. [10]. Survival plates were stained after seven days, and mutagenesis plates after 14 days, with a medium change at day seven. Thioguanine-resistant mutants were selected according to the method of Chang et al. [11], using the reptating technique. A seven-day e~press~on time was used. Mutation frequencies for this marker were calculated as mutants per 106 replated s~rvivors. Bacterial strains and media. E. coli strains WP2~ (trpE, uvrA) and WP44,-NF (trpE, uvrA, tifJ/sli-) were obtained from Dr. Evelyn Witkin, Rutgers Un'iversity. WP44s-NF amp r was isolated in our laboratory as a thermally-induced ampicillin resistant mutant of WP44s-NF. The lysogen WP2s 00 was isolated in our laboratory using a wild type )~ obtained from Dr. Evelyn Witkin. Cultures were grown in MST broth, consisting of Minimal Broth Davis (Difco) containing 0.2 per cent glucose and 20 Ixg/ml tryptophan. Nutrient plates contained nutrient broth solidified with 1.5 per cent agar. Soft agar containing 0.01 M MgSO, and 6.5 g Difco agar per liter of distilled water was used to plate phage and bacteria. Induction o] )~ prophage. The lysogen WP2~ (k) and indicator strain WP44~-NF amp~ were grown in MST to mid-exponential phase. After treatment ef the tysogen, infectious centers were assayed by adding 0.1 ml of a I0 -z dilution of lysogen, and 0.1 ml of undiluted indicator to 2.5 ml soft agar. This is poured onto nutrient broth plates containing 10 Ixg/ml ampicillin and incubated at 37 °. Since the indicator strain WP44~-NF amp r carries .the tif-1 mutation, lysogeny is not maintained at this temperature and clear plaques result, allowing ease ef scoring. BIOCHIMIE, 1982, 64, n ° 8-9.

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Fro. 1. - - The eHects of a 16 hour treatment with diJIerent concentrations ol CdR (©), UdR (0), TdR (A), GdR (11), AdR (cl), and t t U (&) on clonal survival.

(fig. 1). A s n o t e d p r e v i o u s l y [12], a 16 h o u r treatm e n t with 2 m M H U is very toxic, yielding a cloning efficiency of only 20 p e r cent. Of the deoxynucleosides, d e o x y a d e n o s i n e s h o w e d the m o s t toxicity. F r o m this data, it w a s d e t e r m i n e d that a 16 h o u r t r e a t m e n t with 1 m M of each of the d e o x y n u c l e o s i d e s or 0.2 m M h y d r o x y u r e a yields 8 0 - 1 0 0 p e r cent of c o n t r o l survival. This concent r a t i o n (1 m M ) of A d R and G d R h a d b e e n shown to inhibit D N A synthesis in other systems [4, 5]. W e f o u n d that t r e a t m e n t of V 7 9 cells for 16 hours with 1 m R A d R or G d R resulted in r e d u c t i o n s

Mutagenesis in Chinese hamster cells. of 97 and 79 per cent, respectively, of the rate of [3H]thymidine incorporation into cold, acid precipitable material. In addition, treatments with 1 m M C d R or U d R produced a similar inhibition (85 and 97 per cent, respectively). Inhibition of D N A synthesis by 1-2 m M thymidine has already been demonstrated in a variety of m a m m a l i a n cell lines, including Chinese hamster cells [8, 13]. We recently reported that 0.2 m M H U resulted in 95 per cent inhibition of D N A synthesis in V 7 9 cells [12].

811

EfJects of deoxynucleosides and hydroxyurea on mutagenesis in V79 cells. Table 1 shows the effects of a 16 hour treatment with 1 m M deoxynucleosides on mutagenesis using either resistance to ouabain ( O U A r) or 6-thioguanine ( 6 T G r) as genetic markers. In the ouabain selection system, only treatments with T d R and with G d R showed significant (p < 0.01) mutagenicity. W h e n the 6-thioguanine resistance m a r k e r was used, treatment with deoxyguanosine was

TABLE I.

Mutagenesis of deoxynucIeosides (16 hours, i mM) in V79 cells. Treatment

Per cent survival

Total number of mutants

Experiment I : OUA r marker None 100.0 (')UV (2 Jim "~) 85.4 CdR 100.0 UdR 100.0 AdR 75.1 GdR 98.3 TdR 63.6

0 45 0 0 2 17 12

Experiment II : 6TG~ marker None 100.0 (') UV (2 J/re'-') 90.3 CdR 95.5 TdR 88.7 UdR 100.0 AdR 72.6 GdR 75.0

1 527 4 37 65 67 173

Mutants / 10~ survivors

< 0.38 19.74 ~ 0.38 ~ 0.38 1.00 6.46 7.06

0 45

264.00 1.90 18.88 21.70 41.90 104.20

(*) Ultraviolet light (UV) was used as a positive control for mutagenesis in these experiments. TABLE II.

EJ[ects of thymidine and hydroxyurea on ouabain ~ mutagenesis in V79 cells. Treatment Experiment 1 None 0.2 mM TdR 0.5 mM TdR 0.1 mM HU 0.2 mM HU 0.3 mM HU

Per cent survival

Total number of mutants

Mutants/106 survivors

100.0 93.0 72.0 100.0 72.8 45.5

1 10 27 1 2 1

0.31 3.36 11.74 0.31 0.85 0.68

Experiment 2 None

0.2 mM TdR 0.5 mM TdR 0.025 mM CdR 0.2 mM TdR d0.025 mM CdR 0.5 mM TdR q0.025 mM CdR

BIOCHIMIE, 1982, 64, n ° 8-9.

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812

T. G. Rossman and D. S. Stone-Wol]].

highly mutagenic, and significant mutagenicity was also obtained with AdR, UdR and TdR. Chinese hamster cells exposed for a 16 hour treatment with even lower concentrations of T d R show an increase in the mutation frequency at the OUA ~ locus (table II, expt. 1). However, treatment with different concentrations of H U up to 0 . 3 m M had no significant effect on mutageuesis. Similar results were found at the H G P R T locus in Chinese hamster ovary cells [14]. 600

500

strains [16] suggesting induction of the < system. In confirmation of an earlier report [17], is our finding that a 20 minute exposure to H U causes the induction of X prophage in a dosedependent manner (fig. 2). Thymidine does not induce k prophage (fig. 2), nor does it inhibit DNA synthesis of growth at any concentration tested, probably due to its cleavage to thymidine phosphorylase [18]. A 20 minute exposure to 75 m M H U (an inhibitory concentration), followed by a fluctuation test [19] resulted in a significant increase in Trp + reversion (data not shown). The fluctuation test was more sensitive than agar plate methods for detecting Trp + revertants in cultures exposed to HU. High concentrations of T d R failed to increase Trp ÷ revertants in any protocol (data not shown).

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FIG. 2. - - E//ects of hydroxyurea and thymidine on the induction of )~ prophage. E. coli WP2~ (X) in exponential growth was e:~posed for 25 minutes to different concentrations of HU (0) or TdR (O), and assayed as in Methods. Thymidine is believed to inhibit DNA synthesis by depleting the deoxycytidine pools in the cell. The inhibition of DNA synthesis can be reversed by the addition of CdR to the medium [3]. Experiment 2 of table II shows that the addition of 0.025 mM CdR is not mutagenic alone, but has the effect of blocking the mutagenicity of thymidine. Effects of hydroxyurea and thymidine on E. coli SOS ]unctions. As a comparison, E. coil was treated with high concentrations of H U and T d R to determine the effects of these compounds on SOS functions. Hydroxyurea has been shown to inhibit DNA synthesis in E. coli [15]. Like other inhibitors of DNA synthesis, H U causes filamentation of IonBIOCHIMIE, 1982, 64, n ° 8-9.

There is evidence from studies in E. coli that a stalled replication fork generates degradation products in the form of oligonucleotides [20] and gapped DNA [21] which can act to derepress genes controlling SOS functions. One of these functions is thought to be a mutagenic mechanism for replication past non-coding DNA lesions [1, 2]. Since agents which can induce the SOS system need not cause damage to DNA, it follows that the resulting mutagenesis can occur on an undamaged template, and is therefore <>

[24]. Hydroxyurea causes induction of SOS functions in E. coli [16, 17] (fig. 2). Results reported here demonstrate that a mutator effect is not induced in Chinese hamster cells by hydroxyurea treatment (table II). This implies that, unlike the case in E. coli, a stalled replication fork is not sufficient to generate an SOS inducing signal in these cells. The mutagenesis seen as a result of excess deoxynucleotides is most likely the result of base imbalance rather than of inhibition of DNA synthesis. A similar conclusion with respect to excess thyrnidine was suggested by Bradley and Sharkey [14]. Our results differ from theirs in that we observe significant mutagenesis at much lower thymidine concentrations and at high levels of cell survival (table II). In addition, we demonstrate mutagenesis by excess UdR, AdR and G d R (table I). Since we are working at high levels of survival, the increased mutagenesis is unlikely to be due to preferential killing of wild-type cells, as suggested by Brennard and Fox [22]. Nucleotide pool imbalance has been shown to cause a decrease in the fidelity of DNA replication [23].

Mutagenesis in Chinese hamster cells.

It m a y be argued that m a m m a l i a n cells d o contain an inducible system l e a d i n g to u n t a r g e t e d m u tagenesis, b u t that this system c a n n o t be i n d u c e d m e r e l y b y inhibition of D N A synthesis. E x p e r i ments using viral p r o b e s have b e e n r e p o r t e d to s u p p o r t the c o n c e p t of an inducible, e r r o r - p r o n e system in m a m m a l i a n cells, b u t these e x p e r i m e n t s r e m a i n c o n t r o v e r s i a l [see review, 25]. C l e a v e r f o u n d no evidence for i n d u c t i o n of an e r r o r - p r o n e system b y X - r a y s in Chinese h a m s t e r o v a r y cells

[261. W e have r e c e n t l y isolated two m u t a n t clones of Chinese h a m s t e r V 7 9 cells lacking activity of h y p o x a n t h i n e guanine p h o s p h o r i b o s y l transferase ( H G P R T ) , which can be r e v e r t e d to H G P R T + specifically b y either base p a i r substitution o r b y frameshift m u t a g e n s (Stone-Wolff and R o s s m a n , in p r e p a r a t i o n ) . I n some p r e l i m i n a r y studies, we have f o u n d that ultraviolet light fails to m u t a t e either strain. This result suggests that the r e v e r s i o n of these strains is site specific (i.e., targeted) a n d that no general m u t a t o r effect is i n d u c e d in Chinese h a m s t e r V 7 9 cells in response to u l t r a violet irradiation.

Acknowledgments. We wish to thank Ms. Margarita Molina /or her expert technical assistance with the bacterial mutagenesis experiments. Primary funding ]or this research project was provided by the O]]ice o] Research and Development, Environmental Protection Agency, under Grant number R-808482 ; and is part of Center programs supported by Grant ES-00260 ]rom the National Institute o[ Environmental Health Sciences and by Grant CA 13343 ]rom the National Cancer Institute.

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BIOCHIM1E, 1982, 64, n ° 8-9.

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