Absence of interaction between X-rays and UV light in inducing ouabain- and thioguanine-resistant mutants in Chinese hamster cells

247

Mutation Research, 52 (1978) 247--253 © Elsevier/North-Holland Biomedical Press

ABSENCE OF INTERACTION BETWEEN X-RAYS AND UV L I G H T IN INDUCING OUABAIN- AND THIOGUANINE-RESISTANT MUTANTS IN CHINESE H A M S T E R CELLS

JAMES E. CLEAVER

Laboratory of Radiobiology, University of California, San Francisco, Calif. (U.S.A.) (Received 18 January 1978) (Revision received 5 May 1978) (Accepted 15 May 1978)

Summary Chinese hamster ovary cells were irradiated with X-rays at times from 0 to 17 h before being irradiated with ultraviolet (UV) light. No synergism was observed between the t w o radiations for the production of mutants resistant to either ouabain or 6-thioguanine. These experiments were designed to test whether X-rays induced an error-prone repair system that would increase the frequency of mutations produced by UV light, b u t no such system was detected.

Introduction

Most mutations in prokaryotes appear to be produced b y errors in a DNA replication system that is induced by radiations or chemical mutagens [14,16, 18,19,23--25]. Considerable interest has been aroused as to whether mammalian cells contain a similar inducible, error-prone repair system. But because eukaryotic cells do n o t show the characteristic prokaryotic response of DNA degradation after being damaged [2,4], and because the inducible system in prokaryotes appears to be partly involved with this degradation [8], inducible repair, if it exists in eukaryotes, m a y be substantially different from that of prokaryotes. A serious difficulty in designing experiments to detect an inducible errorprone repair system is that the same damage to DNA can be both a potential inducer of the repair system and the damage on which the induced system acts. Therefore, in the experiments described here, I used two different agents: X-irradiation to induce the putative repair system w i t h o u t inducing a large Work p e r f o r m e d

u n d e r t h e auspices o f the U , S . D e p a r t m e n t o f Energy.

248 number of mutants, and ultraviolet (UV) light to cause the damage on which the putative inducible repair system would then act. The effect of X-irradiation at various times before UV irradiation was determined by measuring the frequencies of two kinds of drug-resistant mutants produced by these radiations - - m u t a n t s resistant to ouabain and mutants resistant to 6-thioguanine. It has been suggested that cells can be made resistant to ouabain by point mutations, which can be produced by UV light [1,3,21] but cannot be made resistant to ouabain by deletions or frameshifts, which are the predominant modes of action of X-rays and some chemicals [1,3,7,21]. Cells can be made resistant to 6-thioguanine, however, by many kinds of mutations that result in either loss or changes in function of hypoxanthine-guanine phosphoribosyl transferase. In the experiments reported here, X-rays caused no mutations at one of the loci studied, that of ouabain resistance. Therefore, any synergism between X-rays and UV light in inducing mutations in these experiments could constitute evidence for induction of an error-prone repair system that increases the number of mutations produced by UV light. Materials and methods Chinese hamster ovary (CHO) cells were grown in Eagle's minimum essential medium plus 15% fetal calf serum. Under routine conditions their doubling time was 12 h and the plating efficiency was between 50 and 90%. Stock cultures were maintained by routine subculturing at low density to keep the frequency of spontaneous mutants at very low levels (ouabain-resistant mutants were less than 10 -8 and 6-thioguanine-resistant mutants were less than 2 × 10-6). Cell survival was determined as described elsewhere [10]. For m u t a t i o n experiments with combined X- and UV irradiations, petri dishes containing monolayers of about 106 cells were irradiated with 300 rads of X-rays (100 kVp, Hewlett-Packard Faxitron), allowed to grow for 0--17 h, and then irradiated with 13 J/m 2 of 254-nm UV light (1.3 J/m 2 • sec). The dose rate from this soft X-ray source was estimated by comparing the survival curves for CHO cells irradiated under similar conditions by the Faxitron and by 250kVp from a General Electric Maxitron 300 that had been calibrated with LiF crystals [10]. A dose of 300 rads (100 kVp) caused a growth delay of about 6 h and decreased plating efficiency of single cells to about 85% of the control value. Cultures were supplied with fresh medium, grown for several days, transferred to roller bottles, and maintained in exponential growth by occasional subculturing as required for at least 6 days after the UV irradiation (about 12 cell divisions). By this time, cells had passed through the period of phenotypic lag (expression time), and mutation frequencies had reached constant levels [ 2 2 ] (Table 1). The minimum expression time for ouabain resistance was 2 days and for 6-thioguanine resistance, 6 days (Table 1). Cultures were then trypsinized, diluted to 5 X 10 s cells/ml, and inoculated into 90-mm petri dishes containing 10 ml of medium supplemented with 3 mM ouabain (final density, 10s--106 cells per dish), 0.06 mM 6-thioguanine (final density, 5 X 104--2 × 10 s cells per dish), or drug-free medium (final density, 5--50 cells per dish). After surviving colonies were allowed to develop for 7--8 days, cultures were fixed, stained, and counted. The m u t a t i o n frequency

249 TABLE 1 FREQUENCIES IRRADIATION

OF DRUG-RESISTANT

Time after UV

CHO CELLS RECOVERED

O u a b a i n - r e s i s t a n t cells (3 m M ) a

AT VARIOUS TIMES AFTER UV

6 - T h i o g u a n i n e - r e s i s t a n t cells b

(XIO $)

(XlO s ) 0days(control) 0days(UV) 2 4 6 7 8 9 10

~0.001 0.10±0.03 2.6 ± 0 . 9 1.9 ± 0 . 3 -2.5 ± 0 . 8 --2.0 ± 0 . 4

~0.2 -10.0±3.3 25.0±5.0 35.8±7.7 31.0±6.1 28.5±5.6 22.8±2.2 32.3±3.0

a O n e e x p e r i m e n t p e r f o r m e d a t 15.6 J / m 2. E r r o r c i t e d is c a l c u l a t e d f r o m P o i s s o n f o r m u l a : i f a t o t a l o f N m u t a n t c o l o n i e s w e r e s c o r e d , e r r o r c i t e d is N1/2. b D a t a p o o l e d f r o m m a n y s e p a r a t e e x p e r i m e n t s at 10 J / m 2. E r r o r c i t e d is s t a n d a r d e r r o r c a l c u l a t e d f o r 4 or m o r e s e p a r a t e d e t e r m i n a t i o n s at e a c h tLme p o i n t .

per dish was calculated from the ratio of plating efficiencies of cells grown with ouabain or 6-thioguanine to plating efficiencies of cells grown with drug-free medium [3]. The mutation frequencies per dish decreased at the higher cell concentrations for 6-thioguanine resistance because of density-dependent cell interaction [1]. Only dishes from the range of cell concentrations for which mutation frequencies were constant were used for calculating average mutation frequencies. Average mutation frequencies (F) were calculated by adding together the m u t a n t colonies from all acceptable dishes according to the formula N +- N 1/2 F =

N0 × P.E.

where N = the total number of clonnies scored, N i n = the random sampling error (assuming Poisson statistics), No = the total number of cells plated, and P.E. = the plating efficiency in drug-free medium. F was calculated after X-irradiation, UV irradiation, and X-irradiation followed by UV (Fx, F u , and Fxu, respectively); the effect of prior X-irradiation on UV-induced mutation frequencies (relative mutation frequency, S) was estimated according to the formula S - Fxu -- Fx Fu Error limits for S were obtained by combining the Poisson errors in Fxu, Fx, and Fu; Fx and Fu were determined for every time interval between X- and UV irradiation. Results

Dose--response relationships for both drug-resistant markers were linear for X-rays and UV light separately [3] ; the yields were 1.3 + 0.4 X 10-7/rad and

250

3.6 -+ 0.2 × 10-s/J/m 2 for 6-thioguanine and 1.0 ± 0.2 × 10-6/J/m 2 for ouabain. X-rays were a less efficient mutagen than UV light at comparable levels of survival for both markers. When cells were irradiated with 300 rads of X-rays (85% survival) followed by 13 J/m 2 of UV light (40% survival} with intervals between the dose of up to 17 h, no synergism was detected at any time interval (Fig. 1). The number of points (2 of 15 for ouabain and 1 of 17 for 6-thioguanine) lying above the level expected for no interaction between X-irradiation and UV irradiation was too few to be significant. These results contrast with those obtained by others for cell survival [9] and transformation [6] in which synergism was detected, although these phenomena were n o t directly measured in the current experiments. Presumably the mechanisms involved in cell killing and transformation are different from the mechanisms of mutagenesis.

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HOURS BETWEEN XRAY AND UV IRRADIATION F i g . 1. R e l a t i v e m u t a t i o n frequencies (calculated accozding to the formula shown in Materials and methods) in cultures irradiated with X-rays (300 rads) at various times before irradiation with UV l i g h t ( 1 3 J / m 2 ) . A f t e r U V i r r a d i a t i o n , c u l t u r e s w e r e g r o w n f o r 7 - - 8 d a y s b e f o r e b e i n g s e l e c t e d f o r resist a n c e t o 6 - t h i o g u a n i n e or o u a b a i n . B r o k e n lines r e p r e s e n t s t a n d a r d d e v i a t i o n s f o r m u t a t i o n f r e q u e n c i e s in c e l l s i r r a d i a t e d w i t h U V l i g h t a l o n e .

251 Discussion Within the limitations of these experiments at single doses of X-rays and UV light, the absence of synergism between the two radiations can be interpreted to indicate that there is no inducible error-prone repair system in CHO cells, or at least none that can be induced by X-rays and that acts on UV-induced damage. The question is whether these experiments were capable of detecting an inducible system, if one does exist, or whether the negative result obtained here shows that there is no such system. We need to ask, therefore, whether I chose the right mutagens for optimum detection of an inducible system, whether the doses were appropriate, and whether the time interval between X-ray and UV doses was sufficiently long for repair enzymes to be induced. Although the low mutagenicity of X-rays was useful for inducing the putative error-prone repair system w i t h o u t at the same time inducing a large number of mutants, the low mutagenicity may mean that X-rays are an inherently poor inducer of this repair system; however, X-rays are inducers of the errorprone repair system in microorganisms [19]. But UV light produces so much higher a frequency of m u t a t i o n than X-rays at doses that allow comparable levels of survival that it may have eclipsed any increase in mutations due to X-rays. This question can be resolved to some extent if agents that act as more efficient inducers can be identified. The doses used in these experiments may have affected the efficacy of the mutagens for detecting an inducible repair system, because the function of this system in prokaryotes is dose dependent [23]. My choice of 300 rads of X-rays and 13 J/m 2 of UV light was a compromise between the desire to use doses high enough to give sufficient mutants and low enough to allow considerable survival. Because the survival levels obtained are in a range in which the action of an inducible system is detectable in prokaryotes [18,19,23], the chosen doses seem to have been appropriate. Only a more extensive study at a large number of different survival levels will guarantee that the doses used in this study were appropriate and that there is no detectable inducible system at any dose. A time interval of up to 17 h was allowed between X-irradiation and UV irradiation because this covered one complete cell cycle, including the approximately 6-h division delay caused by the X-ray dose. In a few mammalian cell lines in which induction of a new enzyme or set of enzymes is known to occur, the time for m a x i m u m induction is longer than the time in prokaryotes. For example, m a x i m u m induction of tyrosine amino transferase in mouse cells by exposure to hormones occurs in 3--6 h [13], and m a x i m u m induction of the enzyme system responsible for producing m a m m a r y t u m o r virus RNA occurs in 4--5 h [17]. Also, changes in the rate of post-replication repair seen after UV irradiation, which have been interpreted as being due to an inducible system, occur within 6 h of irradiation [5]. Therefore, in the experiments described here the time interval of 0--17 h between X-irradiation and UV irradiation is longer than the intervals known to be required for induction of new enzymes in mammalian cells. Although the negative results obtained in this limited single-dose experim e n t in CHO cells cannot be generalized w i t h o u t similar investigations with a

252

variety of agents and doses in many cell types, they are consistent with conclusions that can be drawn from mutation dose--response curves in prokaryotes and eukaryotes. The action of the prokaryotic inducible error-prone system seems to result in cumulative (dose-square) mutation frequency curves [12]. For eukaryotic cells, however, when care has been taken to recover all mutants and avoid density-dependent artefacts, the dose--response curves are linear for a variety of agents, including X-rays [3,20] and UV light [ 3 , 1 1 , 1 5 , 2 2 ] . Thus, the mechanisms by which mutations are produced by errors in replication and repair in eukaryotic cells may be constitutive, in contrast to the mechanisms of prokaryotes. Acknowledgements I am grateful to Mr. W. Charles for excellent technical assistance and Drs. Painter, Wolff, and Bochstahler for discussions about the significance of the results. References 1 A r l e t t , C . F . , D. T u r n b u l l , S.A. H a r c o u x t , A . R . L e h m a n n a n d C.M. Colella, A c o m p a r i s o n o f t h e 8-azaguanine and ouabain-resistance systems for the selection of induced mutant Chinese hamster cells, M u t a t i o n R e s . , 3 3 ( 1 9 7 5 ) 2 6 1 - - 2 7 8 . 2 C l e a v e r , J . E . , R e p a i r r e p l i c a t i o n a n d d e g r a d a t i o n o f b r o m o u r a c i l - s u b s t i t u t e d D N A in m a m m a l i a n cells a f t e r i r r a d i a t i o n w i t h u l t r a v i o l e t l i g h t , B i o p h y s . J.~ 8 ( 1 9 6 8 ) 7 7 5 - - 7 9 1 . 3 Cleaver, J.E., I n d u c t i o n o f t h i o g u a n i n e - a n d o u a b a i n - r e s i s t a n t m u t a n t s a n d s i n g l e - s t r a n d b r e a k s in t h e D N A o f C h i n e s e h a m s t e r cells b y 3 H - t h y m i d i n e , G e n e t i c s , 8 7 ( 1 9 7 7 ) 1 2 9 - - 1 3 8 . 4 Cleaver, J . E . , a n d J . E . T r o s k o , D N A - d e g r a d a t i o n p r o d u c t s f r o m m a m m a l i a n cells i r r a d i a t e d w i t h u l t r a v i o l e t l i g h t , I n t . J. R a d i a t . Biol., 1 5 ( 1 9 6 9 ) 4 1 1 - - 4 2 4 . 5 D ' A m b r o s i o , S . M , a n d R . B . S e t l o w , E n h a n c e m e n t o f p o s t r e p l i c a t i o n r e p a i r in C h i n e s e h a m s t e r cells, P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 7 3 ( 1 9 7 6 ) 2 3 9 6 - - 2 4 0 0 . 6 D i P a o l o , J . A . , a n d P . J . D o n o v a n , In v i t r o m o r p h o l o g i c t r a n s f o r m a t i o n o f S y r i a n h a m s t e r cells b y U.V.i r r a d i a t i o n is e n h a n c e d b y X - i r r a d i a t i o n a n d u n a f f e c t e d b y c h e m i c a l c a r c i n o g e n s , I n t . J. R a d i a t . Biol., 30 (1976) 41--53. 7 F r i e d r i c h , U., a n d P. C o f f i n o , M u t a g e n e s i s in $ 4 9 m o u s e l y m p h o m a cells: I n d u c t i o n of r e s i s t a n c e t o o u a b a i n , 6 - t h i o g u a n i n e , a n d d i b u t y r y l c y c l i c A M P , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 7 4 ( 1 9 7 7 ) 6 7 9 - 683. 8 G u d a s , L . J . , T h e i n d u c t i o n o f p r o t e i n X in D N A r e p a i r a n d cell d i v i s i o n m u t a n t s o f Escherichia coli, J. Mol. Biol., 1 0 4 ( 1 9 7 6 ) 5 5 6 - - 5 8 7 . 9 H a n , A., a n d M.M. E l k i n d , A d d i t i v e a c t i o n o f i o n i z i n g a n d n o n - i o n i z i n g r a d i a t i o n s t h r o u g h o u t t h e C h i n e s e h a m s t e r c e l l - c y c l e , I n t . J. R a d i a t . Biol., 31 ( 1 9 7 7 ) 2 7 5 - - 2 8 2 . 1 0 H a r r i s , J . W . , J . A . P o w e r a n d C.J. K o c h , R a d i o s e n s i t i z a t i o n o f h y p o x i c m a m m a l i a n cells b y d i a m i d e , I. E f f e c t s o f e x p e r i m e n t a l c o n d i t i o n s o n survival, R a d i a t i o n Res., 6 4 ( 1 9 7 5 ) 2 7 0 - - 2 8 0 . 11 H s i e , A . W . , P . A . B r i m e r , T . J . M i t c h e l l a n d D . G . G o s s l e e , T h e d o s e - - r e s p o n s e r e l a t i o n s h i p f o r u l t r a v i o l e t - l i g h t - i n d u c e d m u t a t i o n s a t t h e h y p o x a n t h i n e - g u a n i n e p h o s p h o r i b o s y l t r a n s f e r a s e l o c u s in Chin e s e h a m s t e r o v a r y cells, S o m a t . Cell G e n e t . , 1 ( 1 9 7 5 ) 3 8 3 - - 3 8 9 . 1 2 Ishii, Y . , a n d S. K o n d o , C o m p a r a t i v e a n a l y s i s o f d e l e t i o n a n d b a s e - c h a n g e m u t a b i l i t i e s o f Escherichia coli B s t r a i n s d i f f e r i n g in D N A r e p a i r c a p a c i t y ( w i l d - t y p e , UVRA-, P O L A , RECA-) b y v a r i o u s m u t a gens, Mutation Res., 27 (1975) 27--44. 1 3 Ivarie, R . D . , W.J.-W. F a n a n d G . M . T o m k i n s , A n a l y s i s of t h e i n d u c t i o n a n d d e i n d u c t i o n o f t y r o s i n e a m i n o t r a n s f e r a s e in e n u c l e a t e d H T C ceils, J. Cell P h y s i o l . , 8 5 ( 1 9 7 5 ) 3 5 7 - - 3 6 4 . 1 4 K o n d o , S., E v i d e n c e t h a t m u t a t i o n s a r e i n d u c e d b y e r r o r s in r e p a i r a n d r e p l i c a t i o n , G e n e t i c s , 73 (1973) Suppl. 109--122. 15 Maher, V.M., and J.J. McCormick, Effect of DNA repair on the cytotoxicity and mutagenicity of UV i r r a d i a t i o n a n d o f c h e m i c a l c a r c i n o g e n s in n o r m a l a n d x e r o d e r m a p i g m e n t o s u m cells, in: J . M . Y u h a s , R . W . T e n n a n t a n d J . D . R e g a n ( E d s . ) , B i o l o g y o f R a d i a t i o n C a r c i n o g e n e s i s , R a v e n Press, N e w Y o r k , 1976, pp. 129--145. 1 6 R a d m a n , M., S O S r e p a i r h y p o t h e s i s ; P h e n o m e n o l o g y o f a n i n d u c i b l e D N A r e p a i r w h i c h is a c c o m -

253

17

18 19

20 21 22 23

24 25

p a n i e d b y m u t a g e n e s l s , in: P.C. H a n a w a l t a n d R . B . S e t l o w ( E d s . ) , M o l e c u l a r M e c h a n i s m s f o r R e p a i r o f D N A , P l e n u m Press, N e w Y o r k , 1 9 7 5 , p p . 3 5 5 - - 3 6 7 . R i n g o l d , G , M . , K . R . Y a m a m o t o , G.M. T o m k i n s , J . M . B i s h o p a n d H . E . V a r m u s , D e x a m e t h a s o n e m e d i a t e d i n d u c t i o n o f m o u s e m a m m a r y t u m o r virus R N A : A s y s t e m f o r s t u d y i n g g l u e o c o r t i c o i d a c t i o n , Cell, 6 ( 1 9 7 5 ) 2 9 9 - - 3 0 5 . S e g w i c k , S . G . , I n d u c i b l e e r r o r - p r o n e r e p a i r in E s c h e r i c h i a coli, P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 7 2 (1975) 2 7 5 3 - - 2 7 5 7 . S e d g w i c k , S . G . , M i s r e p a i r o f o v e r l a p p i n g d a u g h t e r s t r a n d g a p s as a p o s s i b l e m e c h a n i s m f o r U V i n d u c e d m u t a g e n e s i s in U V R s t r a i n s o f E s c h e r i c h i a coli: A g e n e r a l m o d e l f o r i n d u c e d m u t a g e n e s i s b y m i s r c p a i r ( S O S r e p a i r ) o f c l o s e l y s p a c e d D N A lesions, M u t a t i o n Res., 41 ( 1 9 7 6 ) 1 8 5 - - 2 0 0 . T h a c k e r , J., a n d R. C o x , M u t a t i o n i n d u c t i o n a n d i n a c t i v a t i o n in m a m m a l i a n cells e x p o s e d t o i o n i s i n g radiation, Nature (London), 258 (1975) 429--431. T r o s k o , J . E . , a n d C-C. C h a n g , R o l e o f D N A r e p a i r in m u t a t i o n a n d c a n c e r p r o d u c t i o n , in: K . C . S m i t h ( E d . ) , A g i n g , C a r c i n o g e n e s i s , a n d R a d i a t i o n B i o l o g y , P l e n u m Press, N e w Y o r k , 1 9 7 6 , p p . 3 9 9 - 4 4 2 . Van Zeeland, A.A., and J.W.I.M. Simons, Linear dose-response relationships after prolonged express i o n t i m e s in V - 7 9 C h i n e s e h a m s t e r cells, M u t a t i o n R e s . , 3 5 ( 1 9 7 6 ) 1 2 9 - - 1 3 8 . W i t k i n , E.M., R e l a t i o n s h i p a m o n g r e p a i r , m u t a g e n e s l s , a n d survival: O v e r v i e w , in: P.C. H a n a w a l t a n d R . B . S e t l o w ( E d s . ) , M o l e c u l a r M e c h a n i s m s f o r R e p a i r of D N A , P l e n u m Press, N e w Y o r k , 1 9 7 5 , p p . 347--353. W i t k i n , E . M . , U l t r a v i o l e t m u t a g e n e s i s a n d i n d u c i b l e D N A r e p a i r in Escherichia coli, B a c t e r i o l . Rev., 4 0 (1976) 869--907. W i t k i n , E.M., a n d D . L . G e o r g e , U l t r a v i o l e t m u t a g e n e s i s in P O L A a n d U V R A P O L A d e r i v a t i v e s of E s c h e r i c h i a coli B / R : E v i d e n c e f o r a n i n d u c i b l e e r r o r - p r o n e r e p a i r s y s t e m , G e n e t i c s , 7 3 ( 1 9 7 3 ) S u p p l . , 91--108.