63
Mutation Research, 40 (1976) 63--66
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
Short communication 5-BROMODEOXYURIDINE HAMSTER CELLS *
MUTAGENESIS
IN S Y N C H R O N O U S
PAUL M. AEBERSOLD and H. JOHN BURKI Division of Medical Physics and Donner Laboratory, University of California, Berkeley, California 94720 (U.S.A.) (Received July 31st, 1975) (Accepted October 12th, 1975)
The DNA of mammalian cells has been shown to replicate in specific temporal patterns that repeat from generation to generation [9]. Euchromatic DNA replicates earlier than heterochromatic DNA [7], and the ribosomal genes of Chinese hamster cells (of which there are approximately 300 copies) replicate early in the DNA sysnthesis, or S, period [12]. With bacteria it has been possible to construct a replication map of the genome using nitrosoguanidine, which preferentially mutagenizes the replication point region of the chromosome [3], b u t replication mapping of the mammalian genome with nitrosoguanidine has been unsuccessful [10]. In this report we show that 5-bromodeoxuridine (BUdR) apparently does evoke a temporal pattern of mutation in synchronous Chinese hamster cells. Much work has been done on isolation and characterization of cells resistant to purine analogues such as 8-azaguanine and 6-thioguanine (6TG). The resistant phenotypes are generally stable in the absence of selective pressure [14], and a majority of these variants exhibit a low level of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity [ 5]. Recent characterization of the H G P R T enzyme of such variants gives evidence for alternations in the gene coding for H G P R T [16]. This gene is X-linked in humans [8] and most likely also in Chinese hamsters [17], so that diploid cells of male origin would be expected to have only one copy of the gene. B U d R can substitute for thymidine during DNA synthesis. Once incorporated into DNA, B U d R acts as a mutagen by mispairing with guanine during sybsequent replication [4] or by some undefined mechanism. Low levels of BUdR, around 10-SM, can affect the differentiation of cultured cells in regard to enzyme levels [13], but this reversible effect is contingent on the continued presence of B U d R in the growth medium. B U d R is also toxic to cells, particu* This investigation was supported by N I H grant C A 14310 and the U.S. Environmental Research and Development Agency contract W-7405-eng-48.
64
larly when incorporated into early replicating DNA [6]. In these experiments, we exposed cells to only 30 min of 10-4M BUdR, and we found no significant toxicity during any portion of the cell cycle (data not shown}. Chinese hamster V-79 lung fibroblasts were grown at 37°C in a 5% CO2 atmosphere in Eagle's medium supplemented with 10% fetal calf serum. Exponentially growing cells were synchronized by shaking off the mitotic and recently divided cells. Synchrony was improved by 8 h exposure to 1.0 mM h y d r o x y u r e a which is toxic to cells in S period and prohibits other cells from entering S period [11]. H y d r o x y u r e a was removed by rinsing plates twice in pre-warmed saline prior to addition of fresh growth medium. Two measures of the synchrony are shown in Fig. 1: (a) the curve on the left shows that as cells finish DNA synthesis 4.5 h after the rinse, t h e y survive a second exposure to hydroxyurea, and (b) the curve on the right shows an increase in the number of cells as cytokinesis is completed. The number of cells does n o t quite double due to an expected fraction of dead or dying cells. The initial plating of cells in all experiments was kept to less than 10 s cells per 9 cm dish to avoid cross feeding that alters apparent m u t a t i o n induction [15]. The S period was arbitrarily considered as sequential 30-min intervals, during each of which two plates were exposed to 10-4M BUdR. The BUdR was removed by two saline rinses followed by fresh growth medium. After four days incubation to allow for expression of m u t a n t phenotypes [1], cells were exposed to fresh medium containing 5 pg/ml 6TG. To compensate for possible degradation of 6TG at 37 ° C, this selective medium was replaced after five days. Resistant colonies were stained and counted after ten days of selection (Fig. 2, top). To assure that the observed differences in induction of resistant cells were not merely due to the a m o u n t of BUdR incorporated into the DNA, we carried out other experiments under the same conditions but for 1.0 pCi/ml
100
20
~5o
=
--
~=t
-- 1.5
0 ---0 2 4 6 8 Hours following release of hydroxyurea
1.0 10
E
block
Fig. 1. (o o) S u r v i v a l o f s y n c h r o n o u s cells a f t e r 8 h e x p o s u r e to 1 . 0 m M h y d r o x y u r e a , s t a r t i n g at t i m e p l o t t e d . C o n t r o l p l a t e s w e r e n o t givc-- a s e c o n d e x p o s u r e Of h y d r o x y u r e a a f t e r r i n s i n g o f f t h e f i r s t synchronizing e x p o s u r e . D a t a p l o t t e d are t h e a v e r a g e o f t w o s y n c h r o n i z a t i o n s . (A 4) I n c r e a s e in cell n u m b e r o f s y n c h r o n o u s cells. P l a t e s w e r e t r y p s i n i z e d at t i m e s p l o t t e d a n d t h e c e l l s w e r e c o u n t e d b y a C o u l t e r c o u n t e r . D a t a are t h e a v e r a g e o f t h r e e s y n c h r o n i z a t i o n s . Error bars show standard deviations.
65 ¢0 2 0
]
I
]
~ 10
'rm
0
3._=
2
1 ~3 -I-
0
I 1 Hours
I 2
[
I
I
3
4
5
f o l l o w i n g release of h y d r o x y u r e a b l o c k
Fig. 2. (o o) I n d u c t i o n of 6 - t h i o g u a n i n e r e s i s t a n t c o l o n i e s a f t e r i n c o r p o r a t i o n o f B U d R in s y n c h r o n o u s cells. D a t a are t h e a v e r a g e o f 4 e x p e r i m e n t s ; t w o p l a t e s w e r e e x p o s e d t o 10 -4 M B U d R d u r i n g e a c h 30-rnin i n t e r v a l f o r e a c h e x p e r i m e n t . E r r o r b a r s s h o w s t a n d a r d d e v i a t i o n o f t h e m e a n s . D a s h e d line s h o w s 6 T G r e s i s t a n t c o l o n i e s in s y n c h r o n i z e d c o n t z o l p l a t e s r e c e i v i n g n o B U d R . (A A) U p t a k e of B U d R b y s y n c h r o n o u s cells. I n t w o e x p e r i m e n t s , o n e p l a t e was e x p o s e d t o 1 . 0 / ~ C i / m l [ 6 - 3 H ] B U d R in 1 0 - 4 M t o t a l B U d R d u r i n g e a c h 30-rnin interval.
[6-3H] B U d R in the 10-~M total BUdE. When the cells reached the G2 period, the amount of [6-3H] B U d R incorporated into the 10% trichloroacetic acid insoluble material was determined using a liquid scintillation counter (Fig. 2, bottom). The simplest interpretation of our data is that the gene coding for H G P R T is replicated during those intervals in which we find high numbers of 6TG resistant colonies. The width of these time intervals reflects the width of our synchonous cell populations, so that half the cells presumably have replicated the gene within 1.6 h after release of the HOU block. This interpretation implies that B U d E mutagenizes only that portion of DNA into which it is incorporated. X-linked markers such as H G P E T have a single gene in male cells and are easily mutated. To obtain similar mutation rates for autosomal markers, heterozygous cells can be constructed [2]. It thus seems possible to begin a gene replication map o f the mammalian genome.
66
References 1 C a r v e r , J.i.i., W.C. D e w e y a n d L.E. H o p w o o d , X - r a y - i n d u c e d m u t a n t s r e s i s t a n t to 8 - a z a g u a n i n e . I. E f f e c t s o f cell d e n s i t y a n d e x p r e s s i o n t i m e , M u t a t i o n Res., in press. 2 C e r d f i - O l m e d o , E., P.C. H a n a w a l t a n d N. G u e r o l a , M u t a g e n e s i s o f t h e r e p l i c a t i o n p o i n t by nitrosog u a n i d i n e : m a p a n d p a t t e r n of r e p l i c a t i o n of t h e E. coli c h r o m o s o m e , J. Mol. Biol., 33 ( 1 9 6 8 ) 705--719. 3 Chasin, L . A . , M u t a t i o n s a f f e c t i n g a d e 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 ~ a c t i v i t y in Chinese h a m s t e r cells, Cell, 2 ( 1 9 7 4 ) 3 7 - - 4 1 . 4 F i s h b e i n , L., W.G. F l a m m a n d H,L. Falk, C h e m i c a l M u t a g e n s , A c a d e m i c Press, N e w Y o r k , 1 9 7 0 , pp. 13--17. 5 Gfllin, F.D., D.J. R o u f a , A.L. B e a u d e t a n d C.T. C a s k e y , 8 - a z a g u a n i n e r e s i s t a n c e in m a m m a l i a n cells. I. 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 , G e n e t i c s , 72 ( 1 9 7 2 ) 2 3 9 - - 2 5 2 . 6 K a j i w a r a , K. a n d G.C. Mueller, F r a c t i o n a l s y n t h e s i s of D N A w i t h 5 - b r o m o d e o x y u r i d i n e a n d its e f f e c t o n c l o n i n g e f f i c i e n c y , B i o c h i m . B i o p h y s . A c t a , 91 ( 1 9 6 4 ) 4 8 6 - - 4 9 3 . 7 Lima-de-Faxia, A., D i f f e r e n t i a l u p t a k e of t r i t i a t e d t h y m i d i n e i n t o h e t e r o - a n d e u c h r o m a t i n in Melanoplus a n d Secale, B i o p h y s . B i o c h e m . C y t o l . , 6 ( 1 9 5 9 ) 4 5 7 - - 4 6 4 8 M c K u s i c k , V. a n d G. Chase, H u m a n genetics, A n n . Rev. G e n e t . , 7 ( 1 9 7 3 ) 4 3 5 - - 4 7 3 . 9 Mueller, G.C. a n d K. K a j i w a r a , Early- a n d l a t e - r e p l i c a t i n g D N A c o m p l e x e s in H e L a (cell) nuclei, B i o c h i m . Biophys. A c t a , 1 1 4 ( 1 9 6 6 ) 1 0 8 - - 1 1 5 . 10 O r k i n , S.H. a n d J.W. L i t t l e f i e l d , N i t r o s o g u a n i d i n e m u t a g e n i s i s in s y n c h r o n i z e d h a m s t e r cells, Exp. Cell Res., 66 ( 1 9 7 1 ) 6 9 - - 7 4 . 11 Sinclair, W.K., H y d r o x y u r e a : d i f f e r e n t i a l l e t h a l e f f e c t s o n c u l t u r e d m a m m a l i a n cells d u r i n g the cell c y c l e , Science, 1 5 0 ( 1 9 6 5 ) 1 7 2 9 - - 1 7 3 1 . 12 S t a m b r o o k , P.J., T h e t e m p o r a l r e p l i c a t i o n of r i b o s o m a l genes in s y n c h r o n i z e d h a m s t e r cells. J. Mol. Biol., 82 ( 1 9 7 4 ) 3 0 3 - - 3 1 3 . 13 S t e l l w a g e n , R.H. a n d G.M. T o m k i n s , D i f f e r e n t i a l e f f e c t of 5 - b r o m o d e o x y u r i d i n e in the c o n c e n t r a t i o n s of specific e n z y m e s in h e p a t o m a ceils in c u l t u r e , Proc. Natl. A c a c . Sci. (US), 68 ( 1 9 7 1 ) 1 1 4 7 - - 1 1 5 0 . 14 T h o m p s o n , L.H. a n d R.M. Baker, I s o l a t i o n of m u t a n t s o f c u l t u r e d m a m m a l i a n cells, in D.M. P r e s c o t t (ed.), M e t h o d s in Cell P h y s i o l o g y , Vol. VI, A c a d e m i c Press, N e w Y o r k , 1 9 7 3 , pp. 2 1 0 - - 2 8 1 . 15 V a n Z e e l a n d , A . A . , M.C.E. v a n Diggelen a n d J.W.I.M. S i m o n s , T h e role of m e t a b o l i c c o o p e r a t i o n in s e l e c t i o n of H G P R T d e f i c i e n t m u t a n t s f r o m d i p l o i d m a m m a l i a n cell strains, M u t a t i o n Res., 14 ( 1 9 7 2 ) 355--363. 16 Wahl, G.M., S.H. H u g h e s a n d M.R. C a p e c e h i , I m m u n o l o g i c a l c h a r a c t e r i z a t i o n of H G P R T m u t a n t s o f m o u s e L cells: e v i d e n c e f o r m u t a t i o n s a t d i f f e r e n t loci in t h e H G P R T g e n e , J. Cell. Physiol., 85 ( 1 9 7 5 ) 307--320. 17 W e s t e r v e l d , A., P. Visser, M. F r e e k e a n d D. B o o t s m a , E v i d e n c e for linkage of 3 - P G K , H G P R T , a n d G 6 P D loci in Chinese h a m s t e r ceils s t u d i e d by using a r e l a t i o n s h i p b e t w e e n gene m u l t i p l i c i t y a n d enzyme activity, Biochem. Genet., 7 (1972) 33--40.
Mutation Research, 40 (1976) 63--66
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
Short communication 5-BROMODEOXYURIDINE HAMSTER CELLS *
MUTAGENESIS
IN S Y N C H R O N O U S
PAUL M. AEBERSOLD and H. JOHN BURKI Division of Medical Physics and Donner Laboratory, University of California, Berkeley, California 94720 (U.S.A.) (Received July 31st, 1975) (Accepted October 12th, 1975)
The DNA of mammalian cells has been shown to replicate in specific temporal patterns that repeat from generation to generation [9]. Euchromatic DNA replicates earlier than heterochromatic DNA [7], and the ribosomal genes of Chinese hamster cells (of which there are approximately 300 copies) replicate early in the DNA sysnthesis, or S, period [12]. With bacteria it has been possible to construct a replication map of the genome using nitrosoguanidine, which preferentially mutagenizes the replication point region of the chromosome [3], b u t replication mapping of the mammalian genome with nitrosoguanidine has been unsuccessful [10]. In this report we show that 5-bromodeoxuridine (BUdR) apparently does evoke a temporal pattern of mutation in synchronous Chinese hamster cells. Much work has been done on isolation and characterization of cells resistant to purine analogues such as 8-azaguanine and 6-thioguanine (6TG). The resistant phenotypes are generally stable in the absence of selective pressure [14], and a majority of these variants exhibit a low level of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity [ 5]. Recent characterization of the H G P R T enzyme of such variants gives evidence for alternations in the gene coding for H G P R T [16]. This gene is X-linked in humans [8] and most likely also in Chinese hamsters [17], so that diploid cells of male origin would be expected to have only one copy of the gene. B U d R can substitute for thymidine during DNA synthesis. Once incorporated into DNA, B U d R acts as a mutagen by mispairing with guanine during sybsequent replication [4] or by some undefined mechanism. Low levels of BUdR, around 10-SM, can affect the differentiation of cultured cells in regard to enzyme levels [13], but this reversible effect is contingent on the continued presence of B U d R in the growth medium. B U d R is also toxic to cells, particu* This investigation was supported by N I H grant C A 14310 and the U.S. Environmental Research and Development Agency contract W-7405-eng-48.
64
larly when incorporated into early replicating DNA [6]. In these experiments, we exposed cells to only 30 min of 10-4M BUdR, and we found no significant toxicity during any portion of the cell cycle (data not shown}. Chinese hamster V-79 lung fibroblasts were grown at 37°C in a 5% CO2 atmosphere in Eagle's medium supplemented with 10% fetal calf serum. Exponentially growing cells were synchronized by shaking off the mitotic and recently divided cells. Synchrony was improved by 8 h exposure to 1.0 mM h y d r o x y u r e a which is toxic to cells in S period and prohibits other cells from entering S period [11]. H y d r o x y u r e a was removed by rinsing plates twice in pre-warmed saline prior to addition of fresh growth medium. Two measures of the synchrony are shown in Fig. 1: (a) the curve on the left shows that as cells finish DNA synthesis 4.5 h after the rinse, t h e y survive a second exposure to hydroxyurea, and (b) the curve on the right shows an increase in the number of cells as cytokinesis is completed. The number of cells does n o t quite double due to an expected fraction of dead or dying cells. The initial plating of cells in all experiments was kept to less than 10 s cells per 9 cm dish to avoid cross feeding that alters apparent m u t a t i o n induction [15]. The S period was arbitrarily considered as sequential 30-min intervals, during each of which two plates were exposed to 10-4M BUdR. The BUdR was removed by two saline rinses followed by fresh growth medium. After four days incubation to allow for expression of m u t a n t phenotypes [1], cells were exposed to fresh medium containing 5 pg/ml 6TG. To compensate for possible degradation of 6TG at 37 ° C, this selective medium was replaced after five days. Resistant colonies were stained and counted after ten days of selection (Fig. 2, top). To assure that the observed differences in induction of resistant cells were not merely due to the a m o u n t of BUdR incorporated into the DNA, we carried out other experiments under the same conditions but for 1.0 pCi/ml
100
20
~5o
=
--
~=t
-- 1.5
0 ---0 2 4 6 8 Hours following release of hydroxyurea
1.0 10
E
block
Fig. 1. (o o) S u r v i v a l o f s y n c h r o n o u s cells a f t e r 8 h e x p o s u r e to 1 . 0 m M h y d r o x y u r e a , s t a r t i n g at t i m e p l o t t e d . C o n t r o l p l a t e s w e r e n o t givc-- a s e c o n d e x p o s u r e Of h y d r o x y u r e a a f t e r r i n s i n g o f f t h e f i r s t synchronizing e x p o s u r e . D a t a p l o t t e d are t h e a v e r a g e o f t w o s y n c h r o n i z a t i o n s . (A 4) I n c r e a s e in cell n u m b e r o f s y n c h r o n o u s cells. P l a t e s w e r e t r y p s i n i z e d at t i m e s p l o t t e d a n d t h e c e l l s w e r e c o u n t e d b y a C o u l t e r c o u n t e r . D a t a are t h e a v e r a g e o f t h r e e s y n c h r o n i z a t i o n s . Error bars show standard deviations.
65 ¢0 2 0
]
I
]
~ 10
'rm
0
3._=
2
1 ~3 -I-
0
I 1 Hours
I 2
[
I
I
3
4
5
f o l l o w i n g release of h y d r o x y u r e a b l o c k
Fig. 2. (o o) I n d u c t i o n of 6 - t h i o g u a n i n e r e s i s t a n t c o l o n i e s a f t e r i n c o r p o r a t i o n o f B U d R in s y n c h r o n o u s cells. D a t a are t h e a v e r a g e o f 4 e x p e r i m e n t s ; t w o p l a t e s w e r e e x p o s e d t o 10 -4 M B U d R d u r i n g e a c h 30-rnin i n t e r v a l f o r e a c h e x p e r i m e n t . E r r o r b a r s s h o w s t a n d a r d d e v i a t i o n o f t h e m e a n s . D a s h e d line s h o w s 6 T G r e s i s t a n t c o l o n i e s in s y n c h r o n i z e d c o n t z o l p l a t e s r e c e i v i n g n o B U d R . (A A) U p t a k e of B U d R b y s y n c h r o n o u s cells. I n t w o e x p e r i m e n t s , o n e p l a t e was e x p o s e d t o 1 . 0 / ~ C i / m l [ 6 - 3 H ] B U d R in 1 0 - 4 M t o t a l B U d R d u r i n g e a c h 30-rnin interval.
[6-3H] B U d R in the 10-~M total BUdE. When the cells reached the G2 period, the amount of [6-3H] B U d R incorporated into the 10% trichloroacetic acid insoluble material was determined using a liquid scintillation counter (Fig. 2, bottom). The simplest interpretation of our data is that the gene coding for H G P R T is replicated during those intervals in which we find high numbers of 6TG resistant colonies. The width of these time intervals reflects the width of our synchonous cell populations, so that half the cells presumably have replicated the gene within 1.6 h after release of the HOU block. This interpretation implies that B U d E mutagenizes only that portion of DNA into which it is incorporated. X-linked markers such as H G P E T have a single gene in male cells and are easily mutated. To obtain similar mutation rates for autosomal markers, heterozygous cells can be constructed [2]. It thus seems possible to begin a gene replication map o f the mammalian genome.
66
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