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Thymidine resistance in Chinese hamster V79 cells in vitro
143
Mutation Research, 73 ( 1 9 8 0 ) 1 4 3 - - 1 5 6 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
THYMIDINE RESISTANCE IN CHINESE HAMSTER V79 CELLS IN VITRO
JOHN MORROW
and D O U G L A S
STOCCO
Department of Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430 (U.S.A.) ( R e c e i v e d 17 J a n u a r y 1 9 8 0 ) ( R e v i s i o n received 12 M a y 1 9 8 0 ) ( A c c e p t e d 14 M a y 1 9 8 0 )
Summary Thymidine resistance in V79 Chinese hamster cells has been investigated. Phenotypically stable variant resistant lines occurred at a high frequency, and the mutation rate (2.67 X 10 -s per cell per generation) to 400 ~g/ml thymidine resistance as measured by the standard Luria--Delbriick fluctuation analysis was extremely high. Populations of cells maintained for extended periods in F-10 medium spontaneously increased in resistance, possibly as a result of selective pressures due to the thymidine present in F-10 medium since this change was not observed in Dulbecco's medium. The degree of resistance for a given variant was correlated with the amount of thymidine employed in its selection. Metabolic cooperation, resulting in the suppression of the resistant phenotype, was demonstrated in artificial mixtures of sensitive and resistant clonal lines. Clones isolated in high levels of thymidine possessed lowered uptake of [3H]thymidine and the depression in uptake was related to the level of resistance of the particular clohe. Although thymidine kinase specific activity levels were slightly depressed in variant cell lines, growth rate and uridine uptake were unaffected. We conclude that thymidine resistance is due to a genetically controlled depression of external thymidine uptake.
High concentrations of thymidine are toxic to mammalian cells grown in culture (Lee et al., 1977). The mechanism of toxicity is believed to be the inhibition of the synthesis of deoxycytidine triphosphate from cytidine-5'phosphate, due to feedback inhibition (Morse and Potter, 1965). Selection of cells in high levels of thymidine has allowed the isolation of several classes of resistance. Breslow and Goldsby (1969) isolated and characterized avariant of
* This work was supported by a grant f-corn The Public Health Service (I-ROI-CA-16207-01).
144 Don Chinese hamster fibroblasts which was resistant to high levels of thymidine because of a lowered ability to incorporate exogenous thymidine from the medium. In these cells the mutation rate to thymidine resistance was reported to be 2.6 X 10 -4 per cell per generation. In another study (Mezger-Freed, 1972) haploid frog cell variants were isolated which were partially resistant to BrdU, although they contained near wild-type levels of the enzyme thymidine kinase. The absence of this enzyme is usually responsible for resistance to BrdU (Kit et al., 1963). It was proposed that these mutants were resistant to BrdU because of a defect in the BrdU-transport system as measured by the uptake of tritiated thymidine. A further report on this problem (Freed and Mezger-Freed, 1973) stated that the loss of the thymidine-transport system was an obligate intermediate mutation during the selection of a thymidine kinase negative mutant in haploid frog cells. It has been reported (Meuth and Green, 1974) that sublines of the mouse fibrolast line 3T6 have been isolated which are resistant to arabinosyl cytosine and deoxynucleosides of adenine, thymidine and guanine. These altered sublines are characterized by increases in the activity of the enzyme ribonucleotide reductase and a reduced sensitivity of this enzyme to inhibition by dATP. Fox and Anderson (1974) also reported data on the characterization of high thymidine-resistant mouse-lymphoma cells. Working with thymidine-resistant clones of L5178Y and P388 cells, these authors found that in general, thymidine-resistant clones had substantial amounts of thymidine kinase activity but had signigicantly lower levels of phosphorylated thymidine derivatives, thus suggesting the mutants were the result of altered permeability to thymidine. The data of Breslow and Goldsby (1969) have been analyzed by De Mars (1974) who pointed out that because of the large initial number of cells employed in the fluctuation tests pre-existing mutants may have been included. In addition to the controversy over the actual mutation rate to thymidine resistance, two of the reports mentioned previously (Fox and Anderson, 1974; Mezger-Freed, 1972) indicate that not all of the clones resistant to high thymidine were inhibited in their ability to take up exogenous thymidine to the same extent. Because of uncertainties concerning the mutation rate to high thymidine resistance in mammalian cells and in an effort to further clarify the relationship between the degree of thymidine resistance and thymidine uptake, we have reinvestigated this problem in greater detail. Our approach has been to isolate variants resistant to thymidine in V79 Chinese hamster cells at different concentrations of the selecting agent and at several cell densities, and to genetically and biochemically characterize the various classes of mutants thus obtained. Furthermore, we have measured the mutation rate to thymidine resistance using the Luria--Delbriick (1943) fluctuation test. The figures obtained are among the highest reported for any mammalian cell line (Morrow, 1975). In addition, we have described results of experiments which define the parameters of this system. Finally, we present evidence implicating lowered thymidine uptake as being the mechanism responsible for thymidine resistance and relating the degree of exclusion of external thymidine to the level of resistance.
145 Materials and methods
Experimental procedures Culture techniques, measurement of mutation frequencies and evaluation of resistance levels were performed according to previously described procedures (Morrow et al., 1978; Prickett et al., 1975). Briefly, cells were grown in Ham's F-10 medium, made by us from the components (Sigma, St. Louis, MO) or Dulbecco's medium (K.C. Biologicals) to which 10% fetal calf serum (Kansas City Biologicals, Lenexa, KA) was added. Gentamycin (Schering) was added (50 ~g/ml) to prevent bacterial contamination. The cells were transferred with Viokase (GIBCO, Grand Island, NY), and stored frozen in 5% dimethyl sulfoxide in a Revco freezer. The A3 sublcone of the V79 Chinese Hamster cell line (Gillin et al., 1972) was a generous gift from Dr. Donald Roufa, Division of Biology, Kansas State University. The cells were not clones and did not contain other genetic markers. Stock cultures were maintained in glass milk dilution bottles. For some experiments an azaguanine-resistant derivative of the V79 cell was employed. These cells are deficient in hypoxanthine-guanine phosphoribosyl transferase and do not grow in the reverse selective "HAT" medium (Szybalski and Smith, 1959). To evaluate the resistance level of sensitive and resistant lines, cells were harvested and plated at densities of 500 to 103 cells per 60-mm petri dish (Falcon Plastics, Oxnard, CA) with 3 replicates for each concentration. When macroscopically visible clones appeared they were stained with crystal violet, and counted with the aid of a dissection microscope. The time re(tuired for the growth of clones was approx. 1 week; Fig. 1 shows a pilot experiment experi-
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Fig. 1. T h e t h y m i d i n e - r e s i s t a n t m u t a n t V 7 9 / H T C 3 w a s p l a t e d i n F a l c o n p e t r l d i s h e s i n 0, 2 0 0 a n d 4 0 0 #g/m.] a t a d e n s i t y o f 10 3 cells p e r d i s h . A t v a r y i n g t i m e s t h e r e a f t e r , 3 p l a t e s a t a t i m e w e r e s t a i n e d , a n d the clones counted. Vertical bars indicate the range. For the sake of clarity, the points have been shifted s l i g h t l y f r o m t h e a c t u a l c o n c e n t r a t i o n s , e , 5 t h d a y ; a 7 t h d a y ; o , 1 2 t h d a y ; X, 1 4 t h d a y ; ~, 1 7 t h d a y a f t e r plating.
146 ment in which a resistant line (see below) was tested by plating cells in petri dishes and growing the clones for different periods. As can be seen the period of growth had very little effect on the number of clones with the exception of the 5-day, 400/~g/ml point. Cell numbers were determined with a Celloscope electronic particle counter (Particle Data, Inc., Elmwood, IL). Clonal lines were isolated b y removing a portion of the Falcon flask with a hot cork borer and inserting a sterile stainless steel "peni-cylinder" greased with sterile vaseline over the clone. Viokase was added, and the clone transferred to a new culture vessel. Cells were checked for mycoplasma contamination b y t w o methods: a modification of the m e t h o d of Schneider et al. (1974) in which the ratio of [3H]uridine/[ 14C]uracil incorporation was used as an indicator of mycoplasma contamination, and the ability of the cells to grow in H A T medium (Clive et al., 1973). Mutation rates were calculated using the Luria--Delbrfick (1943) fluctuation test. Single cells were grown in Limbro multi-well plates until visible colonies were formed. The colonies were harvested and suspended in 1.0 ml of culture medium. 0.1 ml was plated in F-10 medium and the other 0.9 ml was plated in F-10 plus thymidine. When visible colonies were formed in these plates (approx. 2 weeks) the plates were stained and counted. To obtain the mutation frequency per plate the number o f control colonies was multiplied by 9 and divided into the number o f colonies in thymidine. The mutation rate was then calculated using the median m e t h o d o f Lea and Coulson {1949). Chromosome studies were performed using previously described methods (Colofiore et al., 1973). Thymidine kinase assays Thymidine kinase specific activity was determined as described by Ives, Durham and Tucker (1969). Cells were harvested, washed twice with Hank's balanced salt solution and pelleted b y centrifugation in a clinical centrifuge. The cells were resuspended in 0.25 M sucrose pH 7.6 and homogenized using a glass homogenizer. 100 /~l of this homogenate was mixed with 100 /~l of 2 X reaction mixture. The final concentration of c o m p o n e n t s in the incubation mixture was 50 mM Tris--HC1 pH 8.0; 5 mM ATP; 2.5 mM MgCI: and 1/ICi/ml [3H]thymidine (methyl-3H, 45.58 Ci/mmole, New England Nuclear, Boston, MA). The incubations were performed in glass test tubes at 37°C with gentle shaking. Samples were taken at specific intervals for up to 30 min and the reaction was stopped b y immersing the tubes in a boiling water bath for 2 min. Following centrifugation at 12 000 X g for 5 min to pellet the denatured protein, 50-/~1 aliquots of each supernatant were pipetted directly onto 18-mm DEAE-substituted paper discs (Whatman DE-81). The discs were allowed to dry for 10 min and then washed for 30 min in a beaker containing 30 ml of water per disc with 2 changes of water in order to remove the unreacted substrate. Following the washing, the discs were then placed in polyethylene minivials and 1.0 ml of HC1/KC1 (0.1/0.2 M) was added to each vial to elute the phosphorylated thymidine derivatives from the discs. After 15 min of elution, 4.0 ml of a 75% toluene 25% triton x-114-Omnifluor (New England Nuclear)
147
scintillation fluid was added to each vial and all samples were assayed for radioactivity. The counting efficiency o f this system was 19.8%.
Uptake studies 5 X 104 wild-type or m u t a n t cells were plated into each well of a multi-well plastic dish in F-10 medium plus 10% fetal calf serum. At the end of 24 h, the medium was removed and the cells were washed with Hank's balanced salt solution. Following the wash, fresh Hank's containing 25/~Ci/ml [SH]thymidine or 10 #Ci/ml [SH]uridine 3H(G) (3.66 Ci/mmole, New England Nuclear) was added to the cells. At intervals b e t w e e n 0 and 10 min, the isotope was removed and the cells were quickly washed twice with Hank's. After the incubations the cells were disrupted with 1% sodium d o d e c y l sulphate (SDS). Aliquots of each sample were taken and placed in scintillation vials containing the toluene-triton Omnifluor cocktail described earlier and a portion of the remainder was used for protein determination (Lowry et al., 1951). The counting efficiency in this system was 33%. Cells in wells n o t used for uptake studies were harvested and the cell number determined. Results
Response of V79 cells to thymidine inhibition U p o n introduction into our laboratory V79 cells were transferred from Dulbecco's medium to Ham's F-10. F-10 was chosen because of our desire to duplicate the conditions of Breslow and Goldsby (1969). However, we noted retrospectively that the wild-type level of resistance increased as a function of time in culture. For this reason n e w aliquots of V79 cells were obtained, and the same pattern was observed (Fig. 2). When cells were cultivated only in Dulbecco's medium, the level o f resistance did n o t appear to rise. We susepect that the change in resistance level was due to the selection of partially thymidine-
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F i g . 2. D o u b l e l o g p l o t s h o w i n g c h a n g e i n I D s 0 o f V 7 9 cells as a f u n c t i o n o f t i m e p e r i o d o f c u l t i v a t i o n . Cells w e r e Crown in F - 1 0 m e d i u m (o) D u l b e c c o ' s m e d i u m ( e ) , o r i n o n e c a s e , 3 0 d a y s in F - 1 0 f o l l o w e d b y 1 0 d a y s i n D u l b e c c o ' s m e d i u m (0). I D $ 0 v a l u e s w e r e c a l c u l a t e d f r o m d a t a s i m i l a r t o F i g . 1; t h u s e a c h point represents the amount of thymidine which inhibited Crowth to 50% that of the control plating efficiency.
148
resistant variants by the small quantities of thymidine present in the F-10 medium.
Isolation of thymidine-resistant cell lines In this series of experiments, V 7 9 cells were plated in 50 and 100/~g/ml of thymidine and clones were isolated and retested. As shown in Fig. 3, some of the clones isolated at 50 #g/ml of thymidine were no more resistant than the wild-type parent whereas all clones isolated at 100 #g]ml were substantially more resistant. Furthermore, the existence of intermediate levels of resistance among the clones indicates that wide ranges of thymidine-resistance levels exist, ranging from the clones which are completely unaffected at the 100 ~g/ml concentration to clones that are totally inhibited at this level. (See also Fig. 2.) 3 clones isolated at 50 ~g/ml of thymidine were extensively tested after varying periods of growth in thymidine-free medium. The line V79/HTC4 was tested 10 days after isolation, and retested 51 and 63 days later. It was grown for a total of 231 days at which time it was stored by freezing (see Methods). After several months it was thawed and retested after 1 day of growth {total 232 days). As demonstrated in Fig. 4 the line was resistant to approximately the same concentration on every occasion tested. 2 other clones were tested
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Fig. 3. Plating e f f i c i e n c y o f V 7 9 p a r e n t c l o n e s i s o l a t e d at 5 0 and 1 0 0 # g / m l o f t h y m i d i n e , o , V 7 9 ( w i l d t y p e ) ; ~, c l o n a l lines i s o l a t e d at 5 0 ~ g / m h o c l o n a l lines i s o l a t e d at 1 0 0 # g / m 1 .
149
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Fig. 4. R e s p o n s e o f the line V 7 9 / H T C 4 to t h y m i d i n e . Vertical bazs indicate t h e range. Cells w e r e plated at SO0 cells per dish. e , I 0 d a y s ; o , 6 3 d a y s ; *, 7 1 d a y s ; ~ , 2 3 2 d a y s .
and one o f these retained its resistance to thymidine over a period of 70 days in F-10 medium. The third clone was no more resistant than the wild-type to thymidine.
Effect of phase of growth on resistance Since enzymes involved in the metabolism of thymidine vary as a function of the growth of the cultures (Littlefield, 1966), it was suspected that cells in logarithmic as opposed to the stationary phase of growth would respond differently to the toxic effects of thymidine. Thus cells were harvested under these conditions and plated in different levels of thymidine-containing medium. The results show little variation in toxicity profiles for cells raised under these differing conditions. Furthermore, use of dialyzed serum in the test medium had little influence on the response of the cells, although overall cloning efficiency was considerably lower (Fig. 5).
Effect of cell crowding on mutation frequency In preliminary studies the mutation frequency was found to be substantially lower in dense cultures suggesting the possibility of metabolic cooperation.
150
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This question was investigated by plating artificial mixtures of sensitive and resistant cells in the presence of thymidine. Table 1 shows that the plating efficiency of a thymidine-resistant mutant was substantially depressed as a result of the presence of sensitive cells at densities of l 0 s per 60-mm petri dish. Mutation-rate estimates
Fluctuation tests were performed in order to measure the mutation rate to high thymidine resistance and to establish the random nature of the variation using the standard Luria--Delbrtick approach. These experiments and their control are shown in Tables 2 and 3. T w o independent clones, V 7 9 / H T C 5 2 and V 7 9 / H T C 5 4 were isolated from this experiment and tested for their level of
TABLE 1 E F F E C T OF S E N S I T I V E CELLS ON T H E P L A T I N G DIFFERING CONCENTRATIONS OF THYMIDINE Thymidine concentration
0 100 200 400
EFFICIENCY
O F R E S I S T A N T C E L L S IN
Cell c o m b i n a t i o n s 103 V 7 9 / H T C 4
105 V 7 9
103 V 7 9 / H T C 4 + 105 V 7 9
322 226 184 135
a 28 16 7
a 21 21 9
a N o t p e r f o r m e d . ( N u m b e r s r e p r e s e n t average c l o n i n g n u m b e r s o f triplicate c u l t u r e s . ) T h e plating efficlencies o f V 7 9 in the s e c o n d c o l u m n d o n o t agree w i t h t h e m u t a t i o n - r a t e e s t i m a t e s ( T a b l e s 2 and 3) because of the effects of crowding.
151 TABLE 2 LURIA--DELBRUCK FLUCTUATION TEST Flask n u m b e r 1 2
3 4 5 6 7 8
C l o n e s in c o n t r o l 275 388 980 500 2000 202 1278 348
C l o n e s in t h y m i d i n e
Resistant clones per 6715
3 42 90 102 53 26 46 272
8 81 67 134 19 96 27 587
V 7 9 cells w e r e s e e d e d i n t o L i m b r o Multi-well p l a t e s a t a d e n s i t y o f 1 cell p e r well. T h o s e wells w h i c h y i e l d e d a single c l o n e w e r e h a r v e s t e d , a n d t h e c l o n e w a s s u s p e n d e d in 1 m l o f m e d i u m . 0.1 m l w a s s e e d e d i n t o a p e t r i dish in n o r m a l g r o w t h m e d i u m , a n d t h e o t h e r 0.9 m l w a s p l a t e d i n t o 4 0 0 # g / m l ' o f t h y m i d i n e . A f t e r c l o n e s a r o s e , t h e y w e r e s t a i n e d a n d c o u n t e d . T h e a v e r a g e n u m b e r o f c l o n e s w a s m e a s u r e d in t h e c o n t r o l , a n d this figure was u s e d t o c a l c u l a t e a n a d j u s t e d n u m b e r o f r e s i s t a n t clones. Using t h e m e d i a n m e t h o d (Table 3 of Lea and Coulson, 1949), the m e d i a n n u m b e r of m u t a n t s was calculated, and the mutat i o n r a t e w a s e s t i m a t e d b y d i v i d i n g b y 6 7 1 5 (9 t i m e s t h e a v e r a g e n u m b e r o f c l o n e s in t h e c o n t r o l ) . T h e d a t a f r o m t h e 8 p l a t e s y i e l d e d a m u t a t i o n r a t e o f 2.67 X 1 0 - 3 . T h e h y p o t h e s i s t h a t all 8 s a m p l e s a r o s e f r o m t h e s a m e p o p u l a t i o n , w i t h t h e t r u e p r o p o r t i o n o f m u t a n t s e s t i m a t e d b y t h e p r o p o r t i o n in t h e t o t a l s a m p l e s , w a s r e j e c t e d b y t h e X2 t e s t (×2 ffi 7 8 3 ; D F = 7 ; P ~ 0 . 0 0 5 ) .
resistance, and both were highly and stably resistant to thymidine (data n o t shown).
Chromosome studies Chromosome counts were performed on the V79 parent and 3 thymidineresistant derivatives. The modal chromosome n u m b e r was 19 in each case, and no differences were observed in the overall numerical distribution.
TABLE 3 C O N T R O L D A T A FOR T H E F L U C T U A T I O N TEST; F R E Q U E N C Y OF CLONES R E S I S T A N T TO 400 /~glml' O F T H Y M I D I N E N u m b e r o f c l o n e s p e r flask
N u m b e r o f flasks V 7 9
0 1 2 3 4 5 6 7
4 5 7 1 0 0 0 1
N u m b e r o f v i a b l e cells p l a t e d p e r flask
1.7 × 102
M e a n c o l o n i e s pex flask
1.61
Mutation frequency
9.4 × 1 0 -3
C o n d i t i o n s w e r e t h e s a m e as t h o s e r e p o r t e d in T a b l e 3, e x c e p t t h a t t h e cells w e r e p l a t e d d i r e c t l y i n t o t h e s e l e c t i n g a g e n t ( 4 0 0 /~g/ml t h y m i d i n e ) ; s i m u l t a n e o u s l y a n e q u a l n u m b e r o f cells w e r e p l a t e d i n t o n o r m a l m e d i u m . T h e m u t a t i o n f r e q u e n c y was o b t a i n e d b y dividing t h e n u m b e r of c l o n e s in t h y m l d i n e b y t h e n u m b e r in n o r m a l m e d i u m .
152
TABLE 4 T H Y M I D I N E K I N A S E A C T I V I T Y IN WILD-TYPE A N D V A R I A N T CLONES Cell llne
IDs 0 #g/ml thymidine a
V79 V79/HTC51 V79/HTC9 V79/HTC5
26 >2000 150 150
Specific activity X i0 -3 cpm/min/mg protein 14.5 8.2 13 12
a All clones grown in D M E M .
Thymidine kinase studies Although resistance to thymidine and thymidine analogs such as bromodeoxyuridine may arise through a loss of thymidine kinase activity, our mutants were not found to be significantly affected (Table 4). In all of the variant clones we observed only slight depressions in thymidine kinase activity. Uptake studies Uptake studies were performed using wild-type cells and 2 resistant lines (V79/HTC51; selected at 100 gg/ml thymidine and V79/HTC51/2m selected from V79/HTC51 in 2 mg/ml thymidine). The results shown in Fig. 6 indicate that there is indeed a negative correlation between the capacity o f the mutants
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Fig. 6. [ 3 H ] T h y m i d i n e u p t a k e o n a per cell basis in w i l d - t y p e and m u t a n t V 7 9 cells. Cells w e r e p l a t e d in m u l t i - w e l l flasks and a l l o w e d t o g r o w f o r 2~ h in F - 1 0 m e d i u m . T h e cells w e r e t h e n i n c u b a t e d in t h e p r e s e n c e o f [ 3 H ] t h y m i d i n e f o r p e r i o d s o f t i m e u p t o 1 0 rain. Cells w e r e w a s h e d t h o r o u g h l y and a n a l y z e d for t h y m i d i n e u p t a k e . Cell n u m b e r s w e r e d e t e r m i n e d using w e l l s n o t u s e d f o r u p t a k e . X, V 7 9 ; A V 7 9 / H T C 5 1 ; o, V 7 9 / H T C 5 1 / 2 m . Fig. 7. T h y m i d i n e u p t a k e in w i l d - t y p e a n d m u t a n t V 7 9 cells. T h e s e e x p e r i m e n t s w e r e p e r f o r m e d as t h o s e in Fig. 4 w i t h t h e e x c e p t i o n t h a t a l l q u o t s f r o m e a c h s a m p l e w e r e a n a l y z e d f o r p r o t e i n c o n t e n t , o, V 7 9 ; ~, V 7 9 / H T C 3 ; e , V 7 9 / H T C 5 1 / 2 m 2 ; ~, V 7 9 / H T C 5 1 / 2 m 3 ; , , V 7 9 / H T C 5 1 / 2 m 1 .
153 TABLE 5 U R I D I N E U P T A K E IN W I L D - T Y P E A N D T H Y M I D I N E o R E S I S T A N T
LINES
Cell line
IDs 0 mg/ml thymidine
U r i d i n e u p t a k e c p m / m i n / m g Protein
V79-wild type V79]HTCS1/2m 1 V79/HTC 51/2m 2
26 >2000 > 2000
8620 7036 8859
to take up exogenous thymidine and their level of resistance. These studies were extended to include 3 subclones from the V 7 9 / H T C 5 1 / 2m line already isolated in our laboratory. In Fig. 7 it is shown that all of the variant cell lines exhibit depressed thymidine uptake. It can also be seen that the clones isolated from the V 7 9 / H T C 5 1 / 2 m line displayed slightly less uptake than the variant line V79/HTC3. Uridine uptake experiments were performed in an effort to determine if the uptake lesion observed i n the mutant cells was specific for thymidine or if it affected pyrimidine transport in general. No difference between uridine uptake in the wild-type cells or in any o f the mutants tested was observed (Table 5). Discussion Elevated concentrations of thymidine added to the medium inhibit the growth of V79 hamster cells, and stably resistant clonal isolates can be obtained with a high rate. Over extended periods of time in F-10 medium the base-line resistance of the wild-type population changes probably due to the selecting o u t of variants with slightly augmented growth capacity in the low levels of thymidine provided b y the F-10 medium. This change in the level o f resistance has n o t been observed in Dulbecco's medium (which does not contain added thymidine) thus strengthening this belief. The fact that highly resistant lines did n o t increase even further in their IDs0 probably is the result of a m a x i m u m effect on the part of the F-10 medium. Although cultivation of the cells in F-10 caused their resistance level to increase to 400 ~g/ml after 1 year, further cultivation did not result ih any additional increase in resistance. We have studied a number of factors which might affect the resistance profiles of both wild-type and variant cells. Neither the state or growth of the cells prior to their introduction into the thymidine, nor the source of the serum, nor the time at which the clones were c o u n t e d after the addition of thymidine to the medium resulted in significant changes in resistance levels. Metabolic cooperation does appear, however, to be a factor, as shown b y the reconstruction experiments. This may explain the lower mutation f r e q u e n c y and rate estimates previously reported b y ourselves (Morrow et al., 1975) and Breslow and Goldsby (1969). When mutation rates were measured b y the classical fluctuation test, very high figures were obtained. Above 2 mg/ml of thymidine no variants were encountered in a n u m b e r o f expeiqments in which several million cells were screened. It may be that at high concentrations in the medium, the a m o u n t of thymidine moving passively into the cell is so large as to overcome any resistance mechanisms resulting from genetic variation.
154 Although the mutation rates reported here are extremely high by the criteria of microbial or human genetics (Morrow, 1975, 1977), they are not unprecedented. Terzi and Hawkins (1974) reported rates to aminopterin resistance as high as 10 -2, and Coffino and Scharff (1971) has shown that immunoglobulin variants in myeloma cells occur with rates as high as 10 -3. It could be objected that the pattern of resistance described here may not represent a true genetic variant, but is rather an artifact of the conditions employed in the culturing of the cells. The results of the experiments in which clonal lines were cultivated for long periods in the absence of thymidine and retested for their level of resistance clearly eliminate this possibility. Thus thymidine resistance, while possessing a number of unusual properties, represents a stable, heritable alteration. The data reported in Fig. 3 demonstrate that discrete levels of resistance do not exist, but a whole spectrum of differing resistance levels can be obtained. There have been reports (De Carli et al., 1963; Terzi, 1974) of a causal association between high rates of somatic cell variation and drastic chromosomal alterations. Although we have not observed such alterations in the present studies, more sophisticated analyses using chromosomal banding techniques will be required to resolve this issue. Roufa et al. (1973) have argued, on the basis of mutation-frequency data and levels of thymidine kinase in BrdU-sensitive and -resistant hamster cells, that resistance is due to homozygosity for a mutation which inactivates the thymidine kinase enzyme. However, Freed and Hames (1976) have evidence that there exist in haploid frog cells 2 forms of thymidine kinase, a thermolabile and a thermostable type, and that a transport reaction exists which is dependent upon the thermolabfle enzyme. This suggestion would be in agreement with the results of Plagemann et al. (1976) who have shown that in TKmouse cells transport is by means of facilitated diffusion which has a high Km for Tdr and is non-specific for various ribonucleotides and deoxyribonucleotides. Thus Plagemann et al. (1976) has suggested that either the rate-limiting step in thymidine incorporation in wild-type cells is phosphorylation, or that the cells possess 2 transport systems: facilitated diffusion and substrate transport involving phosphorylation. Although the studies of other workers and those reported here may be interpreted in terms of a membrane defect in the transport of thymidine, it should be cautioned that other alternatives are possible, and these have not been ruled out at this time. Lowered uptake of thymidine could result from an augmentation of the rate of internal pyrimidine synthesis, or a decrease in the breakdown of pyrimidines. In the case of thymidine transport, equilibration is extremely rapid, and is achieved in a matter of seconds through facilitated diffusion. The fact that the thymidine resistant mutants described here are not affected in their uptake of uridine (Table 5) suggests that they may n o t be transport mutants which would be expected to show no specificity with respect to the various nucleosides (Plagemann et al., 1976). Further experiments are being conducted to resolve this issue.
155 References Bresiow, R.E., and R.A. Goldsby (1969) Isolation and characterization of t h y m i d i n e t r a s n p o r t m u t a n t s of Chinese h a m s t e r cells, Exp. Cell Res., 5 5 , 3 3 9 - - 3 4 6 . Clive, D., W.G. F l a m m and J.B. Patterson (1973) Specific locus m u t a t i o n a l assay systems for mous e l ymp h o m a cells, A p p e n d i x II, in: A. Hollander (Ed.), Chemical Mutagens, Vol. 3, Plenum, New Y ork, pp. 100--103. Coffino, P., and M.D. Scharff (1971) Rate of somatic m u t a t i o n in i m m u n o g i o b u l l n p r o d u c t i o n b y mouse m y e l o m a cells, Proc. Natl. Acad. Sci. (U.S.A.), 6 8 , 2 1 9 - - 2 2 5 . Coinfinre, J., J. Morrow and M.K. Patterson Jr. (1973) Asparagine-requiring t u m o r cell lines and t he i r non-requiring variants: cytogenetics, b i o c h e m i s t r y , and p o p u l a t i o n dyna mi c s , Genetics 75, 503--514. De Carll, L., J.J. Malo and F. Nuzzo (1963). Alkaline phos pha t a s e activity a nd c h r o m o s o m e variation in h u m a n cells in culture, J. Natl. Cancer Inst., 31, 1501--1 509. De Mars, R. (1974) Resistance of cultured h u m a n fibroblasts and ot he r cells t o purine and py~unidide analogues in relation to mutagenesis d e t e c t i o n , Mutation Res., 24, 335--364. Fox, M., and D. Anderson (1974) Characteristics of s p o n t a n e o u s and i nduc e d t h y m i d i n e and 5-iodo-2d e o x y u r i d i n e resistant clones of mouse l y m p h o m a cells, Mut a t i on Res., 25, 89--105. Freed, J.J., and I.M. Haines (1976) Loss of a t h e r m o l a b i l e t h y m i d i n e kinase activity in b r o m o d e o x y uridine resistant (transport deficient, kinase positive) h a p l o i d cultured frog cells, Exp. Cell Res., 99, 126--134. Freed, J.J., and L. Mezger-Freed (1973) Origin of t h y m i d i n e kinase deficient (TK-) ha pl oi d frog cells via an i n t e r m e d i a t e t h y m i d i n e t r a n s p o r t deficient (TT-) p h e n o t y p e , J. Cell. Physiol., 82, 199--212. Gillin, F.D., D.J. Roufa, A.L. Beaudett and C.T. Caskey (1972) 8-Azaguanine resistance in m a m m a l i a n cells, I. Hypoxanthine---guanine phosphoribosyitransferase, Genetics 72, 239--252. Ives, D.H., J.P. Duxham and V.S. Tucker (1969) Rapid d e t e r m i n a t i o n of nucleoside kinase and nucleotldase activities with tritium-labeled substrates, Anal. Biochem., 2 8 , 1 9 2 - - 2 0 5 . Kit, S., D.R. Dubbs, L.J. Piekarski and J.C. Hsu (1963) Deletion of t h y m i d i n e kinase activity from L cells resistant to b r o m o d e o x y u r i d i n c , Exp. Cell Res., 31, 297--312. Lea, D.E., and C.A. Coulson (1949) The d i s t r i b u t i o n of t h e n u m b e r s of m u t a n t s in a bacterial p o p u l a t i o n , J. Genet., 4 9 , 2 6 4 - - 2 8 5 . Lee, S.S., B.C. Giovanella and J.S. Stehlin Jr. (1977) Selective lethal effects of t h y m i d i n e on h u m a n and mo use t u m o r cells, J. Cell. Physiol., 9 2 , 4 0 1 - - 4 0 6 . Littlefield, J.W. (1966) The periodic synthesis of t h y m i d i n e kinase i n mouse fibroblasts, Biochim. Biophys. Acta, 1 1 4 , 3 9 8 - - 4 0 3 . Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall (1951) Protein m e a s u r e m e n t w i t h t h e F ol l n Phenol Reagent, J. Biol. Chem., 1 9 3 , 2 6 5 - - 2 7 5 . Luria, S.E., and M. Delbrtlck (1943) Mutations of b a c t e r i a from virus sensitivity t o virus resistance, Genetics 2 8 , 4 9 1 - - 5 1 1 . Meuth, M., and H. Green (1974) Alterations leading to increased r l b o n u c l e o t i d e reductase in cells selected for resistance to deoxynucleosides, Cell, 3, 367--374. Mezger-Freed, L. (1972) Effect of ploidy and m u t a g e n s on b r o m o d e o x y u r i d i n e resistance in haploid and diploid frog cells, Nature (London), New Biol. 2 3 5 , 2 4 5 - - 2 4 6 . Morrow, J. (1975) On the relationship b e t w e e n s p o n t a n e o u s m u t a t i o n rates in vivo and in vitro, Mutation Res., 33, 367--372. Morrow, J. (1977) Gene inactivation as a m e c h a n i s m for t he generation of variability in somatic cells cultivated in vitro, Mutation Res., 44, 391--400. Morrow, J., D. Stocco and D. Minter (1975) V79 Chinese h a m s t e r cells resistant t o high levels of t h y m i dine, In Vitro, 10, 3 6 9 - - 3 7 0 (abstract). Morrow, J., D. Stocco and E. Barton (1978) S p o n t a n e o u s m u t a t i o n rate t o t hl ogua ni ne resistance is decreased in p o l y p o i d h a m s t e r cells, J. Cell. Physiol., 95, 81---86. Morse, Jr.P~A., and V.R. Potter (1965) Pyrimidine m e t a b o l i s m i n tissue culture cells derived from ra t h e p a t o m a s , I. Suspension cell cultures derived from th e N ovi koff h e p a t o m a , Cancer Res., 25, 4 9 9 - 508. Plagemann, P.G.W., R. Marz and J. Erbe (1976) Transport and c o u n t e r t r a n s p o r t in t h y m i d i n e in ATP depleted and t h y m i d i n e kinase-defieient Novikoff rat h e p a t o m a and mous e L cells: evidence for a high Km facilitated diffusion system with wide n u c l e o t i d e specificity, J. Cell Physiol., 89, 1--18. Prockett, M.S., L. Coultrip, M.K. Patterson and J. Morrow (1975) Effect of p l o i d y on s p o n t a n e o u s mut a t i o n rate to asParagine n o n - r e q u i r e m e n t in cultured cells, J. Cell. Physiol., 85, 621--626. Roufa, D.J., B.N. Sadow and C.T. (1978) Derivation of T K - clones from revertant TK + m a m m a l i a n cells, Genetics, 7 5 , 5 1 5 - - 5 3 0 . Schneider, E.L., E.J. Stanbridge and C.J. Epstein (1974) I n c o r p o r a t i o n of 3H-uridine a nd 3H-uracil i nt o RNA, Exp. Cell Res., 84, 311--318.
156
Szybalski, W., and M.J. Smith (1959) Genetics of h u m a n cell lines, I. 8-Azaguanine resistance, a selective "single-step" marker, Proc. Soc. Exp. Biol. Med. 1 0 1 , 6 6 2 - - 6 6 6 . Terzi, M. (1974) C h r o m o s o m a l variation and the origin of drug-resistant m u t a n t s in m a m m a l i a n cell lines, Proc. Natl. Acad. Sci. (U.S.A.), 71, 5027--5031. Terzi, M., and T.S. Hawkins (1974) Chromosal variation, cellular aging and resdstance t o antifolate analogues in a Chinese h a m s t e r cell line, Biochem. Exp. Biol. 1 1 , 2 4 5 - - 2 5 4 .
Mutation Research, 73 ( 1 9 8 0 ) 1 4 3 - - 1 5 6 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
THYMIDINE RESISTANCE IN CHINESE HAMSTER V79 CELLS IN VITRO
JOHN MORROW
and D O U G L A S
STOCCO
Department of Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430 (U.S.A.) ( R e c e i v e d 17 J a n u a r y 1 9 8 0 ) ( R e v i s i o n received 12 M a y 1 9 8 0 ) ( A c c e p t e d 14 M a y 1 9 8 0 )
Summary Thymidine resistance in V79 Chinese hamster cells has been investigated. Phenotypically stable variant resistant lines occurred at a high frequency, and the mutation rate (2.67 X 10 -s per cell per generation) to 400 ~g/ml thymidine resistance as measured by the standard Luria--Delbriick fluctuation analysis was extremely high. Populations of cells maintained for extended periods in F-10 medium spontaneously increased in resistance, possibly as a result of selective pressures due to the thymidine present in F-10 medium since this change was not observed in Dulbecco's medium. The degree of resistance for a given variant was correlated with the amount of thymidine employed in its selection. Metabolic cooperation, resulting in the suppression of the resistant phenotype, was demonstrated in artificial mixtures of sensitive and resistant clonal lines. Clones isolated in high levels of thymidine possessed lowered uptake of [3H]thymidine and the depression in uptake was related to the level of resistance of the particular clohe. Although thymidine kinase specific activity levels were slightly depressed in variant cell lines, growth rate and uridine uptake were unaffected. We conclude that thymidine resistance is due to a genetically controlled depression of external thymidine uptake.
High concentrations of thymidine are toxic to mammalian cells grown in culture (Lee et al., 1977). The mechanism of toxicity is believed to be the inhibition of the synthesis of deoxycytidine triphosphate from cytidine-5'phosphate, due to feedback inhibition (Morse and Potter, 1965). Selection of cells in high levels of thymidine has allowed the isolation of several classes of resistance. Breslow and Goldsby (1969) isolated and characterized avariant of
* This work was supported by a grant f-corn The Public Health Service (I-ROI-CA-16207-01).
144 Don Chinese hamster fibroblasts which was resistant to high levels of thymidine because of a lowered ability to incorporate exogenous thymidine from the medium. In these cells the mutation rate to thymidine resistance was reported to be 2.6 X 10 -4 per cell per generation. In another study (Mezger-Freed, 1972) haploid frog cell variants were isolated which were partially resistant to BrdU, although they contained near wild-type levels of the enzyme thymidine kinase. The absence of this enzyme is usually responsible for resistance to BrdU (Kit et al., 1963). It was proposed that these mutants were resistant to BrdU because of a defect in the BrdU-transport system as measured by the uptake of tritiated thymidine. A further report on this problem (Freed and Mezger-Freed, 1973) stated that the loss of the thymidine-transport system was an obligate intermediate mutation during the selection of a thymidine kinase negative mutant in haploid frog cells. It has been reported (Meuth and Green, 1974) that sublines of the mouse fibrolast line 3T6 have been isolated which are resistant to arabinosyl cytosine and deoxynucleosides of adenine, thymidine and guanine. These altered sublines are characterized by increases in the activity of the enzyme ribonucleotide reductase and a reduced sensitivity of this enzyme to inhibition by dATP. Fox and Anderson (1974) also reported data on the characterization of high thymidine-resistant mouse-lymphoma cells. Working with thymidine-resistant clones of L5178Y and P388 cells, these authors found that in general, thymidine-resistant clones had substantial amounts of thymidine kinase activity but had signigicantly lower levels of phosphorylated thymidine derivatives, thus suggesting the mutants were the result of altered permeability to thymidine. The data of Breslow and Goldsby (1969) have been analyzed by De Mars (1974) who pointed out that because of the large initial number of cells employed in the fluctuation tests pre-existing mutants may have been included. In addition to the controversy over the actual mutation rate to thymidine resistance, two of the reports mentioned previously (Fox and Anderson, 1974; Mezger-Freed, 1972) indicate that not all of the clones resistant to high thymidine were inhibited in their ability to take up exogenous thymidine to the same extent. Because of uncertainties concerning the mutation rate to high thymidine resistance in mammalian cells and in an effort to further clarify the relationship between the degree of thymidine resistance and thymidine uptake, we have reinvestigated this problem in greater detail. Our approach has been to isolate variants resistant to thymidine in V79 Chinese hamster cells at different concentrations of the selecting agent and at several cell densities, and to genetically and biochemically characterize the various classes of mutants thus obtained. Furthermore, we have measured the mutation rate to thymidine resistance using the Luria--Delbriick (1943) fluctuation test. The figures obtained are among the highest reported for any mammalian cell line (Morrow, 1975). In addition, we have described results of experiments which define the parameters of this system. Finally, we present evidence implicating lowered thymidine uptake as being the mechanism responsible for thymidine resistance and relating the degree of exclusion of external thymidine to the level of resistance.
145 Materials and methods
Experimental procedures Culture techniques, measurement of mutation frequencies and evaluation of resistance levels were performed according to previously described procedures (Morrow et al., 1978; Prickett et al., 1975). Briefly, cells were grown in Ham's F-10 medium, made by us from the components (Sigma, St. Louis, MO) or Dulbecco's medium (K.C. Biologicals) to which 10% fetal calf serum (Kansas City Biologicals, Lenexa, KA) was added. Gentamycin (Schering) was added (50 ~g/ml) to prevent bacterial contamination. The cells were transferred with Viokase (GIBCO, Grand Island, NY), and stored frozen in 5% dimethyl sulfoxide in a Revco freezer. The A3 sublcone of the V79 Chinese Hamster cell line (Gillin et al., 1972) was a generous gift from Dr. Donald Roufa, Division of Biology, Kansas State University. The cells were not clones and did not contain other genetic markers. Stock cultures were maintained in glass milk dilution bottles. For some experiments an azaguanine-resistant derivative of the V79 cell was employed. These cells are deficient in hypoxanthine-guanine phosphoribosyl transferase and do not grow in the reverse selective "HAT" medium (Szybalski and Smith, 1959). To evaluate the resistance level of sensitive and resistant lines, cells were harvested and plated at densities of 500 to 103 cells per 60-mm petri dish (Falcon Plastics, Oxnard, CA) with 3 replicates for each concentration. When macroscopically visible clones appeared they were stained with crystal violet, and counted with the aid of a dissection microscope. The time re(tuired for the growth of clones was approx. 1 week; Fig. 1 shows a pilot experiment experi-
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Fig. 1. T h e t h y m i d i n e - r e s i s t a n t m u t a n t V 7 9 / H T C 3 w a s p l a t e d i n F a l c o n p e t r l d i s h e s i n 0, 2 0 0 a n d 4 0 0 #g/m.] a t a d e n s i t y o f 10 3 cells p e r d i s h . A t v a r y i n g t i m e s t h e r e a f t e r , 3 p l a t e s a t a t i m e w e r e s t a i n e d , a n d the clones counted. Vertical bars indicate the range. For the sake of clarity, the points have been shifted s l i g h t l y f r o m t h e a c t u a l c o n c e n t r a t i o n s , e , 5 t h d a y ; a 7 t h d a y ; o , 1 2 t h d a y ; X, 1 4 t h d a y ; ~, 1 7 t h d a y a f t e r plating.
146 ment in which a resistant line (see below) was tested by plating cells in petri dishes and growing the clones for different periods. As can be seen the period of growth had very little effect on the number of clones with the exception of the 5-day, 400/~g/ml point. Cell numbers were determined with a Celloscope electronic particle counter (Particle Data, Inc., Elmwood, IL). Clonal lines were isolated b y removing a portion of the Falcon flask with a hot cork borer and inserting a sterile stainless steel "peni-cylinder" greased with sterile vaseline over the clone. Viokase was added, and the clone transferred to a new culture vessel. Cells were checked for mycoplasma contamination b y t w o methods: a modification of the m e t h o d of Schneider et al. (1974) in which the ratio of [3H]uridine/[ 14C]uracil incorporation was used as an indicator of mycoplasma contamination, and the ability of the cells to grow in H A T medium (Clive et al., 1973). Mutation rates were calculated using the Luria--Delbrfick (1943) fluctuation test. Single cells were grown in Limbro multi-well plates until visible colonies were formed. The colonies were harvested and suspended in 1.0 ml of culture medium. 0.1 ml was plated in F-10 medium and the other 0.9 ml was plated in F-10 plus thymidine. When visible colonies were formed in these plates (approx. 2 weeks) the plates were stained and counted. To obtain the mutation frequency per plate the number o f control colonies was multiplied by 9 and divided into the number o f colonies in thymidine. The mutation rate was then calculated using the median m e t h o d o f Lea and Coulson {1949). Chromosome studies were performed using previously described methods (Colofiore et al., 1973). Thymidine kinase assays Thymidine kinase specific activity was determined as described by Ives, Durham and Tucker (1969). Cells were harvested, washed twice with Hank's balanced salt solution and pelleted b y centrifugation in a clinical centrifuge. The cells were resuspended in 0.25 M sucrose pH 7.6 and homogenized using a glass homogenizer. 100 /~l of this homogenate was mixed with 100 /~l of 2 X reaction mixture. The final concentration of c o m p o n e n t s in the incubation mixture was 50 mM Tris--HC1 pH 8.0; 5 mM ATP; 2.5 mM MgCI: and 1/ICi/ml [3H]thymidine (methyl-3H, 45.58 Ci/mmole, New England Nuclear, Boston, MA). The incubations were performed in glass test tubes at 37°C with gentle shaking. Samples were taken at specific intervals for up to 30 min and the reaction was stopped b y immersing the tubes in a boiling water bath for 2 min. Following centrifugation at 12 000 X g for 5 min to pellet the denatured protein, 50-/~1 aliquots of each supernatant were pipetted directly onto 18-mm DEAE-substituted paper discs (Whatman DE-81). The discs were allowed to dry for 10 min and then washed for 30 min in a beaker containing 30 ml of water per disc with 2 changes of water in order to remove the unreacted substrate. Following the washing, the discs were then placed in polyethylene minivials and 1.0 ml of HC1/KC1 (0.1/0.2 M) was added to each vial to elute the phosphorylated thymidine derivatives from the discs. After 15 min of elution, 4.0 ml of a 75% toluene 25% triton x-114-Omnifluor (New England Nuclear)
147
scintillation fluid was added to each vial and all samples were assayed for radioactivity. The counting efficiency o f this system was 19.8%.
Uptake studies 5 X 104 wild-type or m u t a n t cells were plated into each well of a multi-well plastic dish in F-10 medium plus 10% fetal calf serum. At the end of 24 h, the medium was removed and the cells were washed with Hank's balanced salt solution. Following the wash, fresh Hank's containing 25/~Ci/ml [SH]thymidine or 10 #Ci/ml [SH]uridine 3H(G) (3.66 Ci/mmole, New England Nuclear) was added to the cells. At intervals b e t w e e n 0 and 10 min, the isotope was removed and the cells were quickly washed twice with Hank's. After the incubations the cells were disrupted with 1% sodium d o d e c y l sulphate (SDS). Aliquots of each sample were taken and placed in scintillation vials containing the toluene-triton Omnifluor cocktail described earlier and a portion of the remainder was used for protein determination (Lowry et al., 1951). The counting efficiency in this system was 33%. Cells in wells n o t used for uptake studies were harvested and the cell number determined. Results
Response of V79 cells to thymidine inhibition U p o n introduction into our laboratory V79 cells were transferred from Dulbecco's medium to Ham's F-10. F-10 was chosen because of our desire to duplicate the conditions of Breslow and Goldsby (1969). However, we noted retrospectively that the wild-type level of resistance increased as a function of time in culture. For this reason n e w aliquots of V79 cells were obtained, and the same pattern was observed (Fig. 2). When cells were cultivated only in Dulbecco's medium, the level o f resistance did n o t appear to rise. We susepect that the change in resistance level was due to the selection of partially thymidine-
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F i g . 2. D o u b l e l o g p l o t s h o w i n g c h a n g e i n I D s 0 o f V 7 9 cells as a f u n c t i o n o f t i m e p e r i o d o f c u l t i v a t i o n . Cells w e r e Crown in F - 1 0 m e d i u m (o) D u l b e c c o ' s m e d i u m ( e ) , o r i n o n e c a s e , 3 0 d a y s in F - 1 0 f o l l o w e d b y 1 0 d a y s i n D u l b e c c o ' s m e d i u m (0). I D $ 0 v a l u e s w e r e c a l c u l a t e d f r o m d a t a s i m i l a r t o F i g . 1; t h u s e a c h point represents the amount of thymidine which inhibited Crowth to 50% that of the control plating efficiency.
148
resistant variants by the small quantities of thymidine present in the F-10 medium.
Isolation of thymidine-resistant cell lines In this series of experiments, V 7 9 cells were plated in 50 and 100/~g/ml of thymidine and clones were isolated and retested. As shown in Fig. 3, some of the clones isolated at 50 #g/ml of thymidine were no more resistant than the wild-type parent whereas all clones isolated at 100 #g]ml were substantially more resistant. Furthermore, the existence of intermediate levels of resistance among the clones indicates that wide ranges of thymidine-resistance levels exist, ranging from the clones which are completely unaffected at the 100 ~g/ml concentration to clones that are totally inhibited at this level. (See also Fig. 2.) 3 clones isolated at 50 ~g/ml of thymidine were extensively tested after varying periods of growth in thymidine-free medium. The line V79/HTC4 was tested 10 days after isolation, and retested 51 and 63 days later. It was grown for a total of 231 days at which time it was stored by freezing (see Methods). After several months it was thawed and retested after 1 day of growth {total 232 days). As demonstrated in Fig. 4 the line was resistant to approximately the same concentration on every occasion tested. 2 other clones were tested
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and one o f these retained its resistance to thymidine over a period of 70 days in F-10 medium. The third clone was no more resistant than the wild-type to thymidine.
Effect of phase of growth on resistance Since enzymes involved in the metabolism of thymidine vary as a function of the growth of the cultures (Littlefield, 1966), it was suspected that cells in logarithmic as opposed to the stationary phase of growth would respond differently to the toxic effects of thymidine. Thus cells were harvested under these conditions and plated in different levels of thymidine-containing medium. The results show little variation in toxicity profiles for cells raised under these differing conditions. Furthermore, use of dialyzed serum in the test medium had little influence on the response of the cells, although overall cloning efficiency was considerably lower (Fig. 5).
Effect of cell crowding on mutation frequency In preliminary studies the mutation frequency was found to be substantially lower in dense cultures suggesting the possibility of metabolic cooperation.
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50
,~o ~oo
THYMIDINE CONCENTRATION vg/ml Fig. 5. R e s p o n s e o f p l a t i n g e f f i c i e n c y o f V 7 9 cells in t h y m i d i n e t o g r o w t h p h a s e o f c u l t u r e and u s e o f d i a l y z e d s e r u m in the t e s t m e d i u m . C o n d i t i o n s s a m e as in p r e v i o u s figures, o log p h a s e ; *, s t a t i o n a r y ; a , s t a t i o n a r y , t e s t e d in d i a l y z e d fetal calf s e r u m .
This question was investigated by plating artificial mixtures of sensitive and resistant cells in the presence of thymidine. Table 1 shows that the plating efficiency of a thymidine-resistant mutant was substantially depressed as a result of the presence of sensitive cells at densities of l 0 s per 60-mm petri dish. Mutation-rate estimates
Fluctuation tests were performed in order to measure the mutation rate to high thymidine resistance and to establish the random nature of the variation using the standard Luria--Delbrtick approach. These experiments and their control are shown in Tables 2 and 3. T w o independent clones, V 7 9 / H T C 5 2 and V 7 9 / H T C 5 4 were isolated from this experiment and tested for their level of
TABLE 1 E F F E C T OF S E N S I T I V E CELLS ON T H E P L A T I N G DIFFERING CONCENTRATIONS OF THYMIDINE Thymidine concentration
0 100 200 400
EFFICIENCY
O F R E S I S T A N T C E L L S IN
Cell c o m b i n a t i o n s 103 V 7 9 / H T C 4
105 V 7 9
103 V 7 9 / H T C 4 + 105 V 7 9
322 226 184 135
a 28 16 7
a 21 21 9
a N o t p e r f o r m e d . ( N u m b e r s r e p r e s e n t average c l o n i n g n u m b e r s o f triplicate c u l t u r e s . ) T h e plating efficlencies o f V 7 9 in the s e c o n d c o l u m n d o n o t agree w i t h t h e m u t a t i o n - r a t e e s t i m a t e s ( T a b l e s 2 and 3) because of the effects of crowding.
151 TABLE 2 LURIA--DELBRUCK FLUCTUATION TEST Flask n u m b e r 1 2
3 4 5 6 7 8
C l o n e s in c o n t r o l 275 388 980 500 2000 202 1278 348
C l o n e s in t h y m i d i n e
Resistant clones per 6715
3 42 90 102 53 26 46 272
8 81 67 134 19 96 27 587
V 7 9 cells w e r e s e e d e d i n t o L i m b r o Multi-well p l a t e s a t a d e n s i t y o f 1 cell p e r well. T h o s e wells w h i c h y i e l d e d a single c l o n e w e r e h a r v e s t e d , a n d t h e c l o n e w a s s u s p e n d e d in 1 m l o f m e d i u m . 0.1 m l w a s s e e d e d i n t o a p e t r i dish in n o r m a l g r o w t h m e d i u m , a n d t h e o t h e r 0.9 m l w a s p l a t e d i n t o 4 0 0 # g / m l ' o f t h y m i d i n e . A f t e r c l o n e s a r o s e , t h e y w e r e s t a i n e d a n d c o u n t e d . T h e a v e r a g e n u m b e r o f c l o n e s w a s m e a s u r e d in t h e c o n t r o l , a n d this figure was u s e d t o c a l c u l a t e a n a d j u s t e d n u m b e r o f r e s i s t a n t clones. Using t h e m e d i a n m e t h o d (Table 3 of Lea and Coulson, 1949), the m e d i a n n u m b e r of m u t a n t s was calculated, and the mutat i o n r a t e w a s e s t i m a t e d b y d i v i d i n g b y 6 7 1 5 (9 t i m e s t h e a v e r a g e n u m b e r o f c l o n e s in t h e c o n t r o l ) . T h e d a t a f r o m t h e 8 p l a t e s y i e l d e d a m u t a t i o n r a t e o f 2.67 X 1 0 - 3 . T h e h y p o t h e s i s t h a t all 8 s a m p l e s a r o s e f r o m t h e s a m e p o p u l a t i o n , w i t h t h e t r u e p r o p o r t i o n o f m u t a n t s e s t i m a t e d b y t h e p r o p o r t i o n in t h e t o t a l s a m p l e s , w a s r e j e c t e d b y t h e X2 t e s t (×2 ffi 7 8 3 ; D F = 7 ; P ~ 0 . 0 0 5 ) .
resistance, and both were highly and stably resistant to thymidine (data n o t shown).
Chromosome studies Chromosome counts were performed on the V79 parent and 3 thymidineresistant derivatives. The modal chromosome n u m b e r was 19 in each case, and no differences were observed in the overall numerical distribution.
TABLE 3 C O N T R O L D A T A FOR T H E F L U C T U A T I O N TEST; F R E Q U E N C Y OF CLONES R E S I S T A N T TO 400 /~glml' O F T H Y M I D I N E N u m b e r o f c l o n e s p e r flask
N u m b e r o f flasks V 7 9
0 1 2 3 4 5 6 7
4 5 7 1 0 0 0 1
N u m b e r o f v i a b l e cells p l a t e d p e r flask
1.7 × 102
M e a n c o l o n i e s pex flask
1.61
Mutation frequency
9.4 × 1 0 -3
C o n d i t i o n s w e r e t h e s a m e as t h o s e r e p o r t e d in T a b l e 3, e x c e p t t h a t t h e cells w e r e p l a t e d d i r e c t l y i n t o t h e s e l e c t i n g a g e n t ( 4 0 0 /~g/ml t h y m i d i n e ) ; s i m u l t a n e o u s l y a n e q u a l n u m b e r o f cells w e r e p l a t e d i n t o n o r m a l m e d i u m . T h e m u t a t i o n f r e q u e n c y was o b t a i n e d b y dividing t h e n u m b e r of c l o n e s in t h y m l d i n e b y t h e n u m b e r in n o r m a l m e d i u m .
152
TABLE 4 T H Y M I D I N E K I N A S E A C T I V I T Y IN WILD-TYPE A N D V A R I A N T CLONES Cell llne
IDs 0 #g/ml thymidine a
V79 V79/HTC51 V79/HTC9 V79/HTC5
26 >2000 150 150
Specific activity X i0 -3 cpm/min/mg protein 14.5 8.2 13 12
a All clones grown in D M E M .
Thymidine kinase studies Although resistance to thymidine and thymidine analogs such as bromodeoxyuridine may arise through a loss of thymidine kinase activity, our mutants were not found to be significantly affected (Table 4). In all of the variant clones we observed only slight depressions in thymidine kinase activity. Uptake studies Uptake studies were performed using wild-type cells and 2 resistant lines (V79/HTC51; selected at 100 gg/ml thymidine and V79/HTC51/2m selected from V79/HTC51 in 2 mg/ml thymidine). The results shown in Fig. 6 indicate that there is indeed a negative correlation between the capacity o f the mutants
x
6O '?, o_ × 50
,? o x z IOC
J k~A ~ 40
(3£ n
80~
j0
~ 30
60
j°/
j o
/°
x
40 F /
>-
/o o/
o~
°~
8
JO
i
p u 2
I 4 TIME
I
I
I
6
8
I0
IN M I N U T E S
~ 2
4 TIME
6 IN
MINUTES
Fig. 6. [ 3 H ] T h y m i d i n e u p t a k e o n a per cell basis in w i l d - t y p e and m u t a n t V 7 9 cells. Cells w e r e p l a t e d in m u l t i - w e l l flasks and a l l o w e d t o g r o w f o r 2~ h in F - 1 0 m e d i u m . T h e cells w e r e t h e n i n c u b a t e d in t h e p r e s e n c e o f [ 3 H ] t h y m i d i n e f o r p e r i o d s o f t i m e u p t o 1 0 rain. Cells w e r e w a s h e d t h o r o u g h l y and a n a l y z e d for t h y m i d i n e u p t a k e . Cell n u m b e r s w e r e d e t e r m i n e d using w e l l s n o t u s e d f o r u p t a k e . X, V 7 9 ; A V 7 9 / H T C 5 1 ; o, V 7 9 / H T C 5 1 / 2 m . Fig. 7. T h y m i d i n e u p t a k e in w i l d - t y p e a n d m u t a n t V 7 9 cells. T h e s e e x p e r i m e n t s w e r e p e r f o r m e d as t h o s e in Fig. 4 w i t h t h e e x c e p t i o n t h a t a l l q u o t s f r o m e a c h s a m p l e w e r e a n a l y z e d f o r p r o t e i n c o n t e n t , o, V 7 9 ; ~, V 7 9 / H T C 3 ; e , V 7 9 / H T C 5 1 / 2 m 2 ; ~, V 7 9 / H T C 5 1 / 2 m 3 ; , , V 7 9 / H T C 5 1 / 2 m 1 .
153 TABLE 5 U R I D I N E U P T A K E IN W I L D - T Y P E A N D T H Y M I D I N E o R E S I S T A N T
LINES
Cell line
IDs 0 mg/ml thymidine
U r i d i n e u p t a k e c p m / m i n / m g Protein
V79-wild type V79]HTCS1/2m 1 V79/HTC 51/2m 2
26 >2000 > 2000
8620 7036 8859
to take up exogenous thymidine and their level of resistance. These studies were extended to include 3 subclones from the V 7 9 / H T C 5 1 / 2m line already isolated in our laboratory. In Fig. 7 it is shown that all of the variant cell lines exhibit depressed thymidine uptake. It can also be seen that the clones isolated from the V 7 9 / H T C 5 1 / 2 m line displayed slightly less uptake than the variant line V79/HTC3. Uridine uptake experiments were performed in an effort to determine if the uptake lesion observed i n the mutant cells was specific for thymidine or if it affected pyrimidine transport in general. No difference between uridine uptake in the wild-type cells or in any o f the mutants tested was observed (Table 5). Discussion Elevated concentrations of thymidine added to the medium inhibit the growth of V79 hamster cells, and stably resistant clonal isolates can be obtained with a high rate. Over extended periods of time in F-10 medium the base-line resistance of the wild-type population changes probably due to the selecting o u t of variants with slightly augmented growth capacity in the low levels of thymidine provided b y the F-10 medium. This change in the level o f resistance has n o t been observed in Dulbecco's medium (which does not contain added thymidine) thus strengthening this belief. The fact that highly resistant lines did n o t increase even further in their IDs0 probably is the result of a m a x i m u m effect on the part of the F-10 medium. Although cultivation of the cells in F-10 caused their resistance level to increase to 400 ~g/ml after 1 year, further cultivation did not result ih any additional increase in resistance. We have studied a number of factors which might affect the resistance profiles of both wild-type and variant cells. Neither the state or growth of the cells prior to their introduction into the thymidine, nor the source of the serum, nor the time at which the clones were c o u n t e d after the addition of thymidine to the medium resulted in significant changes in resistance levels. Metabolic cooperation does appear, however, to be a factor, as shown b y the reconstruction experiments. This may explain the lower mutation f r e q u e n c y and rate estimates previously reported b y ourselves (Morrow et al., 1975) and Breslow and Goldsby (1969). When mutation rates were measured b y the classical fluctuation test, very high figures were obtained. Above 2 mg/ml of thymidine no variants were encountered in a n u m b e r o f expeiqments in which several million cells were screened. It may be that at high concentrations in the medium, the a m o u n t of thymidine moving passively into the cell is so large as to overcome any resistance mechanisms resulting from genetic variation.
154 Although the mutation rates reported here are extremely high by the criteria of microbial or human genetics (Morrow, 1975, 1977), they are not unprecedented. Terzi and Hawkins (1974) reported rates to aminopterin resistance as high as 10 -2, and Coffino and Scharff (1971) has shown that immunoglobulin variants in myeloma cells occur with rates as high as 10 -3. It could be objected that the pattern of resistance described here may not represent a true genetic variant, but is rather an artifact of the conditions employed in the culturing of the cells. The results of the experiments in which clonal lines were cultivated for long periods in the absence of thymidine and retested for their level of resistance clearly eliminate this possibility. Thus thymidine resistance, while possessing a number of unusual properties, represents a stable, heritable alteration. The data reported in Fig. 3 demonstrate that discrete levels of resistance do not exist, but a whole spectrum of differing resistance levels can be obtained. There have been reports (De Carli et al., 1963; Terzi, 1974) of a causal association between high rates of somatic cell variation and drastic chromosomal alterations. Although we have not observed such alterations in the present studies, more sophisticated analyses using chromosomal banding techniques will be required to resolve this issue. Roufa et al. (1973) have argued, on the basis of mutation-frequency data and levels of thymidine kinase in BrdU-sensitive and -resistant hamster cells, that resistance is due to homozygosity for a mutation which inactivates the thymidine kinase enzyme. However, Freed and Hames (1976) have evidence that there exist in haploid frog cells 2 forms of thymidine kinase, a thermolabile and a thermostable type, and that a transport reaction exists which is dependent upon the thermolabfle enzyme. This suggestion would be in agreement with the results of Plagemann et al. (1976) who have shown that in TKmouse cells transport is by means of facilitated diffusion which has a high Km for Tdr and is non-specific for various ribonucleotides and deoxyribonucleotides. Thus Plagemann et al. (1976) has suggested that either the rate-limiting step in thymidine incorporation in wild-type cells is phosphorylation, or that the cells possess 2 transport systems: facilitated diffusion and substrate transport involving phosphorylation. Although the studies of other workers and those reported here may be interpreted in terms of a membrane defect in the transport of thymidine, it should be cautioned that other alternatives are possible, and these have not been ruled out at this time. Lowered uptake of thymidine could result from an augmentation of the rate of internal pyrimidine synthesis, or a decrease in the breakdown of pyrimidines. In the case of thymidine transport, equilibration is extremely rapid, and is achieved in a matter of seconds through facilitated diffusion. The fact that the thymidine resistant mutants described here are not affected in their uptake of uridine (Table 5) suggests that they may n o t be transport mutants which would be expected to show no specificity with respect to the various nucleosides (Plagemann et al., 1976). Further experiments are being conducted to resolve this issue.
155 References Bresiow, R.E., and R.A. Goldsby (1969) Isolation and characterization of t h y m i d i n e t r a s n p o r t m u t a n t s of Chinese h a m s t e r cells, Exp. Cell Res., 5 5 , 3 3 9 - - 3 4 6 . Clive, D., W.G. F l a m m and J.B. Patterson (1973) Specific locus m u t a t i o n a l assay systems for mous e l ymp h o m a cells, A p p e n d i x II, in: A. Hollander (Ed.), Chemical Mutagens, Vol. 3, Plenum, New Y ork, pp. 100--103. Coffino, P., and M.D. Scharff (1971) Rate of somatic m u t a t i o n in i m m u n o g i o b u l l n p r o d u c t i o n b y mouse m y e l o m a cells, Proc. Natl. Acad. Sci. (U.S.A.), 6 8 , 2 1 9 - - 2 2 5 . Coinfinre, J., J. Morrow and M.K. Patterson Jr. (1973) Asparagine-requiring t u m o r cell lines and t he i r non-requiring variants: cytogenetics, b i o c h e m i s t r y , and p o p u l a t i o n dyna mi c s , Genetics 75, 503--514. De Carll, L., J.J. Malo and F. Nuzzo (1963). 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Szybalski, W., and M.J. Smith (1959) Genetics of h u m a n cell lines, I. 8-Azaguanine resistance, a selective "single-step" marker, Proc. Soc. Exp. Biol. Med. 1 0 1 , 6 6 2 - - 6 6 6 . Terzi, M. (1974) C h r o m o s o m a l variation and the origin of drug-resistant m u t a n t s in m a m m a l i a n cell lines, Proc. Natl. Acad. Sci. (U.S.A.), 71, 5027--5031. Terzi, M., and T.S. Hawkins (1974) Chromosal variation, cellular aging and resdstance t o antifolate analogues in a Chinese h a m s t e r cell line, Biochem. Exp. Biol. 1 1 , 2 4 5 - - 2 5 4 .