Mutation Research, 27 (1975) 399-406 © Elsevier Scientific Publishing Company, A m s t e r d a m - - P r i n t e d in The Netherlands

399

C Y C L O H E X I M I D E RESISTANCE IN C H I N E S E HAMSTER CELLS I. SPONTANEOUS MUTAGENESIS

H U B E R T P(~CHE, NINA B. V A R S H A V E R * AND E R H A R D G E I S S L E R Academy of Science of the GDR, Central Institute of Molecular Biology, Cell Genetics Department, Berlin-Buch (G.D.R.) and *Kurchatov Institute of Atomic Energy, Biological Department, Moscow (U.S.S.R.) (Received July 9th, 1974) (Revision received September 27th, 1974)

SUMMARY

Resistance to cycloheximide (CHM) was studied in cultured Chinese hamster cells. Concentrations of CHM above 5"1o-7 M were toxic for the cells. At concentrations above 9' lO-7 M no colonies were recovered in selective medium. 15 resistant clones of independent origin were isolated in selective medium containing 7" lO-7 M CHM. Resistance was stable when the cells were cultured under non-selective conditions. The spontaneous mutation rate was determined by the fluctuation test. Mutations to CHM resistance arose spontaneously. The spontaneous mutation rate to CHM resistance was about lO-5.

INTRODUCTION

Spontaneous gene mutagenesis in mammalian cells in vitro has so far been studied for resistance to various anti-metabolites (particularly 8-azaguanine and 6-mercaptopurine), for auxotrophy and for temperature-sensitivity. Chinese hamster cells were mostly used for these studies2,e,9,20-2~, 25. Results of investigations on the resistance of animal cell lines to the antibiotic CHM have not yet been published. This paper shows the extent to which spontaneous resistance to CHM occurs in Chinese hamster cells as well as the magnitude of the spontaneous mutation rate of these cells. The rate of spontaneous mutation was determined by the fluctuation test according to LURIA AND DELBRUCK13 and the method described by LEA AND COULSON (ref. II). CHM inhibits protein synthesis in mammalian cells by affecting the 80S ribosomesl,5,7,1s,~3, ~s. Isolation of CHM-resistant mutant cells could be of importance for further clarifying the precise way in which CHM affects the animal cell and would also provide a new tool for studying protein synthesis in mammalian systems. Abbreviations: CHM, cycloheximide; MEM, minimal essential medium,

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MATERIALS AND METHODS

We used ovarian cells of the Chinese hamster (CHO), obtained from Dr. Bateman (Paterson Laboratories, Christie Hospital, and Holt Radium Institute, Manchester, England) with a modal chromosome number of 21. The cells were cultivated in Eagle-MEM with lO% calf serum (Institut ftir Immunpdiparate und N/ihrmedien, Berlin). The medium contains penicillin (IOO units/ml) and streptomycin (5o/,g/ml). In addition, tetracycline (Ioo units/ml) was added once a month to prevent growth of mycoplasma. Both the lethal CHM concentration and the rate of spontaneous mutation were determined in 60 mm petri dishes. The selective medium used to determine the lethal CHM concentration was Eagle-MEM + 20% foetal bovine serum (Colorado Serum Company, Denver) with a pH of 7.1 to 7.3 and a final CHM (Upjohn Company, Michigan) concentration of 4"1o-7 M to 9 "1o-7 M. The initial inoculum in these experiments was 25 ooo cells per plate. The samples were incubated in a mixture of 5% C02 in air at 37 °. The selective medium was changed every three or four days. The number of surviving colonies was determined after 12 days. i

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100 ceils ~7.5-105 per dish cells per dish Fig. z. O u t l i n e of t h e f l u c t u a t i o n t e s t for d e t e r m i n i n g t h e s p o n t a n e o u s m u t a t i o n rate.

The fluctuation test to determine the spontaneous mutation rate was performed as shown diagrammatically in Fig, I. The experiment was started with io parallel cultures. The initial inoculum was IOO cells per plate. This number ensured a small enough probability of mutant introduction. After IO days the cells numbered about 75oooo per culture. Samples comprising IO plates each were taken from the first three cultures and samples of five plates each were taken from the other seven to ensure satisfactory statistical distribution. The initial inoculum when plated in selective medium was 5oooo cells per plate. The selective medium here was also EagleMEM + 2o% foetal bovine serum (pH 7.1 to 7-3) with a final CHM concentration of 7"Io-v M. Here, too, the selective medium was changed every three or four days. The results were evaluated by the method of LURIA AND DELBRiJCK13 with formula I in Table I and that of LEA AND COULSON11 using formula II in "[able I. The theoretical variances were calculated by the formula (~r2 theor, z

C.a~.nt22f- 1"

401

CYCLOHEXIMIDE RESISTANCE IN HAMSTER CELLS

where r is the mean number oi mutants per sample, C the number of parallel cultures, a the mutation rate and nt the mean number of cells per sample at the moment of transfer to selective conditions. The stability of phenotypic change of CHM-resistant clones isolated from different plates used for the fluctuation test was determined as follows. The cells of each oi 15 clones were grown in selective medium (7 "IO-? M CHM) for 12-14 days. "Ihe cells of these clones were cultivated in selective and normal media alternately Typeof Crone experiment No.

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C Y C L O H E X I M I D E RESISTANCE IN HAMSTER CELLS

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in Fig. 4. The time dependence of the effects of CHM was also studied. The number ot colonies of surviving cells did not decrease significantly after incubation in the selective medium for more than 12 days. The experiments were therefore evaluated up to the I2th day after commencement of selection (see Fig. 4).

(2) Determination of the spontaneous mutation rate Three series of fluctuation test experiments were performed to learn whether the surviving colonies derive from mutant cells and to determine the rate of spontaneous mutation. The values obtained by the methods of LURIA AND DELBRi3CKla and LEA AND COULSON11 (Table I) were in good agreement with each other and resulted in a spontaneous mutation rate of between 0.5 and 1.3" lO-5. Data in Table I make it possible to establish whether spontaneous mutagenesis is involved. Indeed, the values of means and variances for the number of colonies in samples taken 1tom one culture are very close. On the other hand, experimental variances for samples taken from different cultures greatly exceed the values of the means. This proves that the phenomenon observed is spontaneous mutagenesis. This is supported by comparison of experimental and theoretical variances for parallel culture series. Comparison of the theoretical and experimental variarxces showed that Sr2 tbeor. < 6 J exp., which also confirms that colonies grown in selective medium are indeed mutants that arise spontaneously.

(3) Characterization of surviving clones as mutants Fifteen clones of independent origin were recovered from the fluctuation test experiments and incubated in a selective medium with a CHM concentration of 7" 1°-7 M. These clones were further cultivated in normal and selective media alternately as described in Fig. 2. No significant differences in plating efficiency and cell yield were observed in either medium (Table II). Frozen cells, after being thawed and incubated in selective medium, also showed no significant differences when compared with the CHM-sensitive cells in the normal medium. The modal chromosome number remained constant in both the mutants and the wild type cells, and no pronounced chromosome aberrations were found although minor structural changes cannot be excluded as they would be visible only by using banding techniques. Further characteristics will be stated elsewhere. The mutation rate can be raised significantly by treating the cells with mutagens (manuscript in preparation). DISCUSSION

When the spontaneous mutation rates of various resistance markers are compared, it is found that the rates of mutation in vitro are about lO-4 to lO-8 for human and other mammalian cells4,12,14,17,19,~0,24. The values found in our experiments lie between 0.5" lO-5 and 1.3" IO-~. Different extents of resistance to CHM are possible. Cells cultivated in selective medium with concentrations of CHM between 6- lO-7 and 8. lO-7 M have different extents of resistance (see Fig. 4). The mutant clones grown in media with lower CHM concentrations are not simultaneously resistant to the higher concentlations. The statistical evaluation of the mutation rate here refers to the frequency of mutants with a median degree of resistance to CHM (7" lO-7 M).

TABLE I

5 5 5

I 2 3

0.88 8-3 3.1

Variance

2. 5 2.5 2. 5

Sam pl e s f r o m Number of cells per sample ( .2:05)

9.1 20.7 20.0

28.1 329.7 411.1

24. 7 110. 7 82. 5

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10-7 M) AND SPONTANEOUS MUTATION RATE IN CHINESE HAMSTER CELLS

parallel cultures b M e a n number Variance o f colonies Exp. per sample

RESISTANCE (7"

SM SM NM SM NM SM NM

40.0 36.0 40.0 36.7 35-5 36-4 37-3

36.6 33-3 38.5 36.7 38.6 39.0 4 °.0

36.6 26.8 38.2 4 °.0 3 °.o 37.6 38.3

3 31.5 23. 4 38.2 41-6 39.4 35.0 38.3

4 33.2 30.7 41.6 41.6 34.9 23.3 37.7

5 38.5 36.o 38.0 38.6 35.7 36.7 37.4

6 37.8 35.2 38.0 34.5 34 .8 31.6 4 °.0

7 33.2 33-2 4o.0 45 -o 37.4 28.5 36.6

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37 .8 38.6 37.2 38-7 39.5 36.4 36.7

II

39.1 38.6 37.2 46.7 4 °.6 36.4 40.0

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41.7 41.6 36.7 28-5 39.o 37-6 36.5

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"IO-2 M CHM) AND NORMAL M E D I U M ,

36.5 43.4 36-7 3 ° .0 37.0 41.5 36.5

2:5

s The clone a n d p a s s a g e n o t a t i o n s c o r r e s p o n d to t h e n u m b e r i n d i c a t e d in Fig. 2. SM, S e l e c t i v e m e d i u m (7" 1°-7 M CHM) ; NM, n o r m a l m e d i u m .

IX

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Clone No.

No.

OF I 5 C L O N E S OF I N D E P E N D E N T

Passage

PLATING EFFICIENCY (%) RESPECTIVELYa

TABLE II

s The n u m b e r of cells per p l a t e w as 5" lO4 w i t h i o p l a t e s from e a c h c u l t u r e . E x a m p l e s are g i v e n here of d i s t r i b u t i o n s o b t a i n e d in one of t h r e e p a r a l l e l cultures. S im i lar d i s t r i b u t i o n s were o b t a i n e d in t h e o t h e r t w o p a r a l l e l c ul t ure s . b The n u m b e r of cells pe r p l a t e w a s 5" lO4 w i t h 5 p l a t e s from e a c h c u l t u r e . a, M u t a t i o n r a t e p er cell pe r g e n e r a t i o n ; m, m e a n n u m b e r of m u t a t i o n s pe r c u l t u r e ; C, n u m b e r of p a r a l l e l c u l t u r e s in a series; r, m e a n n u m b e r of m u t a n t s p er c u l t u r e ; r0, v a l u e of m e d i a n of t h e r a n g e of t h e n u m b e r of m u t a n t s in a series of p a r a l l e l c u l t u r e s ; No, n u m b e r of cells p e r c u l t u r e a t t h e b e g i n n i n g of t h e e x p e r i m e n t ; Nt, m e a n n u m b e r of cells p e r c u l t u r e a t t h e t i m e of t r a n s f e r t o s e l e c t i v e m e d i u m .

I 6 3

M e a n numb er o f colonies per sample

Samples f r o m one culture s

Number of cells per sample ('1°5)

mental group No.

F L U C T U A T I O N T E S T . M U T A T I O N S TO C Y C L O H E X I M I D E

Experi-

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RESULTS OF

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CYCLOHEXlMIDE RESISTANCE IN HAMSTER CELLS

405

The resistance of m u t a n t cells to CHM m a y be caused either by impairment of the transport of the agent into the cell, by inactivation of CHM, or by changing ribosomal protein(s) as already observed in several fungal systems3,8,1s, le. Our current experiments using CHO cells indicate that CHM-resistant m u t a n t ceils do possess modified ribosomes which do not react with the antibiotic used here. ACKNOWLEDGEMENTS

We wish to express our gratitude to Professor N. I. SHAPIRO and to Dr. M. THEILE from our Institutes for their discussion. REFERENCES I BATTANER, E., AND D. VAZQUES, I n h i b i t o r s of protein synthesis b y ribosomes of the 8oS-type, Biochim. Biophys. Acta, 254 (1971) 316-33 o. 2 CHO, E. H. Y., P. BRIMER, K. B. JACOESON AND E. V. MERRIAM, M a m m a l i a n cell genetics, I. Selection and characterization of m u t a t i o n s a u x o t r o p h i c for L-glutamine or resistant to 8azaguanine in Chinese h a m s t e r cells in vitro, Genetics, 62 (1969) 359-377. 3 COOPER, D., D. V. BANTHORPE ANn D. WILKIE, Modified ribosomes confering resistance to cycloheximide in m u t a n t s of Saccharomyces cerevisiae, J. Mol. Biol., 26 (1967) 347-350. 4 DEMARS, R., AND K. R. HELD, The s p o n t a n e o u s azaguanine-resistant m u t a n t s of diploid h u m a n fibroblasts, Humangenetik, 16 (1972) 87-1io. 5 GROLLMAN, A. P., AND M. Z. MOANS, I n h i b i t o r s of protein synthesis in eucaryotes : tools in cell research, Fed. Proc., 32 (1973) 1673-1678. 6 HARRIS, M., G r o w t h and survival of m a m m a l i a n cells u n d e r c o n t i n u o u s t h e r m a l stress, Exptl. Cell Res., 56 (1969) 382-386. 7 HASELKORN, R., AND L. B. ROTHMAN-DENES, Protein synthesis, Ann. Rev. Biochem., 42 (1973) 397-438 • 8 HAUGLI, F. B., AND W. V. DOVE, Genetics and biochemistry of cycloheximide resistance in Physarum polycephalum, 2~/Iol.Sen. Genet., 118 (1972) 97-1o 7. 9 KAO, FA-TEN, AND T. T. PUCK, Genetics of somatic m a m m a l i a n cells, IV. Properties of Chinese h a m s t e r cell m u t a n t s w i t h respect to the r e q u i r e m e n t for proline, Genetics, 55 (1967) 513-524. IO KHALIZEV, E. A., Some aspects of the use of m e t h o d s for estimation of s p o n t a n e o u s m u t a t i o n rate in m a m m a l i a n somatic cells, Genetika, No. 3 (1969) 157-168 (Russian with English s u m m ary). i i LEA, D. E., AND C. A. COULSON, The distribution of the n u m b e r s of m u t a n t s in bacterial populations, dr. Genet., 49 (1949) 264-285. 12 LIEBERMANN, I., AND P. OVE, E s t i m a t i o n of m u t a t i o n rates with m a m m a l i a n cells culture, Proc. Natl. Acad. Sci. (U.S.), 45 (1959) 872-877. 13 LURIA, S. E., AND M. DELBRUCK, Mutations of bacteria f r o m virus sensitivity to virus resistance, Genetics, 28 (1943) 491-511. 14 MARSHAK, M. I., AND N. B. VARSHAVER, Studies of s p o n t a n e o u s m u t a t i o n rate in diploid and aneuploid h u m a n cells in vitro, Genetika, No. 2 (197 o) 13o-138 (Russian w i t h English s u m m a r y ) . 15 NEUHA-USER A., W. KLINGMULLER AND F. KAUDEWITZ, Selektion Actidion resistenter Mutanten bei Neurospora crassa sowie ihre genetische u n d biochemische Analyse, Mol. Sen. Genet., lO6 (197 ° ) 18o-194. 16 PONGRATZ, M., AND W. KLINGMULLER, Role of ribosomes in cycloheximide resistance of Neurospora m u t a n t s , Mol. Sen. Genet., 124 (1973) 359-363 . 17 PROPPING, P., Comparison of point m u t a t i o n rates in different species with h u m a n m u t a t i o n rates, Humangenetik, 16 (1972) 43-48. i8 SCHLESSINGER, D., Ribosomes: d e v e l o p m e n t of some c u r r e n t ideas, Bacteriol. Rev., 33 (1969) 445-453. 19 SHAPIRO, N. I., O. N. PETROVA AND A. E. KHALIZEV, A s t u d y of s p o n t a n e o u s m u t a t i o n rate in m a m m a l i a n cell culture, Genetika, No. 12 (1966) 5-17, (Russian with English s u m m a r y ) . 20 SHAPIRO, N. I., A. E. KHALIZEV, E. V. L o s s , M. I. MARSHAK, O. N. PETROVA AND N. B. VARSHAVER, Mutagenesis in cultured m a m m a l i a n cells, I. S p o n t a n e o u s gene m u t a t i o n s in h u m a n and Chinese h a m s t e r cells, Mutation Res., 15 (1972) 2o3-214. 21 SMITH, D. B., AND E. H. Y. CHU, Isolation and characterization of t e m p e r a t u r e - s e n s i t i v e mut a n t s in a Chinese h a m s t e r cell line, Mutation Res., 17 (1973) I I 3 12o.

406

L. H., J. L. HARKINS AND C. P. STANNERS, A mammalian cell mutant with a temperature-sensitive leucyl-transfer RNA-synthetase, Proc. Natl. Acad. Sci. (U.S.), 7 ° (1973) 3o94-3o98. TIMBERLAKE, W. E., AND D. H. GRIFFIN, Direct inhibition of t h e u p t a k e of prolin b y cycloheximide, Biochem. Biophys. Res. Commun., 54 (1973) 216-221. VARSHAVER, N. B., L. G. REZNIK, A. M. BAGROVA AND V. G. CHENIKOV, T h e d e t e c t i o n of a u x o t r o p h i c m u t a n t s in m a m m a l i a n cells, Genetika, No. 3 (1971) 89-94 ( R u s s i a n w i t h E n g l i s h summary). VARSHA.VER, N. B., V. G. CHERNIKOV, M. I. MARSHAK AND N. I. SHAPIRO, T e m p e r a t u r e - s e n s i tive m u t a n t s in c u l t u r e d Chinese h a m s t e r cell populations, Genetika, No. 9 (1972) 54 - 6 o (Russian with English summary). VAZQUES, D., E. BATTANER, R. NETH, G. HELLER AND R. E. MONRO, T h e f u n c t i o n of 8oSr i b o s o m a l s u b u n i t s a n d effect of s o m e antibiotics, Cold Spring Harbor Syrup. Quant. Biol., 34 (1969) 369-375 .

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H. P O C H E et al.

Cycloheximide resistance in Chinese hamster cells. I. Spontaneous mutagenesis.

Resistance to cycloheximide (CHM) was studied in cultured Chinese hamster cells. Concentrations of CHM above 5-10-7 M were toxic for the cells. At con...
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