α-Amanitin-resistant mutants of Chinese hamster ovary (CHO) cells

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Mutation Research, 49 (1978) 95--101 © Elsevier/North-Holland Biomedical Press

a-AMANITIN-RESISTANT MUTANTS OF CHINESE HAMSTER O V A R Y (CHO) CELLS

K.C. GUPTA and M.W. TAYLOR

Department of Biological Sciences, Bloomington, Ind. (U.S,A.) (Received 14 February 1977) (Revision received 26 July 1977) (Accepted 4 August 1977)

Summary Spontaneous and EMS-induced ~-amanitin-resistant CHO cells have been isolated and characterized. DNA-dependent R N A polymerase II in cell-free extracts from a mutant (ARM-l) was partially resistant to ~-amanitin. Growing mutants for several generations in the presence or absence of a-amanitin did n o t change the pattern of inhibition. The mutants grew with a lag following transfer to medium with or without a-amanitin. The mutants have an altered RNA polymerase II, and possibly an altered cell membrane.

Introduction a-Amanitin at low concentrations (<1 pg/ml) is a specific inhibitor of eukaryotic DNA-dependent RNA polymerase II. It inhibits chain elongation b y interacting with the enzyme [3,6]. Chan et al. [4] have reported the isolation of an a-amanitin-resistant CHO cell which has an a-amanitin-resistant RNA polymerase II. The enzyme was found to be totally resistant to low concentrations of a-amanitin. S o m e r s e t al. [13] isolated ~-amanitin-resistant rat myoblast cells in which the level of resistant polymerase II could be increased b y growing the cells in a-amanitin-containing medium. With the availability of cells ~ resistant to a-amanitin, studies in transcription and translation control mechanisms in eukaryotic cells should be amenable. In this paper we report the isolation and characterization of an a-amanitinresistant CHO cell which has partially resistant polymerase II. This marker has been introduced into a cell line which is already auxotrophic for proline, asparagine, purine and deficient in adenine phosphoribosyl transferase (pro-, asn-, pur -4 , APRT-). Abbreviations: CHO, Chinese hamster ovary; DAP, 2,6-diaminopurine; EMS, e t h y l methanesulfonate.

96 Materials and methods

Cultivation of cells. All strains of CHO cells were routinely grown as monolayers in Ham's F-12 supplemented with 5% fetal calf serum and antibiotics (50 pg/ml streptomycin and 100 units/ml penicillin). Cell lines used were CHOl b (pro-, asn-, pur-) [7,15] CHO-baP-101 {pro-, asn-, put- and APRT-) [16] and CHO-Ama-1 {resistant to a-amanitin, kindly supplied by Dr. L. Siminovitch). Whenever DAP was used, dialyzed fetal calf serum replaced fetal calf serum. Mutagenesis. a-Amanitin-resistant cells were obtained from baP-101 either spontaneously or after EMS mutagenesis. Spontaneous mutants were isolated by plating a total of 1 X 107 cells in 20 (100-mm) dishes each containing 10 ml of medium with a-amanitin (1 pg/ml). Medium was changed every 5 days. After 7 days microcolonies of cells were present in 10 of the dishes. 10 colonies (ARS-1--ARS-10) from independent dishes were picked, recloned and subsequently tested for their resistance to a-amanitin. EMS mutagenesis was performed by incubating semi-confluent monolayer of cells (1 X 10 ~) in the presence of 500 pg/ml of the mutagen for 18 h. Cells were allowed to recover for 72 h in the normal medium before they were plated in a selective medium containing a-amanitin. 10 clones (ARM-I--ARM-10) were picked from independent dishes recloned and tested for resistance. Resistant cells were recloned, retested and stored at --170°C until further use. Cell growth. Cell growth was measured in the presence of 20 pg/ml of DAP or DAP and 1 pg/ml a-amanitin. Cells plated at 8--15 X 103/60 mm dish, were harvested from duplicate dishes at 24-h intervals and counted in a hemocytometer. Cell growth measurements of baP-101, ARS-7 and ARM-1 were also performed at different concentrations of a-amanitin. Cells were plated in media containing desired concentrations of a-amanitin and after 6 days the cells were counted from duplicate dishes. Cell-growth measurements on colchicine and cordycepin were performed in the same fashion. DNA-dependent RNA polymerase were prepared and assayed as has been described elsewhere [8]. Protein was precipitated from aliquots {50--200 pl) with 2 ml of 10% TCA. The protein was collected by centrifugation at 15 000 g for 10 min and dissolved in 1 ml of 0.1 N NaOH. Aliquots of the resulting solutions were used for protein estimation by the m e t h o d of Lowry et al. [12] utilizing bovine serum albumin V as a standard. Results

Selection of mutants. Spontaneous mutants were detected as microcolonies after incubation for 7 days in the selective medium in 10 out of 20 petri plates {total of 1 X 107 cells). One clone was picked from each dish (ARS-1--ARS-10) and grown in the selective medium. Assuming t h a t the colonies in each independent dish were derived from one mutational event, the occurrence of spontaneous mutation (pre-existing and induced) to ~-amanitin resistance is 1 X 1 0 - 6 . EMS-treated cells (2 X 106) were plated in 20 petri dishes in the selective medium. 11 of these developed resistant colonies in a week. EMS muta-

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Fig. 1. G r o w t h o f p a r e n t a l ( b a P 1 0 1 , o) and m u t a n t cells ( A R S - 7 , • a n d A R M - l , o) a t d i f f e r e n t c o n c e n t r a t i o n s o f ~ - a m a n i t i n . N u m b e r o f cells in m e d i u m w i t h o u t a m a n i t i n is p r e s e n t e d as 1 0 0 % g r o w t h .

genesis gave a b o u t 20% survival of the treated cells, and the number of mutants recovered increased 5-fold (5 X 10-6). 10 colonies of these (ARM-I--ARM-10) were picked from independent dishes and grown on selective medium. One of the mutants, ARM-l, has been used in further biochemical studies. Karyology. Giemsa-stained metaphase preparations of ARS-7, ARS-10 and ARM-1 were examined. 80% of the metaphases had 20 chromosomes and a b o u t 1% had 40 chromosomes. This is also true of the parental cell line (baP-101). Growth at different concentrations of a-amanitin. Fig. 1 illustrates the growth of baP-101 and mutants ARS-7 and ARM-1 in a-amanitin at concentrations up to 5 pg/ml in the medium. The cells were plated in 60-mm dishes containing the desired medium and incubated for 5 days. At the end of the incubation period, cells were harvested from duplicate dishes and counted. Although the mutants were selected at 1 pg/ml of a-amanitin and were recloned in the selective medium, they grew slowly with increasing concentrations of a-amanitin. At 1 pg/ml of a-amanitin none of the parental cells survived while ARM-1 had a b o u t 50% growth and ARS-7 a b o u t 18% growth as compared to their growth in the drug-free medium. Microscopic examination of the mutants revealed that they were healthy. ARM-1 was found to be relatively more resistant than ARS-7.

Pleiotropic effects Since the mutants required a longer trypsinization time, a b o u t twice that of the parantal cell, to remove them from the plastic surface, we suspected a possible change in membrane properties. Preliminary results indicate a slight difference in the response of the m u t a n t to colchicine and cordycepin. The m u t a n t appears to be slightly more sensitive to the effect of these drugs than the wild-type. Growth rate of cells. For these experiments 8--15 X l 0 s cells were inoculated into 60-mm dishes containing 4 ml of medium with or without 1 pg/ml of

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~-amanitin + DAP (20/~g/ml). At 24-h intervals cells from duplicate dishes were harvested and counted. Figs. 2, 3 and 4 present the growth pattern of l b , baP101 and ARM-1 cells on different media. The growth rate of ARM-1 and the parental cells are essentially the same during the log phase of growth. However in all of these experiments the mutant ARM-1 demonstrated a 24--48 h lag before growth began. This lag is also reflected in poor plaing efficiency of the mutant (0.6%), and may indicate a membrane defect. Thus the mutant cell has a little longer doubling time than the parental and the mutant tends to plateau

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Fig. 5. P a t t e r n s o f i n h i b i t i o n o f P o l y m e r a s e s a c t i v i t y in c e l l - f r e e e x t r a c t s by different concentrations of c~-amanitin in t h e r e a c t i o n m i x t u r e . R e a c t i o n s w e r e c a r r i e d o u t a t 2 5 ° C f o r 10 rain d u r i n g w h i c h t h e r e a c t i o n w a s l i n e a r w i t h t i m e a n d p r o t e i n c o n c e n t r a t i o n s . 1 0 0 % a c t i v i t y c o r r e s p o n d s t o 15 0 0 0 - - 2 0 0 0 0 c p m . b a P l O l , o; A R M - l , e ; A r e a - l , m.

99 sooner in DAP containing medium than in F-12. At the end of 5-days incubation period no parental cells were detected in drug~ontaining media. DNA-dependent RNA polymerase II. Cell-free extracts from baP-101, ARM-1 and Ama-1 were employed to measure RNA polymerase activity b y measuring UMP incorporation at different concentrations of a-amanitin. Ama-1 is already shown to have an altered polymerase II whose activity is resistant to low concentrations of a-amanitin. Fig. 5 presents the response of polymerases from different cell isolates to increasing concentrations of a-amanitin in the reaction mixture. Compared to baP-101, ARM-1 polymerases were found to be partially resistant to a-amanitin. While baP-101 had 14%, ARM-1 had 40% remaining polymerase activity in the presence of 10 ng/ml of a-amanitin. Ama-1 had a b o u t 100% activity in the presence of 100 ng/ml of a-amanitin. The inhibition was monophasic in contrast to biphasic curves obtained with rat-myoblast [13] and mousemyeloma [17] enzymes. To test whether the proportion of a-amanitin-resistant polymerases can be increased b y growing the cells in a-amanitin medium as reported b y Somers et al. [13], the cells were grown in a-amanitin for 10 generations and the enzyme preparations again checked in the same fashion as described above. No change was observed in the pattern of inhibition by a-amanitin. Neither was there any change in a-amanitin-inhibition pattern when cells were grown for several generations w i t h o u t a-amanitin in the medium. Discussion The mutants isolated exhibit the following characteristics; slightly altered sensitivity to colchicine and cordycepin; a lag before growth initiated not detectable in parental cells, and a partially a-amanitin-resistant polymerase II. The spontaneous frequency of mutation of 1 × 10 -6 does n o t seem to be especially high since a-amanitin resistance is known to be dominant [1,11]. It seems likely that the lowered plating efficiency and lag in growth is related to some change in the cell membrane. This assumption is also supported by our observations that the mutants require longer trypsinization time than the parental cells. It seems therefore that the mutants might be pleiotropic. Pleiotropic mutants have been reported b y several workers [2,10,14] especially when the mutants were membrane-related. Our preliminary investigations with colchicine and cordycepin effects on CHO suggest that the mutants are more sensitive to these chemicals than the parental cells. Thus the possibility remains that the cell membrane of the mutants might be altered. The a-amanitin-resistant CHO mutants obtained b y us are different from those reported by Chart et al. [4]. Those obtained by Chan et al. have totally resistant RNA polymerase II at lower concentrations of a-amanitin (up to 100 ng/ml), while our mutants have only partially resistant polymerase II. However, it seems the degree of resistance in our different cell lines may differ. Somers et al. [13] found that the level of a-amanitin-resistant polymerase could be increased b y growing the cells in a medium containing a-amanitin. However, such a situation was not simulated b y the mutants isolated in the present studies.

100 Recently Ingles et al. [9] isolated and characterized a number of CHO cell lines resistant to a-amanitin. The mutants varied greatly in their sensitivity to c~-amanitin, but each has a close correlation with the c~-amanitin sensitivity of its endogenous polymerase II. a-Amanitin inhibition curves of polymerase II have been employed to outline the genetic constitution of a resistant m u t a n t as regards the number of alleles coding for a-amanitin-sensitive subunit of polymerase II. A biphasic inhibition curve suggested the presence of more than one allele coding for the a-amanitin sensitive subunit of polymerase II [13,17]. Ingles et al. [9] obtained a monophasic inhibition curve suggesting the presence of only one allele (hemizygous) in the a-amanitin-resistant CHO cells. We also observed a monophasic inhibition curve similar to that obtained by Ingles et al. [9] confirming t h a t there is only a single allele in CHO cells responsible for coding the a-amanitin subunit of RNA polymerase II. However, 30% more resistance of m u t a n t polymerase II could be assumed to have resulted from a change in the structural gene leading to an alteration in a-amanitin binding as has been suggested by Ingles et al. [9]. Relatively low resistance of RNA polymerase II (in vitro) from the m u t a n t as compared to the resistance of the m u t a n t per se can perhaps be explained as being due to the low permeability of a-amanitin through the cell membrane (Wieland, personal communication). Thus the defective enzyme may result in slower cell growth. Wulf and Bautz [17] observed a longer generation time for a-amanitin-resistant mouse m y e l o m a cells in the presence of c~-amanitin. Our mutants appear to grow at the normal rate but with a lag of 24--48 h following plating. This would result in an apparent longer generation time if measurements were made after a few generations. Acknowledgment We are grateful to Prof. T. Wieland for his generous gift of c~-amanitin and Dr. L. Siminovitch for providing us cell line CHO-Ama-1. This work was supported by U.S.P.H.S. Grant GM18924. References 1 Amati, P., F. Blasi, U. di Porzio, A. Riccio, and C. Traboni, Hamster a-amanitin resistant R N A polymerase II able to transcribe p o l y o m a virus g e n o m e in somatic cell hybrids, Proc. Natl. Acad. Sci. (U.S.A.), 72 (1975) 753--757. 2 Bech-Hansen, N.T., J.E. Till, and V. Ling, Pleiotropic phenotype of colchicine resistant C H O cells: cross-resistance and collateralsensitivity,J. Cell Physiol., 88 (1976) 23--31. 3 C h a m b o n , P., Eukaryotic nuclear R N A polymerases, Ann. Rev. Biochem., 44 (1975) 613--638. 4 Chan, V.L., H.F. Whitmore, and L. Siminovitch, M a m m a l i a n cells with altered for of R N A polymerase If, Proc. Natl. Acad. Sci. (U.S.A.), 69 (1972) 3119--3123. 5 Clements, G.B., and J.H. Subak-Sharpe, Metabolic cooperation between biochemicaUy variant hamster cells and heterokaryons formed between the cells and chick erythrocytes, Exp. Cell Res., 95 (1975) 25--30. 6 D u d a , C.T., Plant R N A p o l y m e r a s e s , A n n . Rev. Plant Physiol., 27 ( 1 9 7 6 ) 1 1 9 - - 1 3 2 . 7 F e l d m a n , R., a n d M.W. T a y l o r , Pttrine m u t a n t s of m a m m a l i a n cell lines, I. A c c u m u l a t i o n o f f o r m y l g l y c i n a m i d e r i b o t i d e b y p u r i n e m u t a n t s of Chinese h a m s t e r o v a r y cells, B i o e h e m . G e n e t . , 12 ( 1 9 7 4 ) 393--405. 8 G u p t a , K.C., a n d M.W. T a y l o r , A c o n v e n i e n t a n d r a p i d p r o c e d u r e f o r e x t r a c t i o n of R N A p o l y m e r a s e II f r o m c u l t u r e d m a m m a l i a n ceils, A n a l . B i o e h e m . , 82 ( 1 9 7 7 ) 3 9 6 - - 4 0 3 .

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9 Ingles, C.J., A. Guialis, J. L a m , a n d L. S i m i n o v i t c h , a - A m a n i t i n r e s i s t a n c e of R N A p o l y m e r a s e II in m u t a n t Chinese h a m s t e r o v a r y eeB lines, J. Biol. C h e m . , 251 ( 1 9 7 6 ) 2 7 2 9 - - 2 7 3 4 . 1 0 Ling, V., Drug r e s i s t a n c e a n d m e m b r a n e a l t e r a t i o n in m u t a n t m a m m a l i a n cells, Can. J. G e n e t . Cytol., 17 ( 1 9 7 5 ) 5 0 3 - - 5 1 5 . 11 L o b b a n , P.E., a n d L. S i m i n o v i t e h , a - A m a n i t i n r e s i s t a n c e : a d o m i n a n t m u t a t i o n in CHO ceils, Cell, 4 (1975) 167--172. 12 L o w r y , O.H., N.J. R o s e n b u r g , A.L. Farr, a n d R.J. R a n d a l l , P r o t e i n m e a s u r e m e n t w i t h the foHn p h e n o l r e a g e n t , J. Biol. C h e m . , 1 9 3 ' ( 1 9 5 1 ) 2 6 5 - - 2 7 5 . 13 Somers, D.G., M.L. Pearson, a n d C.J. Ingles, I s o l a t i o n a n d c h a r a c t e r i z a t i o n of a n ~ - a m a n i t i n r e s i s t a n t rat m y o b l a s t m u t a n t cell Line possessing ~ - a m a n i t i n r e s i s t a n t R N A p o l y m e r a s e II, J. Biol. Chem., 2 5 0 (1975) 4825--4831. 14 S u b a k - S h a r p e , J.H., BiochemicaLiy m a r k e d v a r i a n t s of t h e S y r i a n H a m s t e r f i b r o b l a s t cell line BHK 21 a n d its derivative, E x p . Cell Res., 38 ( 1 9 6 5 ) 1 0 6 - - 1 1 9 . 1 5 Taylor, M.W., M. S o u h r a d a , a n d J. McCall, A n e w class of p u r i n e m u t a n t s of Chinese h a m s t e r o v a r y cells, Science, 172 ( 1 9 7 1 ) 1 6 2 - - 1 6 4 . 16 T a y l o r , M.W., J.H. P i p k o r n , M.K. T o k i t o , a n d R.O. P o z z a t t i Jr., C o n t r o l o f p u r i n e b i o s y n t h e s i s in 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 m u t a n t of CHO cells, S o m a t . Cell Genet., 3 ( 1 9 7 7 ) 1 9 5 - - 2 0 6 . 17 Wulf, E., a n d L. Bautz, R N A p o l y m e r a s e B f r o m a n ~ - a m a n i t i n r e s i s t a n t m o u s e m y e l o m a cell line, FEBS L e t t e r s , 69 ( 1 9 7 6 ) 6--10.