Hum. Genet. 50, 297--305 (1979) © by Springer-Verlag 1979
Cyeloheximide-Resistance in Chinese Hamster Ovary Cells and Human Fibroblast Cells Cytogenetic and Biochemical Characterization
H. P6che 1'2 *, K. H. Nierhaus 2, and Sabine Zakrzewski 3 11nstitut ffir Humangenetik, Universit~itsklinikum Essen, Hufelandstral~e 55, D-4300 Essen 1, Federal Republic of Germany 2Max-Planck-Institut ffir molekulare Genetik (Abt. Wittmann), Ihnestrage 63-73, D-1000 Berlin 33 (Dahlem) 3Institut ft~r Hum~ngenetik, Freie Universit~it Berlin, Heubnerweg 6, D-1000 Berlin 19
Summary. Cycloheximide(CHM)-resistant mutant Chinese hamster ovary (CHO) and human cells were induced with N-nitrosomethylurea (NMU) and ethyl methanesulfonate (EMS); the mutants were viable and showed unlimited growth in the presence of C H M (7× 10-7M), whereas this concentration inhibits protein synthesis in vivo as well as in vitro. N o numerical or structural chromosomal aberrations were found in the mutant cells. In vitro analysis shows that the ribosomes confer resistance against cycloheximide.
Introduction Mutagenesis in mammalian cells has generally been studied in so-called established cell lines of mouse, hamster, and human. Primary cultures would be more suitable, however, because they reflect the in vivo conditions more closely (Albertini and De Mars, 1973). Genetic markers used quite frequently are resistant to 6-mercaptopurine, 8-azaguanine, or 6-thioguanine. T h e y are resistant because the enzyme hypoxanthine guanine phosphoribosyltransferase ( H G P R T ) is inactive in the mutants. Thus they are analogous to the Lesch-Nyhan sYndrome, an X-linked genetic disorder. Recently, mutant mammalian cells have been described that are resistant to the protein synthesis inhibitors CHM, emitin, diphtheria toxin, and Pseudomonas exotoxin due to a translational error (P6che et al., 1975 b; Gupta and Siminovich, 1976; Moehring and Moehring, 1977). Such translation mutants could be used to * To whom offprint requests should be sent
0340-6717/79/0050/0297/$ 01.80
298
H. P6che et al.
s t u d y t h e m e c h a n i s m o f C H M a c t i o n . F u r t h e r m o r e , s u c h m u t a n t cells c o u l d a l l o w c h a r a c t e r i z a t i o n o f t h e m e c h a n i s m o f t r a n s l a t i o n in m a m m a l i a n cells, a n d possibly also the structure and function of their ribosomes. CHM inhibits protein s y n t h e s i s i n e u k a r y o t i c cells b y a f f e c t i n g t h e 80S r i b o s o m e s , b u t n o t t h e m i t o c h o n d r i a l p r o t e i n s y n t h e s i s o n 70S r i b o s o m e s (Vfizquez, 1979; P e s t k a , 1977). I n t h i s p a p e r we s h o w h o w r e s i s t a n c e t o C H M in C H O a n d h u m a n cells c a n b e i n d u c e d b y b o t h N M U a n d E M S , a n d we s h o w t h a t n u m e r i c a l o r s t r u c t u r a l a b e r r a t i o n s a r e a b s e n t in t h e m u t a n t cells.
Materials and Methods CeH Cultures We used Chinese hamster ovary cells, CHO-K1-D3, with a modal chromosome number of 22, and a human diploid fibroblast cell culture (46,XX,18p-). Cells were grown as monolayers in Dulbecco's modification of Eagle's minimum essential medium (Eagle MEM) containing 10% or 20% fetal bovine serum (FBS).
Cyto toxicity Assay We inoculated 60-mm petri dishes with 100 cells/dish. After incubation for 24 h the CHO cells were treated with 50, 75,100, and 150 ~tg N M U / m l for 2.5 h. In a parallel cytotoxity assay we used 100,200,300,400, and 500lag E M S / m l for 18h. The mutagen was then removed by washing with serum-free Dulbecco's medium and Dulbecco's medium with 15% FBS was added. After incubation for 10 days in a 5% CO2-air mixture at 37°C without change of medium, the colonies were fixed and stained. The cytotoxicity was expressed as the number of colonies in the treated dishes, as percentage of the number of colonies in the untreated dishes.
Mutagenesis Assay The scheme for induction and selection of CHM-resistant clones has been described elsewhere (P6che et al., 1975 a). This assay was modified as follows: 105 cells/plate were inoculated. After incubation for 24h the cells were treated with 100 or 125lag N M U / m l for 2.5 h. In parallel experiments the cells were treated with 300 or 400 lag E M S / m l for i8 h. After an expression time of 72 h, the cells were inoculated in a selective medium for C H M (7 x 10-7M). The inoculum added to this selective medium was 105, 2.5 x 105, and 5 x 105 cells/plate. Cultures were evaluated 18 days after the selective medium had been added. The control cultures were handled identically, except for the mutagenic treatment.
Cytogenetic Studies Four hours before harvesting, Colcemid (0.21.tg/ml) was added to the culture. The cells were treated with hypotonic KC1 (75mM) and then fixed in ethanol-acetic acid (3: 1), and chromosomal preparations were obtained as usual. For G banding the technique of Wang and Fedoroff (1972) was modified as follows: Three-day-old slides were treated with trypsin solution (1 part 0.25% trypsin in Hanks' solution and 300 parts methanol phosphate buffer solution (1 : 4, p H 6.8)~ washed in distilled water, stained with Giemsa (1 part Giemsa stock solution and 25 parts phosphate buffer, pH 6.8), washed in distilled water, and air-dried.
Preparation of 80S Ribosomes In general, the method described by Falvey and Staehelin (1970) was followed. All operations were carried out at 0--4°C. Cells were harvested by centrifugation at 3,000 x g for 15 min and washed
Cycloheximide-Resistance in Ovary Cells
299
with PBS solution. The washed cells were resuspended in 3 volumes of a buffer A (20 mM TrisHC1, pH 7.5, 130 m M KC1, 7.5 mM MgC12, 5 mM fl-mercaptoethanol) and homogenized in a Potter homogenizer with 20 strokes. The homogenate was centrifuged for 10 min at 12,000 xg. The ribosomes were then pelleted from the upper two-thirds of the resulting postmitochondrial supernatant t h r o u g h 3 ml 30% sucrose in buffer A per tube (75,000 x g for 5 h). The ribosome pellets were resuspended in buffer A.
Preparation of High-speed Supernatant (S 100) and pH 5 Enzyme Fraction The S 100 and pH 5 enzyme fraction was prepared from the postmicrosomal supernatant of rat liver according to the method described by Falvey and Staehelin (1970). Small portions of the S 100 and pH 5 enzymes were stored at - 8 0 ° C .
Poly(U)-directed Polyphenylalanine Synthesis A modification of the assay described by Nierhaus and Dohme (1979) was used. Reactions were carried out in 125-~tI volumes and these were incubated at 30°C for 45min. The final concentrations were 1 i0 ~tM [14C]Phe (about 150,000 cpm per assay), 20 mMTris-HCl, pH 7.6, 15raM NH4C1, 150mM KC1, 12raM MgC12, 1 mM ATP, 0.3raM GTP, 5raM phosphoenolpyruvate, and 6raM fl-mercaptoethanol. Each assay contained 40ktl S 100, 20~tl pH 5 enzyme fraction, 3 ~tg pyruvate kinase, 40 ttg poly(U), and 20 p.g tRNA from calf liver. 80S ribosomes were present at a concentration of 1 A260 unit per sample. After incubation the reaction was stopped by the addition of bovine serum albumin and 2ml 5% trichloroacetic acid. Samples were then incubated for 15rain at 90°C and filtered through glassfiber filters, which were washed twice, with cold 5% trichloroacetic acid and with ether : ethanol (1 : 1), and then dried and counted.
Results
Lethal Effects of NMU and EMS The cytotoxicity of the mutagens was established before mutations could be induced. Figure 1 shows the percentage of survival as a function of the mutagen concentration of NMU and EMS in CHO cells. A mean survival rate of 50% was found at an NMU concentration of 100~tg/ml, and of 35% at an NMU concentration of 1251ag/ml. Compared with NMU, the mutagenic EMS showed
200
B
A
100,
g "6
5C
Z
Fig. 1A and B. Inactivation of CHO cells by N M U (A) and EMS (B) 5O
100
150
100 pg/ml
208
300
400
500
300
H. P6che et al.
Table 1. The relationship between inoculum size and the frequency of mutants, from CHM sensitivity to resistance (7× 10-7m) in Chinese hamster cells and h u m a n cells Compounds (gg/ml)
Cells per dish
100
NMU
125
Chinese hamster
Human
Controls Treated
Controls Treated
105
22
104
10
48
2.5 105
17
40
30
71
5
105
17
32
40
70
105 2.5 10s
26 20
120 83
11 34
64 73
5
105
15
60
36
80
105
18
95
13
47
2.5 105
18
68
28
60 67
300
EMS
Mean number of mutants induced
5
105
18
50
33
105
21
113
13
72
2.5 105 5 105
16 17
93 82
33 35
73 80
400
A 110
110
90
90
70
70
.__
50
E 3O
30
I0 18
r-F,,
2O 22 24 26
, , ,q~,
, ~-
18 20 22 24 26 Chromosomes/cell
Chromosomes/ceil
C
D
50
._m 50
30 10
Z F
42 44 46 48
Chromosomes/celt
I!
....
/.2 44 46 48 Chromosomes/cell
Fig. 2A--D. Frequency distribution of mitotic chromosomes per cell of CHO-K1-D3 (A), CHO-K1-D3-CHMrl (B), human (46,XX,18p-) (C), and h u m a n (46,XX,18p-) CHMrl (D)
Cycloheximide-Resistance in Ovary Cells
301
Fig.3A and B, G-banding patterns of mitotic chromosomes from CHO-K1-D3 (A) and CHOK1-CHMr 1 (B)
Fig. 4A and B. G-banding patterns of mitotic chromosomes from human (46,XX,18p-) (A) and human (46,XX,18p-) CHM r 1 (B)
302
H. P6che et al.
Table 2. Poly(Phe) incorporation by 80S ribosomes from different sources in the presence of cycloheximide (CHM) or chloramphenicol (CAM), respectively 80S Ribosomes from
CHM
CHO-K l-D3
-
+
+
-
-
+
-
-
+
-
+
-
-
+
[14C]Phe polymerized per ribosome
%a
16 5 16 17 11 17 18 18 18
100 31 100 100 65 100 100 100 100 100 11
-
-
+
-
17 2
-
+
16
-
-
+
-
94
16 17 16
100 106 100 100 100
-
+
-
-
+
-
17 17
-
+
16
-
-
+
-
12 12
-
+
5
Human (46,XX,18p-) CHMr~
Controls 70S ribosomes from E. coli b
-
-
Human (46,XX,18p-)
Human (46,XX, 18p-) CHMrl
CAM (10-3M)
-
CHO-K1-D3-CHMrl
CHO-K 1-D3-CHMr2
(10-3M)
94
100 100 42
a The background (minus ribosomes) was subtracted before the percentage values were calculated. All samples contained 2% ethanol due to the limited solubility of chloramphenicol and all tests were performed in duplicate b When 70S ribosomes were tested, the assay contained tRNA and S 100 from E. coli instead those from calf liver
s u r v i v a l r a t e s o f 5 6 % a n d 4 0 % at c o n c e n t r a t i o n s o f 3 0 0 l a g / m l a n d 4 0 0 l a g / m l , r e s p e c t i v e l y . T h e m u t a g e n i c e x p e r i m e n t s w e r e p e r f o r m e d at this c o n c e n t r a t i o n .
Induction o f Mutants with N M U and E M S T a b l e 1 s h o w s t h e results o f t r e a t m e n t w i t h 100 a n d 125 lag N M U / m l a n d 300 a n d 400lag E M S / m l . T h e d i f f e r e n c e in t h e f r e q u e n c y o f r e s i s t a n t m u t a n t s in t h e m u t a g e n - t r e a t e d series as a g a i n s t t h a t o f s p o n t a n e o u s l y o c c u r r i n g m u t a n t s ( c o n t r o l ) was significant. T h e n u m b e r o f r e s i s t a n t c o l o n i e s f r o m C H O cells d e c r e a s e d b y a f a c t o r o f 0.3 w h e n t h e i n o c u l u m sizes w e r e in the r a n g e o f 105 to
Cycloheximide-Resistance in Ovary Cells
303
5 x l0 s cells at a mutagen concentration of 100 gg N M U / m l and by a factor of 0.2 at a concentration of 125gg/ml. In contrast, human cells under identical conditions increased 0.5-fold and 0.3-fold. The same results were obtained with 300 and 400pg EMS/ml.
Cytogenetic Characterization To rule out numerical or structural chromosomal aberrations as possible causes of C H M resistance, we determined the karyotype of the cell clones. The number of chromosomes was analyzed in 150CHO-K1-D3 and C H O - K 1 - D 3 - C H M 1 cells and 50 human (46,XX,18p-) and 50 human (46,XX,18p-)CHM 1 cells. No difference was observed between the number of chromosomes in the CHO-K1-D3 cells and that in their cycloheximide-resistant mutants. Both had a modal number of 22 chromosomes, with a range of 19 to 24 chromosomes per metaphase, and the human cells had 46 (Fig.2). The G-banding patterns were the same in the original Chinese hamster ovary cells and the human cells as in their respective mutants (Figs. 3 and 4).
Preliminary Biochemical Characterization Ribosomes of the C H O and human wild types and of four cycloheximideresistant clones (two of each C H O and human cells) were tested in a poly(U)directed polyphenylalanine synthesis system in the presence and the absence of cycloheximide and, as a control, chloramphenicol. Cycloheximide inhibited the wild-type cells significantly (residual activity 30% and 10%, respectively; see Table 2)i In contrast, the poly(Phe)-synthesizing activity of the mutant cell lines was not affected or only scarcely reduced. No 80S ribosomes were inhibited by chloramphenicol, whereas a strong inhibition was seen with this drug in the presence of 70S ribosomes from E. coll. This last finding indicates that the 80S preparations were not contaminated by mitochondrial ribosomes.
Discussion Cells that are resistant t o inhibitors of translation are an important aid in translation studies. Most of the inhibitors described so far are either specific for prokaryotes or affect both pro- and eukaryotes. The effects of C H M have been investigated both in intact cells and in cell-free systems of fungi (Bleyman and Bruns, 1977; McLaughlin, 1974; Pohjanpelto, 1976; Siegel and Sisler, 1965), H e L a cells (Warner, 1974; Willems et al., !969), L cells (Ennis, 1966), and rabbit reticulocytes (McKeehan and Hardesty, 1969). The data presented here allow a comparison of the mutagenic activity of N M U and EMS in hamster and human cells. The number of mutations induced depended on the inoculum size in both cell lines in a different way: while in C H O cells the number of induced mutants decreased, it increased significantly in
304
H. P6che et al.
h u m a n cells (Table 1). S i m i l a r results were f o u n d in C H O cells with 8 - a z a g u a n i n e resistance ( C h u a n d Malling, 1968). It is not clear h o w far these results are an effect o f c o n c e n t r a t i o n , as k n o w n f r o m s p o n t a n e o u s m u t a t i o n s in vitro ( D e M a r s , 1974; H a r r i s , 1971). In a n y event, the m u t a t i o n s we i n d u c e d are n o t related to detectable chromosomal aberrations. T h e resistance in the m u t a n t cells m a y be caused by i m p a i r e d t r a n s p o r t o f C H M into the cell, b y i n a c t i v a t i o n o f C H M , or by a l t e r n a t i o n s in r i b o s o m a l p r o t e i n s as o b s e r v e d in several fungal systems ( C o o p e r et al., 1976; H a u g l i a n d D o v e , 1972; P o n g r a t z a n d Klingmtfller, 1973; S u t t o n et al., 1978). S i m i l a r results have been o b t a i n e d in C H O cells. In a d d i t i o n to C H M (P6che et al., 1975), o t h e r i n h i b i t o r s o f p r o t e i n synthesis have been used, n a m e l y emitin ( G u p t a a n d Siminovitch, 1976), D i p h t h e r i a toxin, a n d Pseudomonas exotoxin A ( M o e h r i n g a n d M o e h r i n g , 1977). It is o f interest t h a t r i b o s o m a l m u t a n t s resistant to C H M c o u l d be i n d u c e d chemically in p r i m a r y h u m a n cell cultures u n d e r the same e x p e r i m e n t a l conditions. It seems likely that C H M resistance is due to the same m o l e c u l a r m e c h a n i s m in b o t h C H O a n d h u m a n cell lines. O n the basis o f the p r e s e n t d a t a o n the resistance to C H M in m a m m a l i a n cells, these m u t a n t s c o u l d be used f o r s t u d y i n g the structure a n d function o f m a m m a l i a n ribosomes.
Acknowledgements. We thank Drs. H. G. Wittmann, R. Brimacombe, E. Passarge, and K. Sperling for discussions and comments.
References Albertini, R. J., DeMars, R.: Somatic cell mutation, detection and quantification of X-rayinduced mutation in cultured, diploid human fibroblasts. Mutat. Res. 18, 199--224 (1973) Bleyman, L. K., Bruns, P. J.: Genetic of cycloheximide resistance in Tetrahymena. Genetics 87, 275--284 (1977) Chu, E. H. Y., Malling, H. V.: Mammalian cell genetics. II. Chemical induction of specific locus mutations in Chinese hamster cells in vitro. Genetics 61, 1303--1312 (1968) Cooper, D., Banthorpe, D. V., Wilkie, D.: Modified ribosomes conferring resistance to cycloheximide in mutants of Saccharomyces cerevisiae. J. Mol. Biol. 26,347--350 (1967) DeMars, R.: Resistance of cultured human fibroblasts and other cells to purine and pyrimidine analogues in relation to mutagenesis detection. Mutat. Res. 24, 335--364 (1974) Ennis, H. L.: Synthesis of ribonucleic acid in L cells during inhibition of protein synthesis by cycloheximide. Mol. Pharmacol. 2, 543--557 (1966) Falvey, A. K., Staehelin, T.: Structure and function of mammalian ribosomes. I. Isolation and characterization of active liver ribosomal subunits. J. Mol. Biol. 53, 1--30 (1970) Gupta, R. S., Siminovitch, L.: The isolation and preliminary characterization of somatic cell mutants resistant to the protein synthesis inhibitor emitine. Cell 9, 213--219 (1976) Harris, M.: Mutation rates in cells at different ploidy levels. J. Cell Physiol. 78, 177--184 (1971) Haugli, F. B., Dove, W. F.: Genetics and biochemistry of cycloheximide resistance in Physarum polycephalum. Mol. Gen. Genet. 118, 97--107 (1972) McKeehan, W., Hardesty, B.: The mechanism of cycloheximide inhibition of protein synthesis in rabbit reticulocytes. Biochem. Biophys. Res. Commun. 36,625--630 (1969) McLaughlin, C. S.: Yeast ribosomes: Genetics. In: Ribosomes, Nomura, M., Tissi~res, A., Lengyel, P. (eds.), pp. 815--827. New York: Cold Spring Harbor Laboratory 1974 Moehring, T. J., Moehring, J. M.: Selection and characterization of cell resistant to Diphtheria toxine and Pseudomonas exotoxine A: Presumptive translational mutants. Cell 11,447--454 (1977)
Cycloheximide-Resistance in Ovary Cells
305
Nierhaus, K. H., Dohme, F.: Total reconstistution of 50S subunits from Escherichia coli ribosomes. Methods Enzymol. 59, 443--449 (1979) Pestka, S.: Inhibitors of protein synthesis. In: Molecular mechanisms of protein biosynthesis, Weissbach, H., Pestka, S. (eds.), pp. 467--553. New York: Academic Press 1977 P6che, H., Varshaver, N. B., Theile, M., Geissler, E.: Cycloheximide resistance in Chinese hamster cells. II. Induction of CHM resistance in Chinese hamster cells by N-nitrosomethylurea. Mutat. Res. 30, 83--88 (1975a) P6che, H., Junghahn, I., Geissler, E., Bielka, H.: Cycloheximide resistance in Chinese hamster cells. III. Characterization of cell-free protein synthesis by polysomes. Mol. Gen. Genet. 138, 173--177 (1975b) Pohjanpelto, P.: Cycloheximide elicits in human fibroblasts a response characteristic for initiation of cell proliferation. Exp. Cell Res. 102, 138--142 (1976) Pongratz, M., Klingmtiller, W.: Role of ribosomes in cycloheximide resistance of Neurospora mutants. Mol. Gen. Genet. 124, 359--363 (1973) Sutton, C. A., Arens, H., Hallberg, R. L.: Cycloheximide resistance can be mediated through either ribosomal subunits. Proc. Natl. Acad. Sci. USA 75, 3158--3162 (1978) V~zquez, D.: Inhibitors of protein synthesis. (Molecular biology, biochemistry and biophysics, Vol. 30.) Berlin-Heidelberg-New York: Springer 1979 Wang, H. C., Fedoroff, S.: Banding in human chromosomes treated with trypsine. Nature New Biol. 235, 52--54 (1972) Warner, J. R.: The assembly of ribosomes in eukaryotes: In: Ribosomes, Nomura, M., Tissi~res, A., Lengyel, P. (eds.), pp. 461--488. New York: Cold Spring Harbor Laboratory 1974 Willems, M., Penman, M., Penman, S.: The regulation of RNA synthesis and processing in the nucleolus during inhibition of protein synthesis. J. Cell Biol. 41, 177--187 (1969)
Received March 26, 1979
Cyeloheximide-Resistance in Chinese Hamster Ovary Cells and Human Fibroblast Cells Cytogenetic and Biochemical Characterization
H. P6che 1'2 *, K. H. Nierhaus 2, and Sabine Zakrzewski 3 11nstitut ffir Humangenetik, Universit~itsklinikum Essen, Hufelandstral~e 55, D-4300 Essen 1, Federal Republic of Germany 2Max-Planck-Institut ffir molekulare Genetik (Abt. Wittmann), Ihnestrage 63-73, D-1000 Berlin 33 (Dahlem) 3Institut ft~r Hum~ngenetik, Freie Universit~it Berlin, Heubnerweg 6, D-1000 Berlin 19
Summary. Cycloheximide(CHM)-resistant mutant Chinese hamster ovary (CHO) and human cells were induced with N-nitrosomethylurea (NMU) and ethyl methanesulfonate (EMS); the mutants were viable and showed unlimited growth in the presence of C H M (7× 10-7M), whereas this concentration inhibits protein synthesis in vivo as well as in vitro. N o numerical or structural chromosomal aberrations were found in the mutant cells. In vitro analysis shows that the ribosomes confer resistance against cycloheximide.
Introduction Mutagenesis in mammalian cells has generally been studied in so-called established cell lines of mouse, hamster, and human. Primary cultures would be more suitable, however, because they reflect the in vivo conditions more closely (Albertini and De Mars, 1973). Genetic markers used quite frequently are resistant to 6-mercaptopurine, 8-azaguanine, or 6-thioguanine. T h e y are resistant because the enzyme hypoxanthine guanine phosphoribosyltransferase ( H G P R T ) is inactive in the mutants. Thus they are analogous to the Lesch-Nyhan sYndrome, an X-linked genetic disorder. Recently, mutant mammalian cells have been described that are resistant to the protein synthesis inhibitors CHM, emitin, diphtheria toxin, and Pseudomonas exotoxin due to a translational error (P6che et al., 1975 b; Gupta and Siminovich, 1976; Moehring and Moehring, 1977). Such translation mutants could be used to * To whom offprint requests should be sent
0340-6717/79/0050/0297/$ 01.80
298
H. P6che et al.
s t u d y t h e m e c h a n i s m o f C H M a c t i o n . F u r t h e r m o r e , s u c h m u t a n t cells c o u l d a l l o w c h a r a c t e r i z a t i o n o f t h e m e c h a n i s m o f t r a n s l a t i o n in m a m m a l i a n cells, a n d possibly also the structure and function of their ribosomes. CHM inhibits protein s y n t h e s i s i n e u k a r y o t i c cells b y a f f e c t i n g t h e 80S r i b o s o m e s , b u t n o t t h e m i t o c h o n d r i a l p r o t e i n s y n t h e s i s o n 70S r i b o s o m e s (Vfizquez, 1979; P e s t k a , 1977). I n t h i s p a p e r we s h o w h o w r e s i s t a n c e t o C H M in C H O a n d h u m a n cells c a n b e i n d u c e d b y b o t h N M U a n d E M S , a n d we s h o w t h a t n u m e r i c a l o r s t r u c t u r a l a b e r r a t i o n s a r e a b s e n t in t h e m u t a n t cells.
Materials and Methods CeH Cultures We used Chinese hamster ovary cells, CHO-K1-D3, with a modal chromosome number of 22, and a human diploid fibroblast cell culture (46,XX,18p-). Cells were grown as monolayers in Dulbecco's modification of Eagle's minimum essential medium (Eagle MEM) containing 10% or 20% fetal bovine serum (FBS).
Cyto toxicity Assay We inoculated 60-mm petri dishes with 100 cells/dish. After incubation for 24 h the CHO cells were treated with 50, 75,100, and 150 ~tg N M U / m l for 2.5 h. In a parallel cytotoxity assay we used 100,200,300,400, and 500lag E M S / m l for 18h. The mutagen was then removed by washing with serum-free Dulbecco's medium and Dulbecco's medium with 15% FBS was added. After incubation for 10 days in a 5% CO2-air mixture at 37°C without change of medium, the colonies were fixed and stained. The cytotoxicity was expressed as the number of colonies in the treated dishes, as percentage of the number of colonies in the untreated dishes.
Mutagenesis Assay The scheme for induction and selection of CHM-resistant clones has been described elsewhere (P6che et al., 1975 a). This assay was modified as follows: 105 cells/plate were inoculated. After incubation for 24h the cells were treated with 100 or 125lag N M U / m l for 2.5 h. In parallel experiments the cells were treated with 300 or 400 lag E M S / m l for i8 h. After an expression time of 72 h, the cells were inoculated in a selective medium for C H M (7 x 10-7M). The inoculum added to this selective medium was 105, 2.5 x 105, and 5 x 105 cells/plate. Cultures were evaluated 18 days after the selective medium had been added. The control cultures were handled identically, except for the mutagenic treatment.
Cytogenetic Studies Four hours before harvesting, Colcemid (0.21.tg/ml) was added to the culture. The cells were treated with hypotonic KC1 (75mM) and then fixed in ethanol-acetic acid (3: 1), and chromosomal preparations were obtained as usual. For G banding the technique of Wang and Fedoroff (1972) was modified as follows: Three-day-old slides were treated with trypsin solution (1 part 0.25% trypsin in Hanks' solution and 300 parts methanol phosphate buffer solution (1 : 4, p H 6.8)~ washed in distilled water, stained with Giemsa (1 part Giemsa stock solution and 25 parts phosphate buffer, pH 6.8), washed in distilled water, and air-dried.
Preparation of 80S Ribosomes In general, the method described by Falvey and Staehelin (1970) was followed. All operations were carried out at 0--4°C. Cells were harvested by centrifugation at 3,000 x g for 15 min and washed
Cycloheximide-Resistance in Ovary Cells
299
with PBS solution. The washed cells were resuspended in 3 volumes of a buffer A (20 mM TrisHC1, pH 7.5, 130 m M KC1, 7.5 mM MgC12, 5 mM fl-mercaptoethanol) and homogenized in a Potter homogenizer with 20 strokes. The homogenate was centrifuged for 10 min at 12,000 xg. The ribosomes were then pelleted from the upper two-thirds of the resulting postmitochondrial supernatant t h r o u g h 3 ml 30% sucrose in buffer A per tube (75,000 x g for 5 h). The ribosome pellets were resuspended in buffer A.
Preparation of High-speed Supernatant (S 100) and pH 5 Enzyme Fraction The S 100 and pH 5 enzyme fraction was prepared from the postmicrosomal supernatant of rat liver according to the method described by Falvey and Staehelin (1970). Small portions of the S 100 and pH 5 enzymes were stored at - 8 0 ° C .
Poly(U)-directed Polyphenylalanine Synthesis A modification of the assay described by Nierhaus and Dohme (1979) was used. Reactions were carried out in 125-~tI volumes and these were incubated at 30°C for 45min. The final concentrations were 1 i0 ~tM [14C]Phe (about 150,000 cpm per assay), 20 mMTris-HCl, pH 7.6, 15raM NH4C1, 150mM KC1, 12raM MgC12, 1 mM ATP, 0.3raM GTP, 5raM phosphoenolpyruvate, and 6raM fl-mercaptoethanol. Each assay contained 40ktl S 100, 20~tl pH 5 enzyme fraction, 3 ~tg pyruvate kinase, 40 ttg poly(U), and 20 p.g tRNA from calf liver. 80S ribosomes were present at a concentration of 1 A260 unit per sample. After incubation the reaction was stopped by the addition of bovine serum albumin and 2ml 5% trichloroacetic acid. Samples were then incubated for 15rain at 90°C and filtered through glassfiber filters, which were washed twice, with cold 5% trichloroacetic acid and with ether : ethanol (1 : 1), and then dried and counted.
Results
Lethal Effects of NMU and EMS The cytotoxicity of the mutagens was established before mutations could be induced. Figure 1 shows the percentage of survival as a function of the mutagen concentration of NMU and EMS in CHO cells. A mean survival rate of 50% was found at an NMU concentration of 100~tg/ml, and of 35% at an NMU concentration of 1251ag/ml. Compared with NMU, the mutagenic EMS showed
200
B
A
100,
g "6
5C
Z
Fig. 1A and B. Inactivation of CHO cells by N M U (A) and EMS (B) 5O
100
150
100 pg/ml
208
300
400
500
300
H. P6che et al.
Table 1. The relationship between inoculum size and the frequency of mutants, from CHM sensitivity to resistance (7× 10-7m) in Chinese hamster cells and h u m a n cells Compounds (gg/ml)
Cells per dish
100
NMU
125
Chinese hamster
Human
Controls Treated
Controls Treated
105
22
104
10
48
2.5 105
17
40
30
71
5
105
17
32
40
70
105 2.5 10s
26 20
120 83
11 34
64 73
5
105
15
60
36
80
105
18
95
13
47
2.5 105
18
68
28
60 67
300
EMS
Mean number of mutants induced
5
105
18
50
33
105
21
113
13
72
2.5 105 5 105
16 17
93 82
33 35
73 80
400
A 110
110
90
90
70
70
.__
50
E 3O
30
I0 18
r-F,,
2O 22 24 26
, , ,q~,
, ~-
18 20 22 24 26 Chromosomes/cell
Chromosomes/ceil
C
D
50
._m 50
30 10
Z F
42 44 46 48
Chromosomes/celt
I!
....
/.2 44 46 48 Chromosomes/cell
Fig. 2A--D. Frequency distribution of mitotic chromosomes per cell of CHO-K1-D3 (A), CHO-K1-D3-CHMrl (B), human (46,XX,18p-) (C), and h u m a n (46,XX,18p-) CHMrl (D)
Cycloheximide-Resistance in Ovary Cells
301
Fig.3A and B, G-banding patterns of mitotic chromosomes from CHO-K1-D3 (A) and CHOK1-CHMr 1 (B)
Fig. 4A and B. G-banding patterns of mitotic chromosomes from human (46,XX,18p-) (A) and human (46,XX,18p-) CHM r 1 (B)
302
H. P6che et al.
Table 2. Poly(Phe) incorporation by 80S ribosomes from different sources in the presence of cycloheximide (CHM) or chloramphenicol (CAM), respectively 80S Ribosomes from
CHM
CHO-K l-D3
-
+
+
-
-
+
-
-
+
-
+
-
-
+
[14C]Phe polymerized per ribosome
%a
16 5 16 17 11 17 18 18 18
100 31 100 100 65 100 100 100 100 100 11
-
-
+
-
17 2
-
+
16
-
-
+
-
94
16 17 16
100 106 100 100 100
-
+
-
-
+
-
17 17
-
+
16
-
-
+
-
12 12
-
+
5
Human (46,XX,18p-) CHMr~
Controls 70S ribosomes from E. coli b
-
-
Human (46,XX,18p-)
Human (46,XX, 18p-) CHMrl
CAM (10-3M)
-
CHO-K1-D3-CHMrl
CHO-K 1-D3-CHMr2
(10-3M)
94
100 100 42
a The background (minus ribosomes) was subtracted before the percentage values were calculated. All samples contained 2% ethanol due to the limited solubility of chloramphenicol and all tests were performed in duplicate b When 70S ribosomes were tested, the assay contained tRNA and S 100 from E. coli instead those from calf liver
s u r v i v a l r a t e s o f 5 6 % a n d 4 0 % at c o n c e n t r a t i o n s o f 3 0 0 l a g / m l a n d 4 0 0 l a g / m l , r e s p e c t i v e l y . T h e m u t a g e n i c e x p e r i m e n t s w e r e p e r f o r m e d at this c o n c e n t r a t i o n .
Induction o f Mutants with N M U and E M S T a b l e 1 s h o w s t h e results o f t r e a t m e n t w i t h 100 a n d 125 lag N M U / m l a n d 300 a n d 400lag E M S / m l . T h e d i f f e r e n c e in t h e f r e q u e n c y o f r e s i s t a n t m u t a n t s in t h e m u t a g e n - t r e a t e d series as a g a i n s t t h a t o f s p o n t a n e o u s l y o c c u r r i n g m u t a n t s ( c o n t r o l ) was significant. T h e n u m b e r o f r e s i s t a n t c o l o n i e s f r o m C H O cells d e c r e a s e d b y a f a c t o r o f 0.3 w h e n t h e i n o c u l u m sizes w e r e in the r a n g e o f 105 to
Cycloheximide-Resistance in Ovary Cells
303
5 x l0 s cells at a mutagen concentration of 100 gg N M U / m l and by a factor of 0.2 at a concentration of 125gg/ml. In contrast, human cells under identical conditions increased 0.5-fold and 0.3-fold. The same results were obtained with 300 and 400pg EMS/ml.
Cytogenetic Characterization To rule out numerical or structural chromosomal aberrations as possible causes of C H M resistance, we determined the karyotype of the cell clones. The number of chromosomes was analyzed in 150CHO-K1-D3 and C H O - K 1 - D 3 - C H M 1 cells and 50 human (46,XX,18p-) and 50 human (46,XX,18p-)CHM 1 cells. No difference was observed between the number of chromosomes in the CHO-K1-D3 cells and that in their cycloheximide-resistant mutants. Both had a modal number of 22 chromosomes, with a range of 19 to 24 chromosomes per metaphase, and the human cells had 46 (Fig.2). The G-banding patterns were the same in the original Chinese hamster ovary cells and the human cells as in their respective mutants (Figs. 3 and 4).
Preliminary Biochemical Characterization Ribosomes of the C H O and human wild types and of four cycloheximideresistant clones (two of each C H O and human cells) were tested in a poly(U)directed polyphenylalanine synthesis system in the presence and the absence of cycloheximide and, as a control, chloramphenicol. Cycloheximide inhibited the wild-type cells significantly (residual activity 30% and 10%, respectively; see Table 2)i In contrast, the poly(Phe)-synthesizing activity of the mutant cell lines was not affected or only scarcely reduced. No 80S ribosomes were inhibited by chloramphenicol, whereas a strong inhibition was seen with this drug in the presence of 70S ribosomes from E. coll. This last finding indicates that the 80S preparations were not contaminated by mitochondrial ribosomes.
Discussion Cells that are resistant t o inhibitors of translation are an important aid in translation studies. Most of the inhibitors described so far are either specific for prokaryotes or affect both pro- and eukaryotes. The effects of C H M have been investigated both in intact cells and in cell-free systems of fungi (Bleyman and Bruns, 1977; McLaughlin, 1974; Pohjanpelto, 1976; Siegel and Sisler, 1965), H e L a cells (Warner, 1974; Willems et al., !969), L cells (Ennis, 1966), and rabbit reticulocytes (McKeehan and Hardesty, 1969). The data presented here allow a comparison of the mutagenic activity of N M U and EMS in hamster and human cells. The number of mutations induced depended on the inoculum size in both cell lines in a different way: while in C H O cells the number of induced mutants decreased, it increased significantly in
304
H. P6che et al.
h u m a n cells (Table 1). S i m i l a r results were f o u n d in C H O cells with 8 - a z a g u a n i n e resistance ( C h u a n d Malling, 1968). It is not clear h o w far these results are an effect o f c o n c e n t r a t i o n , as k n o w n f r o m s p o n t a n e o u s m u t a t i o n s in vitro ( D e M a r s , 1974; H a r r i s , 1971). In a n y event, the m u t a t i o n s we i n d u c e d are n o t related to detectable chromosomal aberrations. T h e resistance in the m u t a n t cells m a y be caused by i m p a i r e d t r a n s p o r t o f C H M into the cell, b y i n a c t i v a t i o n o f C H M , or by a l t e r n a t i o n s in r i b o s o m a l p r o t e i n s as o b s e r v e d in several fungal systems ( C o o p e r et al., 1976; H a u g l i a n d D o v e , 1972; P o n g r a t z a n d Klingmtfller, 1973; S u t t o n et al., 1978). S i m i l a r results have been o b t a i n e d in C H O cells. In a d d i t i o n to C H M (P6che et al., 1975), o t h e r i n h i b i t o r s o f p r o t e i n synthesis have been used, n a m e l y emitin ( G u p t a a n d Siminovitch, 1976), D i p h t h e r i a toxin, a n d Pseudomonas exotoxin A ( M o e h r i n g a n d M o e h r i n g , 1977). It is o f interest t h a t r i b o s o m a l m u t a n t s resistant to C H M c o u l d be i n d u c e d chemically in p r i m a r y h u m a n cell cultures u n d e r the same e x p e r i m e n t a l conditions. It seems likely that C H M resistance is due to the same m o l e c u l a r m e c h a n i s m in b o t h C H O a n d h u m a n cell lines. O n the basis o f the p r e s e n t d a t a o n the resistance to C H M in m a m m a l i a n cells, these m u t a n t s c o u l d be used f o r s t u d y i n g the structure a n d function o f m a m m a l i a n ribosomes.
Acknowledgements. We thank Drs. H. G. Wittmann, R. Brimacombe, E. Passarge, and K. Sperling for discussions and comments.
References Albertini, R. J., DeMars, R.: Somatic cell mutation, detection and quantification of X-rayinduced mutation in cultured, diploid human fibroblasts. Mutat. Res. 18, 199--224 (1973) Bleyman, L. K., Bruns, P. J.: Genetic of cycloheximide resistance in Tetrahymena. Genetics 87, 275--284 (1977) Chu, E. H. Y., Malling, H. V.: Mammalian cell genetics. II. Chemical induction of specific locus mutations in Chinese hamster cells in vitro. Genetics 61, 1303--1312 (1968) Cooper, D., Banthorpe, D. V., Wilkie, D.: Modified ribosomes conferring resistance to cycloheximide in mutants of Saccharomyces cerevisiae. J. Mol. Biol. 26,347--350 (1967) DeMars, R.: Resistance of cultured human fibroblasts and other cells to purine and pyrimidine analogues in relation to mutagenesis detection. Mutat. Res. 24, 335--364 (1974) Ennis, H. L.: Synthesis of ribonucleic acid in L cells during inhibition of protein synthesis by cycloheximide. Mol. Pharmacol. 2, 543--557 (1966) Falvey, A. K., Staehelin, T.: Structure and function of mammalian ribosomes. I. Isolation and characterization of active liver ribosomal subunits. J. Mol. Biol. 53, 1--30 (1970) Gupta, R. S., Siminovitch, L.: The isolation and preliminary characterization of somatic cell mutants resistant to the protein synthesis inhibitor emitine. Cell 9, 213--219 (1976) Harris, M.: Mutation rates in cells at different ploidy levels. J. Cell Physiol. 78, 177--184 (1971) Haugli, F. B., Dove, W. F.: Genetics and biochemistry of cycloheximide resistance in Physarum polycephalum. Mol. Gen. Genet. 118, 97--107 (1972) McKeehan, W., Hardesty, B.: The mechanism of cycloheximide inhibition of protein synthesis in rabbit reticulocytes. Biochem. Biophys. Res. Commun. 36,625--630 (1969) McLaughlin, C. S.: Yeast ribosomes: Genetics. In: Ribosomes, Nomura, M., Tissi~res, A., Lengyel, P. (eds.), pp. 815--827. New York: Cold Spring Harbor Laboratory 1974 Moehring, T. J., Moehring, J. M.: Selection and characterization of cell resistant to Diphtheria toxine and Pseudomonas exotoxine A: Presumptive translational mutants. Cell 11,447--454 (1977)
Cycloheximide-Resistance in Ovary Cells
305
Nierhaus, K. H., Dohme, F.: Total reconstistution of 50S subunits from Escherichia coli ribosomes. Methods Enzymol. 59, 443--449 (1979) Pestka, S.: Inhibitors of protein synthesis. In: Molecular mechanisms of protein biosynthesis, Weissbach, H., Pestka, S. (eds.), pp. 467--553. New York: Academic Press 1977 P6che, H., Varshaver, N. B., Theile, M., Geissler, E.: Cycloheximide resistance in Chinese hamster cells. II. Induction of CHM resistance in Chinese hamster cells by N-nitrosomethylurea. Mutat. Res. 30, 83--88 (1975a) P6che, H., Junghahn, I., Geissler, E., Bielka, H.: Cycloheximide resistance in Chinese hamster cells. III. Characterization of cell-free protein synthesis by polysomes. Mol. Gen. Genet. 138, 173--177 (1975b) Pohjanpelto, P.: Cycloheximide elicits in human fibroblasts a response characteristic for initiation of cell proliferation. Exp. Cell Res. 102, 138--142 (1976) Pongratz, M., Klingmtiller, W.: Role of ribosomes in cycloheximide resistance of Neurospora mutants. Mol. Gen. Genet. 124, 359--363 (1973) Sutton, C. A., Arens, H., Hallberg, R. L.: Cycloheximide resistance can be mediated through either ribosomal subunits. Proc. Natl. Acad. Sci. USA 75, 3158--3162 (1978) V~zquez, D.: Inhibitors of protein synthesis. (Molecular biology, biochemistry and biophysics, Vol. 30.) Berlin-Heidelberg-New York: Springer 1979 Wang, H. C., Fedoroff, S.: Banding in human chromosomes treated with trypsine. Nature New Biol. 235, 52--54 (1972) Warner, J. R.: The assembly of ribosomes in eukaryotes: In: Ribosomes, Nomura, M., Tissi~res, A., Lengyel, P. (eds.), pp. 461--488. New York: Cold Spring Harbor Laboratory 1974 Willems, M., Penman, M., Penman, S.: The regulation of RNA synthesis and processing in the nucleolus during inhibition of protein synthesis. J. Cell Biol. 41, 177--187 (1969)
Received March 26, 1979
