MGG

Molee. gen. Genet. 175, 181-185 (1979)

cD by Springer-Verlag 1979

Resistance against Cycloheximide in Cell Lines from Chinese Hamster and Human Cells is Conferred by the Large Subunit of Cytoplasmic Ribosomes Hubert P6che 1, Sabine Zakrzewski 2, and K n u d H. Nierhaus t 1 Max-Planck-Institutfi3r Molekulare Genetik, Abt. Wittmann, Ihnestr. 63-73, D-1000 Berlin-Dahlem z Institut ffir Humangenetik, Freie Universitfit, Heubnerweg6, D-1000 Berlin 19

Summary. Cell lines from Chinese hamster ovary [CHO-K1-D3] and human fibroblast cells [46, XX, 18p-] were mutagenized with N-nitrosomethylurea followed by a selection for cycloheximide resistance. Two mutants resistant against the durg were selected from either wildtype. 80S ribosomes and their ribosomal subunits were isolated from all mutant and wildtype cells. 80S ribosomes reassociated from the isolated subunits were as active as isolated 80S couples in the poly (U) dependent poly (Phe) synthesis. Hybrid 80S ribosomes constructed from subunits of the various cell lines of the same species were fully active, whereas the interspecies 80S hybrids were not active at all in poly (Phe) synthesis. Hybrid 80S ribosomes from subunits of mutant and the ocrresponding wildtype cells were tested in the poly (U) assay in the presence and absence of cycloheximide. The results strikingly indicate that in all four mutant cell lines the resistance against cycloheximide is conferred by the large subunit of cytoplasmic ribosomes.

Introduction Cycloheximide has become the standard drug for specific inhibition of eukaryotic protein synthesis performed by 80S ribosomes in contrast to systems containing mitochondrial or prokaryotic ribosomes (for reviews see Pestka, 1977, a n d Vazques, 1979). In spite of intensive research the molecular mechanism of the cycloheximide induced inhibition remains K.H. Nierhaus CHM, Cycloheximide;CHO, Chinese hamster ovarien; FBS, foetal bovine serum; Eagle MEM, Eagle mimmal essential medium; EMS, Ethyl-metansulfonate;NMU, N-nitrosomethylurea

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obscure. An inhibition of the initiation, elongation (Lin et al., 1966; Obrig et al., 1971) and termination (Godchaux et al., 1967; Rajalakshmi et al., 1971) has been reported. An interference with the ribosomal P-site has been discussed (McKeehan and Hardesty, 1969) where the drug possibly affects the G T P binding and/or hydrolysis (Rajalakshmi et al., 1971). In support of this hypothesis it was shown recently that cycloheximide inhibits all energy consuming steps in translation (Oleinick, 1977). Resistant mutants have been isolated from various organisms including yeast (Cooper et al., 1967; Rao and Grollmann, 1967; Jiminez et al., 1972), Neurospora (Pongratz and Klingmfiller, 1973) Physarum (Haugli and Dove, 1972), Tetrahymena (Sutton et al., 1978) and cell lines from Chinese hamster ovary (P6che et al., 1975). In all these cases the resistance was conferred by the 80S ribosome, and the three groups analyzing the mutants in yeast reported that the resistance was a property of the 60S subunit. Surprisingly, in Tetrahymena the resistance apparently can be conferred by either ribosomal subunit (Sutton et al., 1973). Here we report an analysis of resistant mutants derived from mammalian cells. The resistance of four mutants obtained from Chinese hamster ovary and human cells, respectively, is mediated through the large ribosomal subunit.

Material and Methods Cell Cultures. The cells used for the investigation were ovary cells of Chinese hamster, CHO-K1-D3, with a modal chromosome number of 22, and a diploid cell culture of human fibroblasts (46, XX, 18p-). This human cell line was used because it grew well. The abnormal karyotype is unrelated to the problem studied. All cells grow as monolayersin Dulbecco's modification of Eagle's medium, containing 10% or 20% fetal bovine serum (FBS).

0026-8925/79/0175/0181/$01.00

182

H. P6che et al. : 60S Ribosomal Subunit Confers Cycloheximide Resistance

MutagenesisAssay. The procedure was a modification of that previously described (P6che et al., 1975). Exponentially growing cells were inoculated with 105 cells/plate. After incubation for 24 h, the cells were treated with 100 I-tg/ml N-nitrosomethylnrea (NMU) for 2.5 h. The mutagen was then removed by washing with serumfree Dulbecco's medium and subsequent addition of Dulbecco's medium with 15% FBS. After an expression time of 72 h, the cells were inoculated into a selective medium for CHM (7× 10 -7 M). The inocnlum added to this selective medium was either 105, 2.5 x 10s or 5 x 105 cells/plate. The control experiments were identical, but without mutagenic treatment. The selective medium was changed every 3 or 4 days, and the cultures were incubated at 37°C in a 5% CO2-air mixture for 18 days. Colonies to be cultured were taken by Pasteur pipette from unstained plates. The isolated human ceil clones grow as monolayers and the Chinese hamster ovary cell clones as spinner cultures in a selective medium at7x10 7MCHM.

~120

.~_~ e0 I

o::U . ./I

E

g

ol / I

105

Preparation of 80S Ribosomes and High-Speed Supernatant. The preparation procedure followed essentially the method described by Falvey and Staehelin (1970). All operations were carried out at 0 ~ t° C. Cells were harvested by centrifugation at 3,000 x g for 15 min and washed with a PBS-solution. The washed cells were resuspended in 3 vol. of buffer A (20 ram Tris-HC1, pH 7.6, 130 mM KC1, 7.5 mM MgCI2, 5 mM/~-mercaptoethanol) and homogenized in a Potter homogenizer with 20 strokes. The homogenate was centrifuged for 10 min at 12,000 x g. The ribosomes from the upper two-thirds of the resulting post-mitochondrial supernatant were underlayered with 3 ml of 30% sucrose in buffer A and sedimented for 5 h centrifngation at 75,000 x g. The ribosome pellets were resuspended in buffer A. The upper two-thirds of the clear supernatant (S-150 enzymes) was removed and immediately frozen in small samples and stored at - 8 0 " C.

Preparation of Ribosomal Subunits. The washed celIs were resuspended in 3 vol. of buffer B (20 mM Tris-HC1, pH 7.6, 250 mM KC1, 2.5 mM MgC12, 5 mM/~-mercaptoethanol) and homogenized in a Potter homogenizer. The homogenate was centrifuged for 10 rain at 12,000 x g. The upper two-thirds of the supernatant was incubated in the presence of 1 mM puromycin and 1 mM GTP for 30 min at 37 ° C. After incubation the suspension was cooled in ice and centrifuged for 10 min at 12,000 x g to remove aggregates formed during the incubation. The ribosomal subunits were separated by centrifugation of the clarified suspension on 10-30% (w/v) sucrose gradients in buffer C (20 mM Tris-HC1, pH 7.6, 400 mM KC1, 3 mM MgC12, 5 mM /3-mercaptoethanol; Beckman SW27 rotor; 8 h at 83,000 x g). Gradients were analyzed at 290 rim, appropriate fractions were pooled, the subunits were sedimented (18 h at 250,000 x g) and resuspended in buffer A.

Poly (U)-directed Polyphenylalanine Synthesis. The assay was a modification of that of Nierhaus and Dohme (1978). Reactions were carried out in 125 gi volumes and incubated at 30°C for 45 min. The final concentrations were 20 mM Tris-HC1, pH 7.6, 15 mM NH4C1, i50 mM KC1, 12 mM MgC12, 1 mM ATP, 0.3 mM GTP, 5raM phosphoenolpyruvate, 6 m M /~-mercaptoethanol, 20 gg tRNA from calf liver, 3 gg pyruvatekinase and 60 gl S-150 enzymes. Each assay contained 70 gM [14C] phenylalanine and 40 pg poly (U). The 80S ribosomes were present in amounts of 1 Az60 unit per assay, and the 60S and 40S subunits were also each present in 1 A260 unit per assay, respectively. After incubation one drop of a bovine serum albumin solution (1% w/v) and 2 ml of 5% trichloroacetic acid were added. Samples were then incubated for 15 min at 90 ° C, then filtered through glassfiber filters which were washed twice with cold 5% trichloracetic acid, and once with ether:ethanol (1 : 1), dried and counted.

I

2,5 × 105 Cells/p[efe

I

5 xlO 5

Fig, 1. Dependence of the number of mutants on inoculum size, in the presence and absence of mutagen (100 gg/ml NMU). • • CHO cells without mutagen; z x - - ± , CHO cells with mutagen; e - - e , human cells without mutagen; o - - © , human cells with mutagen

Results

Selection of Cycloheximide Resistant Mutants N-nitrosomethylurea (NMU) was used as mutagen ( 1 0 0 ~tg/ml), 7 x 1 0 - T M cycloheximide was present in the selective medium. Figure 1 demonstrates that 1) t h e m u t a g e n i n c r e a s e s 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 s i g n i f i c a n t l y as c o m p a r e d to the spont a n e o u s l y o c c u r r i n g m u t a n t s , a n d 2) t h e C h i n e s e h a m s t e r o v a r y ( C H O ) cells a n d t h e h u m a n f i b r o b l a s t cells s h o w e d o p p o s i n g r e s p o n s e s t o a n i n c r e a s e o f t h e i n o c u l u m size. T h e l a t t e r d e p r e s s e d t h e n u m b e r o f r e s i s t a n t C H O c o l o n i e s b y a f a c t o r o f 0.3, w h e r e a s i n c o n t r a s t , t h e n u m b e r o f r e s i s t a n t h u m a n cell c o l o n i e s w a s s t i m u l a t e d b y a f a c t o r 1.5. T w o r e s i s t a n t c l o n e s f r o m e a c h cell l i n e w e r e chosen. These mutants were bred in the presence of 7x10-TM cycloheximide, both wildtypes without t h e d r u g ( f o r d e t a i l s see E x p e r i m e n t a l P r o c e d u r e s ) .

Poly (Phe) Synthesis of Ribosomes Derived from Sensitive and Resistant Cell Lines, Respectively 80S r i b o s o m e s w e r e i s o l a t e d f r o m t h e f o u r m u t a n t s and the respective wildtypes and tested for poly (Phe) synthesis in the presence of their respective S-150 enz y m e s f r o m w i l d - t y p e cells a t v a r i o u s c y c l o h e x i m i d e concentrations (Fig. 2 A a n d B) B o t h w i l d t y p e s s h o w e d a s i m i l a r r e s p o n s e t o t h e d r u g . T h e C H O cells w e r e s l i g h t l y m o r e s e n s i t i v e t h a n t h e h u m a n cells, the residual activity at 10-3M cycloheximide being

H. P6che et al. : 60S Ribosomal Subunit Confers Cycloheximide Resistance

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concentration of 10 3 M. Therefore, this concentration was used for testing cycloheximide sensitivity in the following assays. In order to assign the resistance property to a ribosomal subunit, it was necessary to dissociate the 80S ribosomes into their subunits without losing activity. After a series of systematic trials we achieved excellent dissociation and separation of highly active subunits, by a modification of the method described by Falvey and Staehelin (1970). 40S and 60S subunits were recombined and compared with the corresponding undissociated 80S particles in kinetic measurements of poly (Phe) synthesis. All (40S+60S) ribosomes were highly active and showed 70-90% of the activity of the original 80S ribosomes (data not

,2. 6o Table 2. Cycloheximide sensitivity of hybrid 80S couples

40

Cell line

20-

B

~/1

Resistance type of subunits

Cycloheximide

cpm

[14C]Phe incorp. per ribosome

[MI -6

10-5

10-~-

10-3

M Fig. 2A and B. Poly (Phe) synthesis by nondissociated 80S ribosomes at various cycloheximide concentrations. A ribosomes derived from CHO cells; z s - - A , wildtype; o - - o , CHO-CHMM; o - - o , CHO-CHM'-2. B ribosomes from h u m a n fibroblast cells; A A, wildtype; o - - e , H F C - C H M r - I ; © - - o , H F C - C H M ' - 2

Table l, Activities of homologous and heterologous 80S hybrids (poly(U) system) Source of subunits 60S

40S

CHO human CHO human

CHO human human CHO

[14C]Phe incorporated per 80S ribosome

38 35 0.2 0.5

The background (minus particle, 725 cpm) was subtracted before the amount of incorporated Phe was calculated. 38 Phe per ribosome is equivalent to 65,775 cpm

15 and 30%, respectively. In contrast, none of the ribosomes derived from resistant strains were affected by either drug concentration except the ribosomes from C H O - C H M M . The latter ribosomes were inhibited by about 35% at the lowest drug concentration tested (10 -6 M), but this inhibition level remained constant at higher concentrations. The different response to cycloheximide between sensitive and resistant ribosomes is most easily detected at a drug

Chinese hamster ovary cells [CHO]

60S

40S

WT WT

WT WT

10 -3

68,500 6,250

39 4

WT WT

CHMr-I CHMr-1

10-3

37,800 6,100

22 3

CHMr-1 CHMM

WT WT

10 -3

64,500 64,400

37 37

CHMr-1 CHMr-1

CHMr-I CHMM

10 .3

66,400 67,700

38 39

WT WT

CHMr-2 CHMr-2

10 -3

44,200 5,300

25 3

CHMr-2 CHM~-2

WT WT

10 ~

44,600 41,400

26 24

CHMr-2 CHMr-2

CHMr~2 CHMr-2

10 3

38,500 20,100

22 12

WT WT

10 -3

62,000 23,600

35 13

CHMr-1 CHMr-1

10 -3

66,000 5,500

38 3

CHMr-1 CHMr-1

WT WT

10-3

61,000 57,000

35 33

CHMr-1 CHMr-I

CHMr-1 CHMM

10 -3

65,000 62,000

37 35

WT WT

CHM'-2 CHM~-2

10 3

63,400 5,000

36 3

CHMr-2 CHMr-2

WT WT

10 . 3

65,300 62,000

37 35

CHMr-2 CHM~-2

CHMr2 CHMr-2

10 3

61,800 62,300

35 37

Human WT fibroblast WT cells WT WT

184

H. P6che et al. : 60S Ribosomal Subunit Confers Cycloheximide Resistance

shown). In contrast, heterologous 80S hybrids constructed f r o m hamster and h u m a n subunits were not active at all (Table 1). In the next experiment, h o m o l o g o u s 80S hybrids were constructed f r o m the subunits o f each m u t a n t and those o f the respective wildtype. The 80S hybrid ribosomes were tested in the absence and presence o f 1 0 - 3 M cycloheximide. It is clear f r o m Table 2 that in all cases where a 60S subunit f r o m the m u t a n t was c o m b i n e d with a 40S one f r o m wildtype cells, a resistance pattern was f o u n d (no inhibition with cycloheximide). In contrast, the reversed c o m b i n a t i o n o f wildtype 60S and m u t a n t 40S subunit revealed a sensitive response to cycloheximide (drastic inhibition by the drug). This experiment strikingly demonstrates that in all m u t a n t s f r o m b o t h Chinese hamster ovary and h u m a n fibroblast cells, the resistance against cycloheximide is located in the 60S subunit.

Discussion Antibiotics inhibiting protein synthesis play an important role in the analysis o f the ribosomal functions, by allowing a functional intermediate to accumulate which might otherwise be difficult or even impossible to isolate. F u r t h e r m o r e , drugs have been used to assign ribosomal c o m p o n e n t s to specific partial functions o f the ribosome. In the latter case, m u t a n t s resistant against the drug are isolated and the altered c o m p o n e n t identified, which can then be related to that function which is inhibited by the antibiotic. In eukaryotes, only a few resistant m u t a n t s have been well documented. Emetine resistance is located in the 40S ribosomal subunit ( G u p t a and Siminovitch, 1977), and a protein p r o b a b l y responsible for the 40S alteration has been identified (Boersma et al., 1979). The p h e n a n t h r e n alkaloids cryptopleurine, tylocrebine and tylophorine p r o v o k e resistant m u t a n t s which also have an altered 40S subunit (Skogerson et al., 1973 ; G r a n t et al., 1974). Cycloheximide resistance seems to affect exclusively the 60S subunit, since three different groups have been able to assign the resistance p r o p e r t y to the 60S subunits o f yeast m u t a n t s ( R a o and Grollmann, 1967; C o o p e r etal., 1967; Himinez etal., 1975). A n altered 60S protein has been identified in a cycloheximide resistant m u t a n t f r o m the fungus Pod o s p o r a anserina. However, the authors did not dem o n s t r a t e that the resistance was mediated by the 60S subunit (Begueret et al., 1977). Surprisingly, a recent report d e m o n s t r a t e d that in the ciliated p r o t o z o a n T e t r a h y m e n a thermophila the resistance can be conferred by either ribosomal subunit (Sutton et al., 1978). The m u t a n t s carrying

an altered 60S subunit showed a p r o n o u n c e d resistance in vivo in contrast to those with an altered 40S subunit, However, in the poly (Phe) synthesizing system all the m u t a n t s revealed a low level o f resistance; 10-4 M cycloheximide drastically depressed the activity o f b o t h wildtype and m u t a n t ribosomes. Here we have analyzed four m u t a n t s resistant against cycloheximide and derived f r o m two different animal cell lines, namely Chinese hamster ovary and h u m a n fibroblast cells. In the poly (Phe) synthesizing system all m u t a n t s are resistant against high doses o f cycloheximide. A t 10-3 M cycloheximide the wildtype ribosomes are severely inhibited, whereas the m u t a n t ribosomes show either no effect or a low inhibition. In all cases the resistance is mediated by the 60S subunit. Thus, it is possible that high-dose resistance against cycloheximide is linked exclusively to an alteration in the large ribosomal subunit, whereas a low-dose resistance can be conferred by either subunit.

Acknowledgements. We thank Drs. H.G. Wittmann, R. Brimacombe, K. Sperling and in particular E. Passarge for criticisms and discussions.

References Begueret, J., Perrot, M., Crouzet, M. : Ribosomal proteins in the fungus Podospora anserina: Evidence for electrophoretically altered 60S protein in a cycloheximide resistant mutants. Mol. Gen. Genet. 156, 141-144 (1977) Boersma, D., McGII1,S.M., Mollenkamp, J.W., Roufa, D.J. : Emitine resistance in Chinese hamster cells is linked genetically with an altered 40S ribosomal subunit protein, $20. Proc. Natl. Acad. Sci.U.S.A. 76, 415-419 (1979) Cooper, D., Banthorpe, D.V. and Wilkie, D. : Modified ribosomes conferring resistance to cycloheximide in mutants of Saccharomyces cerevisiae. J. Mol. Biol. 26, 347 350 (1967) 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) Godchanx, W., Adamson, S.D., Herbert, E. : Effect of cycloheximide on polyribosome function in reticulocytes. J. Mol. Biol. 27, 57-72 (1967) Grant, P., Sfinchez, L., Jim6nez, A. : Crytopleurine resistance: Genetic locus for a 40S ribosomal component in Saccharomyces cerevisiae. J. Bacteriol. 120, 1308-1314 (1974) Gupta, R.S., Siminovitch, L. : The molecular basis of emitine resistance in Chinese hamster ovary cells : Alteration in the 40S ribosomal subunit. Cell 10, 61-66 (1977) Haugli, F.B., Dove, W.F., Jimenez, A. : Genetics and biochemistry of cycloheximide resistance in Physarum polycephalum. MoI. Gen. Genet. 118, 97-107 (1972) Jim6nez, A., Sanchez, L., Vazquez, D.: Location of resistance to the alkaloid narciclasine in the 60S ribosomal subunit. FEBS Lett. 55, 53-56 (1975) Lin, S.Y., Mosteller, R.D., Hardesty, B.: The mechanism of sodium fluoride and cycloheximide inhibition of haemoglobin biosynthesis in cell free reticulocyte system. J. Mol. Biol. 21, 51-69 (1966) McKeehan, W., Hardesty, B.: The mechanism of cycloheximide inhibition and protein synthesis in rabbit reticulocytes. Biochem. Biophys. Res. Comm. 36, 625-630 (1969)

H. P6che et al.: 60S Ribosomal Subunit Confers Cycloheximide Resistance Nierhaus, K.H., Dohme, F. : Total reconstitution of 50S subunits from Escherichia coli ribosomes. In: Methods in enzymology LIX, pp. 443-449. New York: Academic Press 1978 Obrig, T.G., Culp, W.J., McKeehan, W.L., Hardesty, B.: The mechanism by which cycloheximide and related glutaramide antibiotics inhibit peptide synthesis and reticulocyte ribosomes. J. Biol. Chem. 246, 174-181 (1971) Olelnick, N.L : Initiation and elongation of protein synthesis in growing cells : Differential inhibition by cycloheximide and emitine. Arch. Biochem. Biophys. 182, 17l 180 (1977) Pestka, S.: Inhibitors of protein synthesis. In: Molecular mechanisms of protein biosynthesis (H. Weissbach, S. Pestka, eds.), pp. 467-553. New York: Academic Press 1977 P6che, H., Varshaver, N.B., Theile, M., Geissler, E. : Cyeloheximide resistance in Chinese hamster cells. II. Induction of CHM resistance in Chinese hamster cells by N-nitrosomethylurea. Mutat. Res. 30, 83 88 (1975) P6che, H., Jnnghahn, 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 (1975) Pongratz, M., Klingmiiller, W. : Role of ribosomes in cycloheximide resistance of Neurospora mutants. Mol. Gen. Genet. 124, 359 363 (1973)

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Rajalakshmi, S., Liang, H., Sarma, D.S.R., Kisilevsky, R., Farber, E. : Cycloheximide, an inhibitor of peptide chain termination or release in liver in vivo and in vitro. Biochem. Biophys. Res. Commun. 42, 259-265 (1971) Rao, S.S., Grollmann, A.P.: Cycloheximide resistance in yeast: Property of the 60S ribosomal subunit. Biochem. Biophys. Res. Commun. 29, 696-704 (1967) Skogerson, L., McLaughlin, C., Wakatama, E.: Modification of ribosomes in cryptopleurine-resistant mutants of yeast. J. Bacteriol. 116, 818-822 (1973) Sutton, C.A., Ares, M., Hallberg, R.L. : Cycloheximide resistance can be mediated through either ribosomal subunits. Proc. Natl. Acad. Sci. U.S.A. 75, 3158-3162 (1978) Vazquez, D.: Inhibitors of protein synthesis, In: Molecular biology, biochemistry and biophysics 30 (A. Kleinzeller, G.F. Springer, H.G. Wittmann, eds.), pp. 155-158. Berlin-Heidelberg-New York: Springer 1979

Communicated

b y E. B a u t z

Received June 11, 1979