INDUCTION OF FOUR PROTEINS IN EUKARYOTIC ... - Science Direct

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Biochimica et Biophysica Acta, 518 ( 1 9 7 8 ) 4 0 1 - - 4 1 2 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

BBA 99153

INDUCTION OF FOUR PROTEINS IN EUKARYOTIC CELLS BY KETHOXAL BIS(THIOSEMICARBAZONE )

WARREN

L E V I N S O N *, H E R M A N

O P P E R M A N N , and J E A N J A C K S O N

Department of Microbiology, University of California, San Francisco, Calif. 94143 (U.S.A.) (Received August 22nd, 1977)

Summary Kethoxal bis(thiosemicarbazone) induces the synthesis of four proteins (100 000, 70 000, 35 000 and 25 000 daltons) in normal chick embryo cells. The 70 000 dalton species is produced at the fastest rate 2 h after exposure to the compound. Pulse-chase experiments revealed neither precursors nor products of these proteins and both actinomycin and cycloheximide inhibited their synthesis. Neither of the two substituents of the inducer, kethoxal or thiosemicarbazide, were active. The four proteins were induced in several other species, but human cells produced only three proteins (100 000, 70 000 and a different 30 000 dalton form).

Introduction

Kethoxal bis(thiosemicarbazone) is a metal binding ligand with anti-tumor activity [1]. This compound inhibits Walker 256 carcinosarcoma growth in rats and the addition of copper in the diet enhances its activity [2]. The preformed drug. copper(II) complex has similar anti-tumor activity [3]. The synthesis of DNA in both murine Sarcoma 180 cells [4] and normal chick embryo cells [5] is more sensitive to the drug and its copper complex than is RNA or protein synthesis. Recently, it was shown that the respiration of Ehrlich ascites cells is inhibited by the drug. copper complex [6]. We have been studying the anti-viral effects of kethoxal bis(thiosemicarbazone) and other thiosemicarbazones. We have shown that this class of compounds can inactivate, on contact in vitro, the transforming ability of Rous sarcoma virus by inhibiting its RNA dependent DNA polymerase [7]. The activity of the drug • copper complex in this regard is greater than the effect of the free ligand [8]. In addition, we have found that this compound inhibits, * To w h o m c o m m u n i c a t i o n s s h o u l d be addressed.

402 intracellularly, the replication of vesicular stomatitis virus [5]. In an experim e n t designed to determined the effect of the drug on vesicular stomatitis virus protein synthesis, we found that the synthesis o f four proteins was greatly enhanced in uninfected chick embryo cells. This report documents and explores this observation. Materials and Methods

Chemicals. Chemicals were obtained from the following sources: kethoxal bis(thiosemicarbazone) and kethoxal from Nutritional Biochemicals, thiosemicarbazide from Eastman Kodak and CuSO4 from Matheson, Coleman and Bell. Dimethyl sulfoxide (spectral quality) obtained from Matheson, Coleman and Bell was used as the solvent for the stock solutions. Cell cultures. Secondary cultures of chick e m b r y o cells were prepared as previously described [9]. In addition, h u m a n foreskin cells, mouse embryo cells, duck embryo cells, Chinook salmon cells, and Rous sarcoma virus-transformed chick embryo cells were propagated in a similar manner. Medium 199 plus 4% calf serum was used as growth medium and the cultures were incubated in plastic dishes in a humidified atmosphere with sufficient CO2 to keep the medium at pH 7.4. The temperature was 38°C except with the salmon cells for which it was 15°C. Induction of proteins. 5 • 10 s cells were seeded onto 35-mm dishes in 2.5 ml growth medium and incubated at 38°C overnight. The medium was discarded, the cultures washed twice with 2 ml methionine-free medium 199, and 2.5 ml methionine-free 199 was replaced. The drug was then added. In our standard induction procedure, 1 pl of 7 • 10 -4 M (200 pg/ml) solution was added for a final concentration of 2 . 8 - 1 0 - T M (80 ng/ml). The final concentration of dimethyl sulfoxide solvent was 0.04%. The cultures were incubated at 38°C (salmon cells at 15°C). [3SS]Methionine (spec. act. 275 Ci/mmol) was added at a final concentration of 2 pCi/ml. The cultures were incubated at 38°C for 1 h, the medium discarded, and the cultures washed once with 2 ml Medium 199. The cells were scraped into 2 ml Medium 199, centrifuged at 1500 Rev./min for 10 min at 4°C in a Sorvall RC-3 centrifuge and the supernatant completely removed. The tube was allowed to drain and wiped dry. 50 pl of solubilizing buffer containing 0.07 M Tris ( p H 6 . 8 ) / 1 0 % glycerol/2% sodium dodecyl sulfate (SDS)/5% 2-mercaptoethanol was added. The sample was boiled for 2 min and stored at--70°C. Electrophoretic analysis of proteins. Samples were analysed by electrophoresis on polyacrylamide slab gels (4 X 15 × 0.15 cm with 1 cm wells) using an apparatus as described by Studier [10]. Equal amounts of proteins were loaded onto the gel. Proteins were separated on 12% gels (Crosslinker = ~) in a discontinuous buffer system as described by Laemmli [ 11], with a constant current of 30 mA for approx. 4 h. The gels were then exposed to Kodak RP Royal X-omat X-ray film, incubated at room temperature for 2 days and developed.

403 Results

Kinetics of synthesis Uninfected chick embryo cells were exposed to kethoxal bis(thiosemicarbazone) (80 ng/ml, 2.8- 10 -7 M) then, at 2-h intervals, labelled with [35S]methionine for 30 min. The cell extracts were analyzed by slab acrylamide gel electrophoresis. Fig. 1 illustrates the induction of four proteins whose molecular weights are estimated at 100 000, 70 000, 35 000 and 25 000. The estimates are based on T4 phage protein markers. The synthesis of the 70 000 dalton species is induced at the fastest rate. The 25 000 dalton protein appears last but, by 12 h, the 70 000 and 25 000 dalton species appear to be synthesized to approximately the same extent. In the 10- and 12-h samples, a fifth induced protein appears at approximately 55 000 daitons. Cells not treated with the drug contain little or none of these proteins either at 2 h {Fig. 1) or at 12 h {unpublished).

Concentration of inducer Uninfected chick embryo cells were exposed to varying concentrations of drug for 3 h and then labelled with [3SS]methionine for 1 h. The gel analysis of the cell extracts shown in Fig. 2A demonstrates that cultures given 800 ng/ml drug produce the 25 000 dalton species but synthesize less than normal amounts of protein overall. At 240 ng/ml the 70 000 and 25 000 dalton species are visible and at 80 ng/ml all four species are synthesized. The data in Fig. 2B shows that cultures exposed to 80 ng/ml and 40 ng/ml produced all four proteins but those exposed to 20 ng/ml and 10 ng/ml did not. Cultures treated with 0.04% dimethyl sulfoxide, the solvent for the drug, show no increase in synthesis of these proteins.

Deinduction of synthesis Uninfected chick embryo cells were exposed to 80 ng/ml drug for 2 h, then the drug containing medium was removed, the cultures washed twice and fresh growth medium without drug was replaced. At 2-h intervals, the cultures were exposed to [3SS]methionine for 30 min and the cell extracts analysed by gel electrophoresis. The data in Fig. 3 illustrates that the four proteins continue to be made until 12 h when decreased amounts of the 100 000, 70 000 and 35 000 dalton species are produced. The 25 000 dalton species continues to be made at a high rate during the 12 h period. In a similar experiment, the medium containing 80 ng/ml drug was removed after a 4 h exposure, incubated in fresh medium for a further 20 h labelled with [aSS]methionine for 30 min and analyzed. None of the induced proteins could be visualized on the gels after 20 h.

Precursor-product analysis Pulse and pulse-chase experiments were performed to determine precursorproduct relationships. When cultures exposed to 80 ng/ml drug for 3 h were exposed to [3SS]methionine for 5 min, the four species were observed, indicating that it is unlikely that high molecular weight precursors of these proteins are made. When parallel cultures were exposed to [aSS]methionine for 15 min,

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Fig. 1. K i n e t i c s o f s y n t h e s i s . C h i c k e m b r y o cells w e r e e x p o s e d t o k e t h o x a l b i s ( t h i o s e m i c a r b a z o n e ) ( K T S ) ( 8 0 n g / m l ) a n d a t 2oh i n t e r v a l s l a b e l l e d w i t h [ 3 5 S ] m e t h i o n i n e f o r 3 0 r a i n . T h e cells w e r e e x t r a c t e d in s o i n b i l i z i n g b u f f e r c o n t a i n i n g 2% S D S a n d a n a l y z e d b y p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s . T h e p r o t e i n s w e r e d e t e c t e d b y a u t o r a d i o g r a p h y u s i n g X - r a y film. a, N o k e t h o x a l b i s ( t h i o s e m i c a r b a z o n e ) ; b, k e t h o x a l b i s ( t h i o s e m i c a r b a z o n e ) a t 2 h; c, 4 h ; d, 6 h~ e, 8 h~ f~ 1 0 h ; g, 1 2 h. A, a c t i n .

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then washed and incubated in Medium 199 containing 30/~g/ml methionine in the absence of both drug and [3SS]methionine for 2 h, the same a m o u n t of the four proteins were seen. This indicates that these proteins are n o t precursors of other species and that they are n o t degraded significantly during a 2 h period.

Possibility of artefact Several types of experiments were done to test whether the appearance of these proteins was due to a technical artefact. First, cultures were labelled with 3H-labelled protein hydrolystate instead of [3SS]methionine. The same four proteins were seen, indicating that the observation is n o t an artefact of the isotope. Second, cultures were exposed to either actinomycin (4 pg/ml) or cycloheximide (80 ng/ml) for 1 h prior to adding the inducer. Both drugs inhibited induction, indicating that cellular RNA and protein synthesis is required and that the proteins are not the result of adsorption of [3SS]methionine onto preexisting proteins: Third, the cultures were labelled with [3SS]methionine for 2 h in the absence of the inducer, then the isotope was removed prior to exposing the cells to the drug for 3 h. No induced proteins were seen indicating that these proteins do not form by aggregation of other proteins. Fourth, cells were exposed and labelled with [3SS]methionine in Medium' 199 containing normal amounts of methionine and the same four proteins were seen. This indicates that the proteins are n o t an artefact of the absence of methionine.

Induction by kethoxal bis(thiosemicarbazone) substituents Chick cells were exposed to the two substituents, kethoxal and thiosemicarbazide, for 3 h, then labelled with [3SS]methionine and analysed by gel electrophoresis. In addition, since the inducer is known to avidly bind copper [3], exposure to CuSO4 was tested for its inducing ability. Neither kethoxal, thiosemicarbazide nor CuSO4 induced the proteins.

Induction in cells of different species Cells from several species were compared to uninfected chick e m b r y o cells for their ability to synthesize these four proteins in the presence of 80 ng/ml inducer. The data in Fig. 4A shows that there are t w o proteins in human foreskin fibroblasts which are similar to the 100 000 and 70 000 dalton proteins in chick cells. However, in contrast to the 35 000 and 25 000 dalton proteins in chick cells, only one protein of approx. 30 000 daltons is seen. Fig. 4B illustrates that Rous sarcoma virus-transformed chick e m b r y o cells produce the same proteins as do uninfected chick cells. However, Chinook salmon cells produce an increased a m o u n t only of the 70 000 species. In contrast to the chick cells, the untreated salmon cells have a significant amount of protein which comigrates with the induced species. Secondary cultures of mouse and duck e m b r y o cells produce the same four proteins, as do chick e m b r y o cells when exposed to 80 ng/ml inducer (unpublished).

Attempts to determine possible function of induced proteins In an effort to gain s o m e insight regarding the possible function of these proreins, several experiments were performed: (1) Since it is known that the drug or its copper complex have t w o signifi-

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cant effects on cells, e.g., inhibition of nuclear DNA synthesis [4] and inhibition of respiration in the mitochondria [6], we tested whether cytosine arabinoside (2.5 pg/ml), fluorodeoxyuridine (10pg/ml), NaCN ( 1 0 0 p g / m l ) or dinitrophenol (80 pg/ml) induced these proteins. No induction was seen. (2) We determined whether these proteins were made on mitochrondrial ribosomes by exposing the cells to chloramphenicol (50 pg/ml) for 1 h prior to adding drug. The induced proteins were made normally in the presence of chloramphenicol.

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Fig. 2. C o n c e n t r a t i o n o f k e t h o x a l b i s ( t h i o s e m i c a r b a z o n e ) . C h i c k e m b r y o cells w e r e e x p o s e d t o 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 k e t h o x a l b i s ( t h i o s e m i e a r b a z o n e ) f o r 3 h, a n d t h e n l a b e l l e d w i t h [ 3 5 S ] m e t h i o n i n e f o r I h. T h e cells w e r e e x t r a c t e d in s o i n b i l i z i n g b u f f e r c o n t a i n i n g 2% S D S a n d a n a l y z e d b y p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s . T h e p r o t e i n s w e r e d e t e c t e d b y a u t o r a d i o g r a p h y u s i n g X - r a y f i l m . A: a, N o k e t h o x a l b i s ( t h i o s e m i c a r b a z o n e ) ; b , k e t h o x a l b i s ( t h i o s e m i c a r b a z o n e ) a t 8 0 n g / m l ; e, 2 4 0 n g / m l ; d, 8 0 0 n g / m l . B: a, N o k e t h o x a l b i s ( t h i o s e m i c a r b a z o n e ) ; h, k e t h o x a l b i s ( t h i o s e m i c a r h a z o n e ) a t 8 0 n g / m l ; e, 4 0 n g / m l ; d, 2 0 n g / m l ; e, 1 0 n g / m l ; f, M e 2 S o .

(3) We tested whether two inducers of the cytochrome P-450 microsomal oxidase enzymes would induce these proteins [12]. Neither sodium phenobarbitol (250 gg/ml) nor ethyl alcohol (3 • 10 -a %) were active.

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O Fig. 3. D e i n d u c t i o n o f s y n t h e s i s . C h i c k e m b r y o cells w e r e e x p o s e d t o k e t h o x a l b i s ( t h i o s e v a i c a r b a z o n e ) ( 8 0 n g / v a l ) f o r 2 h a n d t h e n t h e d r u g - c o n t a i n i n g m e d i u m w a s d i s c a r d e d a n d f r e s h m e d i u m r e p l a c e d . A t 2-h i n t e r v a l s t h e c u l t u r e s w e r e e x p o s e d t o [ 3 5 S ] v a e t h i o n i n e f o r 3 0 vain. T h e cells w e r e e x t r a c t e d in solubilizing b u f f e r c o n t a i n i n g 2% S D S a n d a n a l y z e d b y p o l y a c r y l a v a i d e gel e l e c t r o p h o r e s i s . T h e p r o t e i n s w e r e d e t e c t e d b y a u t o r a d i o g r a p h y u s i n g X - r a y f i l m . a, k e t h o x a l b i s ( t h i o s e v a i c a ~ b a z o n e ) a t 4 h; b, 6 h; e, 8 hl d , 10h;e, 12h.

(4) It is possible that these induced proteins are those of an endogenous C-type R N A virus. Although this possibility seemed unlikely since the molecular weights of the induced proteins do n o t correspond to those of RAV-O

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Fig. 4. A .

(our p r o t o t y p e endogenous virus of chicken cells), we attempted to determine whether [3H]uridine-labelled particles with p = 1.16 were produced in drug treated cells. Isopynic sucrose gradients analysis revealed no particles at p = 1.16 in the medium for untreated or treated cells in contrast to the peak of [3H]uridine-labelled particles recovered from parallel cultures of Rous sarcoma virus-infected chick embryo cells.

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a

13

c

cl

e

f

Fig. 4. I n d u c t i o n in cells of o t h e r species. C h i c k e m b r y o cells, R o u s s a r c o m a virus t r a n s f o r m e d c h i c k e m b r y o cells, h u m a n f o r e s k i n cells a n d C h i n o o k s a l m o n cells w e r e e x p o s e d to k e t h o x a l b i s ( t h i o s e m i c a r b a z o n e ) ( 8 0 n g / m l ) f o r 3 h a n d t h e n l a b e l l e d f o r 1 h w i t h [ 3 $ S ] m e t h i o n i n e . T h e cells w e r e e x t r a c t e d in solubllizing b u f f e r c o n t a i n i n g 2% SDS a n d a n a l y z e d b y p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s . T h e p r o t e i n s were d e t e c t e d b y a u t o r a d i o g r a p h y u s i n g X - r a y film. A: a, c h i c k , n o k e t h o x a l b i s ( t h i o s e m i c a r h a z o n e ) ; b, chick, k e t h o x a l b i s ( t h i o s e m i e a r h a z o n e ) i e, h u m a n , n o k e t h o x a l b i s ( t h i o s e m i c a r b a z o n e ) ; d, h u m a n , b i s ( t h i o ~ e m i c a r b a z o n e ) . B: a, c h i c k , n o k e t h o x a l b i s ( t h i o s e m i c a r b a z o n e ) ; b, c h i c k , k e t h o x a l h i s ( t h i o s e m i c a r b a z o n e ) ; c, R o u s s a r c o m a cells, n o k e t h o x a l b i s ( t h i o s e m i c a x b a z o n e ) ; d, R o u s s a r c o m a cells, k e t h o x a l b i s ( t h i o s e m i e a r b a z o n e ) ; e, s a l m o n cells, n o k e t h o x a l b i s ( t h i o s e m i c a r b a z o n e ) ; f, s a l m o n cells, kethoxal bis(thio semicarbazone).

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Discussion The major point of interest regarding these induced proteins is their function(s). Several possibilities can be entertained: (1) they are enzymes produced to circumvent a blocked metabolic pathway; (2) they are proteins produced when the cell is dying, that is, they may be enzymes with a catabolic function; (3) they may be drug detoxifying enzymes other than those of the microsomal oxidase system; (4) they may be involved in DNA repair (see below). Our preliminary attempts to ascribe some function related to inhibition of DNA synthesis or of mitochondrial respiration were unsuccessful. In addition, they do n o t appear to be microsomal oxidase enzymes or proteins of an induced endogenous virus. The induction of these proteins is closely coordinated. Fig. 1 shows that the kinetics of synthesis of the 100 000, 70 000 and 35 000 dalton proteins is the same with only the 25 000 species retarded at the 2 h point. They appeared to be similar, if not identical, in a variety of species with the exception of salmon cells in which only the 70 000 dalton protein was observed under these experimental conditions and human cells in which a 30 000 dalton species was seen and the 35 000 and 25 000 dalton were not. However, there is one aspect in which these proteins do differ e.g., their response to removal of the drug. Fig. 3 shows that the synthesis of the 25 000 protein does n o t decrease 10 h after deinduction in contrast to the other three species. There are several possibilities regarding the mechanism of induction. Since both actinomycin and cycloheximide inhibit induction, it appear that de novo RNA and protein synthesis are required. One possibility is that inducer binds to the repressor of an operon which is derepressed as a consequence. The second possibility is that drug • copper complexes bind to DNA and these proteins are induced as a result. This hypothesis is based on our observation that drugcopper complexes b u t not drug alone bind to DNA and RNA in vitro [13]. Another possible mechanism is that the inducer inhibits an enzyme which results in a decreased amount of product thereby stimulating the synthesis of more enzyme. Since keth'oxal bis(thiosemicarbazone) is a copper-binding ligand, it is possible that this activity may play a role in induction. Our studies of other copperchelating agents indicate that no consistent correlation can be made since antabuse (disulfiram) induces the same four proteins in chick cells b u t 8-hydroxyquinoline and isonicotinic acid hydrazide do not. It was suggested above that these proteins may be synthesized in response to toxicity caused by the inducer. It is clear from the gel analysis presented here that overall protein synthesis is normal. In addition, visual observation under 30X and 500X phase microscopy shows no alteration of the cells. We have explored possibility of toxicity further, by determining the effect of the drug on the incorporation of [3H]thymidine, [3H]uridine and 3H-labelled amino acids into acid-precipitable materials. We found that cells exposed to 80 ng/ml drug for 4 h incorporated 75, 28 and 24% less thymidine, uridine, and amino acids, respectively, than did untreated cells [5]. It is possible, therefore, that the effect of drug on DNA synthesis may be related to the protein induction. Our attempt to mimic the induction with other inhibitors of DNA synthesis failed.

412 Since one effect of the drug in mouse cells is inhibition of thymidylate synthetase [4], it is interesting that another inhibitor of that enzyme, flurodeoxyuridine, does n o t include these proteins. However, the drug is known to inhibit other enzymes involved in DNA synthesis such as 5,10-methylene-tetrahydrofolate dehydrogenase and dihydrofolate reductase. It is possible that this pathway may be involved. The enzymes d e o x y t h y m i d i n e mono- and diphosphate kinase and DNA nucleotidyltransferase were n o t inhibited. In regard to inhibition of DNA synthesis, Gudas and Pardee [14] have shown that a 40 000 dalton protein (protein X) is induced in Escherichia coli by a variety of inhibitors of DNA synthesis. Protein X has been related to DNA repair since recA- mutants do not produce protein X and are deficient in postreplication DNA repair and recombination [ 15]. In view of these observations, we speculate that drug or drug • copper complexes may damage DNA and the induced proteins may be part of the DNA repair process.

Acknowledgements This work was supported by Public Health Service grant CA 12705 from the National Cancer Institute, American Cancer Society grant VC-70, and Public Health Service contract N.I.H. NO1 CP 33293 within the Virus Cancer Program of the National Cancer Institute. H.O. was supported by a Leukamia Society Postdoctoral fellowship. References 1 Van Giessen, G. and Petering, H. (1968) J. Med. Chem. 11,695---699 2 Petering, H. and Van Giessen, G. (1966) The Biology of Copper (Peisach, J., ed.), pp. 197--209, Academic Press, New York 3 Crim, J. and Petering, H. (1967) Cancer Res. 27, 1 2 7 8 - - 1 2 8 5 4 Booth, B., Johns, D., Bertino, J. and SartoreUi, A. (1968) Nature 217, 250--251 5 Levinson, W., Opperman, H. and Jackson, J. (1977) J. Gen. Virol. 37, 183--190 6 Chan-Stier, C., Minkel, D. and Petering, D. (1976) Bioinorganic Chem. 6, 203--217 7 Levinson, W., Faras, A., Woodson, B., Jackson, J. and Bishop, J.M. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 164--168 8 Kaska, W., Carrano, C., Michalowski, J., Jackson, J. and Levinson, W. (1977) Bioinorganie Chem., in t h e press 9 Levinson, W. (1967) Virology 32, 47--83 10 Studier, F. (1973) J. Mol. Biol. 79, 237--248 11 Laemmli, V. (1970) Nature 227, 6 8 0 - - 6 8 5 12 Weihel, F., Selkirk, J., Gelboin, H., Haugen, D., V a n D e r Hoeven, T. and Coon, M. (1975) Proc. Natl. A c a d . Sci. U.S. 72, 3 9 1 7 - - 3 9 2 0 13 Mikelens, P., Woodson, B. and Levinson, W. (1976) Biochem. Pharmacol. 25, 821--827 14 Gudas, L. and Pardee, A. (1975) I>roc. Natl. Acad. Sci. U.S. 72, 2330--2334 15 Gudas, L. (1976) J. Mol. Biol. 104, 567--587