Heat shock, protein synthesis and ribosomal protein S6 phosphorylation in vitro in Yoshida AH 130 ascites hepatoma cells

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S6 SYNTHESIS RIBOSOMAL PROTEIN AND HEAT SHOCK, PROTEIN PHOSPHORYLATION IN VITRO IN YOSHIDA AH 130 ASCITES HEPATOMA CELLS. R. Comolli, M. Frigerio and P. Alberti Sezione Dipartimento di Fisiologia e Biochimica Generali, Patologia Generale, Universith di Milano, and Centro sullo Studio della Patologia Cellulare de1 CNR, I-20133 Milano, Italy.

di

ABSTRACT An activated 40s ribosomal protein S6 kinase has been demonstrated as previously in cytosolic extracts froT proliferating as well resting cells of a very undifferentiated rat ascites hepatoma cell line (Yoshida AH 130), grown in vivo (Cell Biol. Int. Rep., 1986, In the present report we present evidence of 10, 821-831). unmodified activity of this kinase and S6 phosphorylation in vitro in cells submitted to a physiological stress such as a szlethal elevation (heat shock: 42 OC for 2 h). The heat temperature decline in the number of active treatment causes a3frogressive ribosomes and of L- S methionine incorporation into total protein, of cellular proteins suggesting drastically decreased synthesis Cells recovering from heat shock show the under these conditions. induced synthesis of a protein with an apparent Mr of 50 kDa. Spontaneous high expression of heat shock proteins (HSP 70, 89, loo), without heat shock, occurs in these tumor cells. INTRODUCTION The S6 phosphoprotein is a major phosphorylated protein in mammalian ribosomes that appears to have a regulatory role in the initiation of protein synthesis (Traugh and Pendergast, 1986). The by a specific cytosolic S6 kinase phosphorylation of this protein (Novak-Hofer and Thomas, 1984; Tabarini et al., 1987; Mizuta et al. 1987) is temporally associated with the increase of the protein synthetic rate in cells induced to grow by serum, hormones or whereas low or lacking phosphorylation is observed growth factors, in resting cells. Activation of S6 kinase and elevated levels of phosphorylated S6 protein could characterize the uncontrolled growth of neoplastic as shown in cells transformed by tumor viruses (Blenis and cells, hepatoma (Comolli et Erikson, 1984), in the Yoshida AH 130 ascites al., 1986) and in 4-dimethylaminoazobenzene-induced liver tumor tissue (Comolli et al., 1988). In the present report we have investigated the eventual effects of a sublethal heat treatment (heat shock: 42 OC for 2h) on S6 kinase and S6 phosphoprotein phosphatase activities in cytosolic extracts from proliferating as well as stationary phase Yoshida ascites tumor cells. Actually, exposure of eukaryotic cells to a heat stress induces significant changes in the phosphorylation levels of specific translational components such as the ribosomal protein S6 (Scharf and Nover, 1982; Glover, 1982; Kennedy et al., 1984). Rapid synthesis of a particular set of cellular proteins, the heat-shock proteins (HSP) and a concomitant inhibition in the synthesis of 0309-1651/66/100907-11/$03.00/o

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other proteins occur under these conditions (Subjeck and Shyy, 1986; Burdon, 1986). The S6 phosphorylation has been measured in vitro according to the procedure of Novak-Hofer and Thomas (1984)Tandthe effects of heat shock on protein synthesis have been determined. The results demonstrate that the in vitro phosphorylation of ribosomal protein S6 by the specific cytosolic kinase is not significantly modified by heat stress in Yoshida ascites hepatoma in spite of significant reductions in active ribosomes and cells, in protein synthesis under these conditions. Spontaneous high expression of heat shock proteins (HSP 70, 89, loo), without heat Cells recovering from heat shock showed the shock, was observed. synthesis of a protein with an apparent Mr of 50 kDa.

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MATERIALS AND METHODS hapatoma cells (AH CELL GROWTHAND HARVESTING. Yoshida ascites 130) were propagated by injection (6.0-7.0 x 10' cells) into the of female albino rats of the Wistar peritoneal cavity strain maintained in a light-controlled room (lights on from 7:00-&9:00) and stationary phase at a temperature of 23 + 1 OC. Proliferating ascites cells were harvested on day 4 and 12 after inoculation Cells were washed to remove the ascitic (Comolli et al., 1986). fluid and contaminating erythrocytes with a solution (Medium A) containing 147mM NaCl, 6mM KCl, 1mM MgS04 and 25mM Na2HP04-NaH PO4 buffer, pH 7.4. The cells were then counted and their viabi 3. ity (Comolli et al., 1984). determined PREPARATION OF CELL EXTRACTS (S6 KINASE) IN CONTROL AND HEAT SHOCKED CELLS. Proliferating and stationary phase cells, purified as described, were suspended in ice-cold extraction buffer (80mM beta-glycerophosphate buffer, pH 7.3, 20mM EGTA *, 15mM magnesium acetate), washed twice in the cold and homogenized in a Dounce homogenizer. The homogenate was centrifuged at 100,000 x g at 2OC for 120 min and the supernatant was immediately frozegat -80 'C. cells were To examine the effects of heat shock, 5.0 x 10 preincubated in Medium A at 37 OC or 42 OC for 2h. At the end of the incubation period cells were chilled, centrifuged, washed twice with the extraction buffer and homogenized as reported above. PREPARATION OF 40s RIBOSOMAL SUBUNITS. These were prepared from the liver of fasted rats as previously described (Comolli et al., 1986). 40s ribosomes were stored frozen at -80 OC. * Abbreviations: 1,2-di(2-aminoethossi)-ethan-N,N,I~',N'-tetra-acetic EGTA, MOPS, 3-(N-morpholino)propane sulphonic acid; SDS, sodium dodecyl sulphate; beta-MSH, beta-mercaptoethanol; Tris, 2-amino-2(hydroxymethyl)propane-1,3-dial; TCA, trichloroacetic acid; TEA, triethanolamine; PMSF, phenylmethane sulphonyl fluoride.

acid;

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S6 PHOSPHORYLATION IN VITRO: ASSAY FOR PROTEIN KINASE ACTIVITY. This was performed according to the procedure of Novak-Hofer and Thomas (1984). The phosphorylation reaction was carried out for 30 min at 30 OC using a reaction mixture containing: 50mM MOPS pH 7.0, 1OmM msnesium acetate, 1mM Dithiothreitol, 60uM ATP, 2.5 uCi of 5000 Ci/mof) and 1.5 A gamma- P ATP (Amersham, sp. act. units of 40s ribosomes in a total volume of 100 ul. The reac'!n z was initiated by the addition of cell extracts containing 60 ug of protein and stopped by adding 10 ul of 20% SDS-50% beta-MSH. The and analysed by samples were boiled for 2 min SDS-gel electrophoresis. DETERMINATION OF PHOSPHATASE ACTIVITY. The assay was performed according to the procedure of Novak-Hofer and Thomas (1984). S6 PHOSPHORYLATION: SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS. Electrophoresis was carried out for 6.5 h at 150 V as described (Laemmli, 1970). The polyacrylamide gel consisted of a separating gel of 15% acrylamide, 0.087% bisacrylamide, 0.38M Tris-HCl pH 8.7, 0.1% SDS and a stacking gel consisting of 5% acrylamide, 0.3% bisacrylamide, 0.125M Tris-HCl pH 6.8, 0.1% SDS. The running buffer was 0.025M Tris-HCl pH 8.3, 0.19M glycine, 0.1% SDS. Gels were stained with 0.1% Coomassie brilliant blue G250 in 20% ethanol-7% acetic acid, destained in the same mixture and exposed to Kodak XAR-5 films at -80 "C using intensifying screens. The protein band was cut out of ma1 protein S6 (Mr = 31,500) "t~sre,s,p~~~in,s,Itoa~~o~~P incorporated was quantitated by liquid scintillation spectroscopy. HEAT SHOCK TREATMENT: DETERMINATION OF ACTIVE RIBOSOMES. The proportion of ribosomes active in protein synthesis was determined according to Martin (1973) and Storb and Martin (1972). 3.0 x 10 ascites cells were incubated for 10, 30, 60 and 120 min at 42 OC in Medium A. Then cells were lysed by homogenization in 25mM Tris-HCl buffer pH 7.6, 40mM KCl, 7.5mM magnesium acetate and 1OmM beta-MSH containing Triton sodium x100 and deoxycholate, 1% final concentration. After centrifugation at 10,000 x g for 5 min the supernatant was diluted to 50-60 A ~ougyy/wmas ~;~eJanA'f',",","ig ribonuclease (Boehringer Biochemia), min at O-4 OC, 0.25 vol. of 2.5M KC1 were' then added and the samples immediately layered onto 5-30% linear sucrose density gradients, containing 800mM KCl, 15mM magnesium acetate, 50mM Tris-HCl buffer, pH 7.6 and centrifuged at 27,000 rpm in a Spinco SW 27 rotor for 150 min at 4 OC. The gradients were monitored at 260 nm and the absorbance profiles, recorded and quantitated directly by weighing cutouts of the peaks. HEAT SHOCK TREAT NT: DETERMINATION OF THE RATE OF PROTEIN Y SYNTHESIS. cells suspended in Medium A were incubated with 50 uCi 5'03$-~~thionine L(Amersham ; sp. act. 1000 Ci/mmol) for 5, 10, 20, 30, 60, 90 and 120 min. at 37 OC or 42 OC, directly chilled in ice and separated from the Medium by centrifugation (100 x g for 10 min). The collected cells were washed twice and suspended in lml of Lysis buffer (10mM TEA-HCl pH 7.4; 1OmM NaCl; 1mM MgCl 2; 5% beta-MSH and 1mM PMSF) (Dean and Atkinson, 1985).

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Each sample was sonicated for 30 sec. Cell lysates were clarified by35centrifugation (8,000 x g for 5 min) and the amount of L- S-Methionine incorporated into 10% TCA-precipitable material was determined in a Packard Tri Carb A300 spectrometer using a toluene based scintillator. HEAT SHOCK TREATMENT: DETERMINATION OF HSP SYNTHESIS. 5.0 x lo7 cells suspended in Medium A were incubated at 37 OC or 42 OC for lh or 2h. Following this itial incubation cells were labeled for 2h with 100 uCi of L- @S - Methionine at 37 OC except for cells preincubated for lh or 2h at 42 OC that received 300 uCi of radioactive material. At the end of the last incubation period cells were chilled in ice and total protein was extracted as reported above (Dean and Atkinson, 1985). Cell lysates were used directly for SDS-gel electrophoresis. HEAT SHOCK TREATMENT: SDS POLYACRYLAMIDE GEL ELECTROPHORESIS. Sodium dodecyl sulphate gel electrophoresis was performed for 7h at 35 mA according to the method of Laemmli (1970). The separating gel contained 1.5M Tris-HCl pH 8.8, 10% acrylamide, 0.2% bisacrylamide, 10% SDS. The stacking gel consisted of 0.5M Tris-HCl pH 6.8, 10% acrylamide, 0.2% bisacrylamide, 10% SDS and the running buffer contained 0.05M Tris-HCl, 0.38M glycine, 0.1% SDS. Following electrophoresis the gels were stained with 0.2% Coomassie brilliant blue G250 in 20% ethanol-7% acetic acid, and then destained3in the (FN HANCE, same mixture. The gels were processed for fluorography NEN) dried under vacuum and exposed to Kodak XAR-5 film at -80 OC for one month. Films were quantified using a Ultroscan laser densitometer (LKB, mod.2202). PROTEIN ESTIMATION. Protein was estimated by the method of Lowry at al. (1951) RESULTS AND DISCUSSION S6 KINA~ACTIVITY FROM ASCITES CELL EXTRACTS AND EFFECTS OF HEAT extracts from proliferating and stationary SHOCK. The cytosolic phase ascites cells preincubated for 2h at 37“C or 42OC, tested for S6 kinase activity (Table 1 and Figure l), were capable to ribosomal protein S6 to the same extent. ;;-&;fYl;;; gosft$j P incorporated into S6 was only slightly modified'by heat shock and the difference was statistically not Table 2 a slight increase of significant. As reported in phosphatase activity was observed under these conditions. HEAT SHOCK TREATMENT: DETERMINATION OF ACTIVE RIBOSOMES AND OF of the proportion of ribosomes PROTEIN SYNTHESIS. The analysis active in protein synthesis, as studied by measuring the ribosome dissociation into subunits at high ionic strength after RNAse reported in Figure 2, showed rapid decline in active treatment, shock in proliferating cells, due to ribosomes during heat inhibition of protein synthesis initiation. Cells recovering from 1 h heat shock demonstrated an increase of ribosomes active in protein synthesis.

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activity -Table -1: S6 kinase ascites cells incubated at from 7 separate experiments.

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in cytosolic extracts from Yoshida 37 OC and 42 OC for 2 h. Results are

P incorporated into 37 oc

cells

S6 (cpm + S.E.) 42 'T

1309+112

1149+ 55

1030+ 71 Stationary phase __-----_-------~_------~-------~---------~------~~-------~~~~~--~-~

946+ 71

ar-

A

B

c

D

E

-F

i3

H

of S6 (arrow) phosphorylating activity of Figure 1: Autoradiogram extracts from proliferating and stationary phase Yoshida cytosolic at 37 or 42OC for 2 h. ascites CErlls incubated from proliferating cells (37V); Lane A: extract from proliferating cells (37OC); Lane B: 40s ribosomes+extract Lane C: extract from proliferating cells (42OC); Lane D: 40s ribosomes+extract from proliferating cells (42OC); from stationary phase cells (37V); Lane E: extract from stationary phase cells (37V); Lane F: 40s ribosomes+extract Lane G: extract from stationary phase cells (42*C); Lane H: 405 ribosomes+extract from stationary phase cells (42OC). Each lane was loaded with equal numbers of cell equivalents.

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Table 2: S6 Phosphatase -ascites cells incubated

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activity in cytosolic at 37OC and 42X for

extracts 2 h.

from

1988

Yoshida

--___---_____------_----------------------------------------------32

liver 40s ribosomes (cpm 2 S.E.) 37 oc 42 "C _____-________---___----------------------------------------------Proliferating

P retained

cells

in labeled

178+14.3 -

163+7.8

Stationary phase 173+ 6.5 ---_-------__------------------------------------------------------

163+8.4

active ribosomes in Figure 2: Per cent incubated at 42V for different times. direction of sedimentation. A: cells not incubated B: cells incubated for 1Omin C: cells incubated for 30min D: cells incubated for 120min E: cells incubated for 60min+120min

Yoshida ascites cells The arrow indicates = = = = at 37OC =

58%; 41%; 35%; 15%; 35%.

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35 Accordingly, the incorporation of L- S methionine into total cell protein was significantly reduced (-70%) by 1 h heat shock in both proliferating and stationary phase cells ( FiguL'_e35;) . me:;y;;--L during the first 5-10 min incubation the incorporation into total protein was less dramatically affected by heat stress in stationary phase cells, compared to proliferating cells, due to their lower efficiency in amino acid incorporation under basal conditions. This could be due to several (-80%) factors: the decrease in the initiation factor(s) activity in stationary phase cells (Comolli and Bardella, 1981, 1982), the decrease of the intracellular pH (Cqmolli et al., 1984) and of the Na+/H+ exchange (Comolli et al., 1985) under these conditions, possibly affecting the Na+-dependent System A transport of the amino acid (Inui and Christensen, 1966). In stationary phase cells, where protein degradation could exceed synthesis, the less efficient Na+-independent System L transport of methionine could possibly be utilized preferentially.

wo-

400.. ~

____

-------a

_-’

Figure 3: Incorporation of L35S-Methionine in proliferating and stationary phase Yoshida ascites cells incubated at 37OC and 42OC. 0 = proliferating cells incubated at 37OC; () = proliferating cells incubated at 42OC; a= stationary phase cells incubated at 37OC; n = stationary phase cells incubated at 42OC.

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35 Figure 4: SDS-polyacrylamide gel patterns of S-Methionine-labeled total polypeptides. Lane A: cells incubated for 1 h at 37 OC; Lane B: cells incubated for 2 h at 37 OC; Lane C: cells incubated for 1 h at 42 OC; Lane D: cells incubated for 2 h at 42 OC. The arrow indicates the 50 kDa protein. Each lane was loaded with equal numbers of cell equivalents. HEAT SHOCK TREATMENT: HSP SYNTHESIS. The synthesis of heat shock proteins in the Yoshida ascites tumor was examined in proliferating and stationary phase cells exposed to he a.& (42 OC) for 1 or 2 h and Comparable, then labeled at 37 OC for 2 h with L- S methionine. results were obtained in both conditions. As shown in Figure 4, no significant differences between cells exposed to normal or elevated temperature were detectable as far as the synthesis of the major heat shock proteins (HSP 70, 89 and 100) is concerned, suggesting higher basal HSP levels in these tumor cells. Similar results have been obtained with mouse embryonal carcinoma cells (Bensaude and Morange, 1983) and SV40-transformed mouse embryo cells (Omar and Lanks, 19841, where spontaneous high expression of heat shock proteins has been observed. The induction by hepatocarcinogens and during liver regeneration of heat shock gene expression has also

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described (Carr et al., 1986). most notable difference in the shock treatment was the increased of approximately 50 kDa. (Figure

tracing Figure 5: Densitometric 4: a = 37OC; b = 42OC; A = actin.

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pattern of translation expression of a protein 4, arrow and Figure 5).

autoradiograph

shown

in

after with

figure

The present experiments suggest that in cells characterized by a rather uncontrolled growth such as the Yoshida AH 130 rat ascites hepatoma there is no close correlation between the rate of protein as determined by measurs synthesis, the proportion of active ribosornes and the incorporation of L- S methionine into total cell and the levels of ribosomal protein S6 phosphorylation in protein, vitro by the specific cytosolic kinase. This agrees well with results reported previously in the same tumor (Comolli et al., 1986). Yoshida ascites hepatoma cells submitted to heat stress show an increased expression of a protein (Mr 50 kDa) that appears unrelated to the major classes of the heat shock proteins of eukaryotic 'This protein appears possibly similar cells. to a protein (Mr 48-50 kDa) induced in chick embryo fibroblasts by a hyperosmolar stress (Petronini et al., 1987). The synthesis of a particular set of heat shock proteins of low abundance or nonexistent in unstressed cells, that do not correspond to the "classic" heat shock proteins, has been described in mammalian cells (Reiter and Penman, 1983; Maytin et al., 1985). It is worth noting that the ffSP 70, 89 and 100 are constitutively synthetized and highly expressed in this very undifferentiated tumor cell line.

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Acknowledgement:

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REFERENCES. BENSAUDE 0. and MORANGEM. (1983). Spontaneous high expression of heat-shock proteins in mouse embryonal carcinoma cells and ectoderm from day 8 mouse embryo. The EMBO Journal, 2, 173-177. BLENIS J. and ERIKSON R.L. (1984). Phosphorylation of the ribosomal protein S6 is elevated in cells transformed by a variety of tumor viruses. Journal of Virology, 50, 966-969. BURDON, R.H. (1986). Heat Shock and the heat shock proteins. Biochemical Journal, 240, 313-324. Induction CARR B.I., HUANG T.H., BUZIN C.H. and ITAKURA K. (1986). expression without heat of heat shock gene shock by hepatocarcinogens and during hepatic regeneration in rat liver. Cancer Research, 46, 5106-5111. COMOLLI R. and BARDELLA L. (1981). Correlation between growth rate, activity of the ribosomal dissociation factor and proportion of in protein synthesis in Yoshida ascites ribosomes active hepatoma AH-130 cells grown in vivo. Cancer Biochemistry and Biophysics, 5, 239-246. COMOLLI R.,and BARDELLA L. (1982). eIF-2 initiation factor activity cells hepatoma AH 130 and in in Yoshida ascites 4-dimethylaminoazobenzene-induced liver tumor tissue during growth. Cancer Biochemistry and Biophysics, 6, 119-124. Amiloride and glucose COMOLLI R., CASALE A. and MARIOTTI 1). (1984). effects on the intracellular pH of Yoshida rat ascites hepatoma AH-130 cells grown in vivo. Cell Biology International Reports, 8, 297-307. Amiloride COMOLLI R., ZANONI L., MAURI C. and LEONARD1 M.G. (1985). inhibits protein synthesis and lowers the intracellular pH in exponential growing Yoshida rat ascites hepatoma (AH 130) for a role of the Na+/H+ exchanger. Cell cells : evidence Biology International Reports, 9, 1017-1025. COMOLLI R., LEONARD1 M.G., ALBERT1 P. and FRIGERIO M. (1986). ribosomal protein S6 phosphorylation in Protein synthesis, SDS gel electrophoresis vitro and the effects of amiloride: studies in the Yoshida ascites tumor (AH 130) grown in vivo. Cell Biology International Reports, 10, 821-831. of a COMOLLI R., ALBERT1 P. and FRIGERIO M. (1988). Activation S6 during protein kinase the ribosomal hepatocarcinogenesis. 4-dimethylaminoazobenzene-induced rat Cancer Letters, in press. DEAN R.L. and ATKINSON B.G. (1985). Synthesis of heat shock following brief, blood cells proteins in quail red physiologically relevant increases in whole body temperature. Comparative Biochemistry and Physiology, 81B, 185-191. GLOVER C.V.C. (1982). Heat shock induces rapid dephosphorylation of Proceedings of the National a ribosomal protein in Drosophila. Academy of Sciences USA, 79, 1781-1785. KENNEDY I.M., BURDON R.H. and LEADER D.P. (1984). Heat shock causes the phosphorylation of ribosomal changes in the diverse proteins of mammalian cells. FEBS Letters, 169, 267-273.

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STORB U. and MARTIN T.E. (1972). Number and activity of free and membrane-bound spleen ribosomes during the course of the immune response. Biochimica Biophysics Acta, 281, 406-409. SUBJECK J.R. and SHYY T.T. (1986). Stress protein systems of mammalian cells. American Journal of Physiology, 250, Cl-C17. TABARINI D., GARCIA de HERREROSA., HEINRICH J. and ROSEN O.M. (1987). Purification of a bovine liver S6 kinase. Biochemical and Biophysical Research Communications, 144, 891-899. TRAUGH J.A. and PENDERGAST A.M. (1986). Regulation of protein synthesis by phosphorylation of ribosomal protein S6 and aminoacyl-tRNA synthetases. Progress in Nucleic Acid Research and Molecular Biology, 33, 195-230. Received

16.11.88

Accepted

23.8.88