Translational inhibition in extracts from serum-deprived animal cells

Biochimica et Biophysica Acta, 325 (1973) 545-553

~) Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands BBA 97839

T R A N S L A T I O N A L I N H I B I T I O N IN EXTRACTS F R O M S E R U M - D E P R I V E D A N I M A L CELLS

JOHN A. HASSELL* and DEAN LEE ENGELHARDT* Microbiology Section, The University of Connecticut, Storrs, Conn. 06268 (U.S.A.)

(Received June 12th, 1973)

SUMMARY When Vero M 3 cells, a line of African green monkey kidney cells, are deprived of serum during exponential growth they display a decreased net rate of intracellular protein synthesis. Cytoplasmic extracts prepared from these serum-starved cells have a diminished capacity to promote protein synthesis mediated by endogenous m R N A when compared to their non-deprived counterparts. This diminished translational capacity is not due to a limitation in m R N A since the amount of polyribosomes remains constant during the time of serum starvation used in this work. These same extracts also have a reduced capacity to promote polyphenylalanine synthesis mediated by exogenously added polyuridylate. By supplementing extracts with a postribosomal supernatant fraction from HeLa cells polyphenylalanine synthesis mediated by polyuridylate can be stimulated ten-fold for extracts prepared from serum-deprived cells, but only two-fold for control extracts. These observations can be explained by postulating that serum deprivation alters the protein synthetic machinery of the cell by producing either an inhibitor or deficiency in some component required for translation of mRNAs.

INTRODUCTION On removing serum from the media of animal cells in culture an ensemble of events ensues which has been termed the negative pleiotypic response. The response is said to be negative when growth promoting agents are withdrawn and positive when these agents are returned to the culture media. The negative response is characterized by a coordinate decrease in the overall rate of RNA and protein synthesis, the rate of uptake of nucleic acid precursors and glucose, and an increase in the rate of protein degradation. Cell lines which have been virally transformed have a decreased capacity for the pleiotypic response, suggesting a causal relationship between these two events 1. Our study was undertaken to determine if the decreased intracellular rate of protein * Present address: Columbia University, College of Physicians and Surgeons, Department of Microbiology, New York, N.Y. 10032, U.S.A.

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synthesis elicited by serum deprivation is accompanied by a corresponding decreased capacity for protein synthesis of cytoplasmic extracts of these cells. The results indicate that serum deprivation of Vero cells leads to a clear depression in the protein synthesizing activity of cytoplasmic extracts and suggest two possible mechanisms by which this translational control is effected. MATERIALS AND METHODS

Sources of materials The sources of materials required for cell maintenance, in vitro and in vivo protein synthesis, and RNA and protein isolations were indicated previously 2. Cell culture For the experiments reported here a line of African green monkey kidney cells (Vero) was utilized. The cells were cloned in this laboratory, and the clone used for this work is designated Vero M a. A stock of low-density Vero M a cells were maintained by weekly transfer of 107 cells to a roller bottle (840 cm 2 Bellco Corp.). The cells were grown in Dulbecco's modified Eagle Medium (DME medium) a supplemented with calf serum to l0 % (v/v). A stock of Vero M a cells was frozen, and periodically frozen cells were thawed and used as stock for subsequent experiments to insure uniformity. (a) Preparation of extracts. Large quantities of Vero M3 cells to be used in making cytoplasmic extracts were grown in roller bottles. The cells were seeded at 1 • 107 per roller bottle. Cells were deprived of serum while growing exponentially (2-107--3 • 107 cells per roller bottle). It is essential to deprive these cells of serum while growing logarithmically, since the protein synthetic capacity of extracts is directly related to the growth rate of the cells from which they are obtained. Serum deprivation of non-growing cells resulted in a small decrease (70 ~ as active as controis) in the protein synthetic capacity of extracts prepared from these cells. However, when growing cells were deprived of serum they yielded extracts which were generally 30 ~o as active in protein synthesis compared to non-serum-deprived controls (Hassell, J. A., unpublished). To serum deprive cultures the old media was poured off, and the cell layer rinsed twice with 50 ml vol. of prewarmed D M E medium (37 °C). 250 ml of D M E medium was added to half the cultures (serum-deprived cells) while the other half received 250 ml of DME medium supplemented with calf serum to 10 % (v/v) (Controls). Cytoplasmic extracts were prepared according to the method of Marcus and Salb 4 at varying times after serum deprivation. This method was as follows. The cells were trypsinized from the roller bottle, resuspended in phosphate-buffered saline and pelleted. The cells were then washed in phosphate-buffered saline, pelleted, and resuspended in 0.01 M KC1--0.01 M MgCI2--0.01 M Tris, pH 7.4 (KCI--MgCI2-Tris buffer) at a concentration of 5.107 cells/ml and allowed to swell for 10 min. The cells were then broken with thirteen strokes of a stainless steel fight-fitting (0,015 inch) Dounce homogenizer. The homogenate was centrifuged at 1500 rev./min for 10 min at 0 °C. The supernatant, termed cytoplasmic extract was generally assayed for protein synthetic activity immediately following preparation, but could be stored in small (0.2 ml) aliquots at -70 °C for several weeks without loss of activity. Extracts were never frozen and thawed more than once prior to assay.

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(b) Cell growth and in vivo protein synthesis. Cells were grown at 37 °C in a humidified incubator under a 5 % CO 2 atmosphere. The growth of Vero M3 cells was monitored daily as follows. Cells were seeded onto Falcon plastic Petri dishes (19.6 cm 2) and 4 ml of DME supplemented with calf serum to 10 % (v/v) added. Cell number was determined daily in duplicate by removing the cells with T. E. (0.005 ~o trypsin, 0.005 M EDTA in saline D [GIBCO]) and counting an aliquot with a hemocytometer. To determine the in vivo rate of protein synthesis exponentially growing cultures of Vero M 3 cells in 8.1 cm 2 plastic Petri dishes were deprived of serum as previously described. The cultures were incubated in 1 ml of serum-free DME medium and the cells labelled for 60 min periods with 2.5 /~Ci/ml of L-[14C]-leucine (312 Ci/mole) at intervals after serum starvation. At the end of the labelling period the media was aspirated off and the cell layer washed twice with 2-ml vol. of ice-cold phosphate-buffered saline. The cells were then dissolved with I ml of a 0.1% sodium dodecyl sulphate solution, aminoacyl-tRNAs were saponified with NaOH (final concentration 1 M), the sample precipitated with trichloroacetic acid at a final concentration of 10 % and the precipitate collected on Whatman glass fibre paper filters (GF/A). The intracellular rate of protein synthesis was calculated per cell and the results expressed as the percentage of a control culture incubated under identical conditions in the presence of DME medium supplemented with dialyzed calf serum (10 v/v final concentration). (c) In vitro assay for protein synthesis. The standard reaction mixture volume for in vitro protein synthesis was 0.1 ml. The reaction mixture contained 0.2 M TES, pH 7.6, (20 °C); 5 mM dilithium ATP; 5 mM phosphoenol pyruvate; 0.01 unit phosphoenol pyruvate kinase; 0.l mM dithiothreitol; 0.5 mM disodium GTP; 50 mM KC1; 8 mM MgC12; 2 mM CaC12; 20 mCi of [14C]phenylalanine (spec. act. 460 Ci/mole), and 1 mg/ml of tRNA. When polyuridylate was added for polyphenylalanine synthesis, it was at a final concentration of 100/~g/ml of reaction mixture. The protein synthesis reaction was incubated for 60 rain at 35 °C. After incubation 0.5 ml of sodium hydroxide (1.0 M) was added to the standard reaction mixture. This mixture was incubated for 10 rain at 35 °C, then precipitated by addition of 3 ml of 10 (w/v) trichloroacetic acid. The precipitate was collected, on Whatman GF/A filters, washed six times with 5 ml vol. of 5 % trichloroacetic acid and counted in a liquid scintillation counter. (d) Preparation of sucrose gradients. The polysome profiles reported in Fig. 5 were obtained by sedimenting cytoplasmic extracts made 1% in sodium deoxycholate through a 12 ml linear 15 to 50 % sucrose gradient in buffer containing 0.01 M Tris, pH 7.6, 0.01 M KCI and 0.0115 M MgC12. The run was performed using an SB283 head in an IEC B50 centrifuge at 25 000 rev./min for 6 h at 4 °C. The elution profile was obtained using an ISCO gradient collector and ultraviolet monitor. (e) Techniques reported elsewhere. Techniques used in this study which are not described in this section have been previously described 2. These techniques include, the growth and maintenance of HeLa suspension cells, the preparation of 30--70 (NH4)2 SO4 fractions of postribosomal supernatants, and the isolation of tRNA. One change has been made in that the 30-- 70 % (NH4)2 SO4 fraction of the postribosomal supernatant is resuspended in buffer containing 0.05 M Tris--HCl, pH 7.6, 0.03 M KC1 and 0.001 M MgCI 2 rather than Buffer A.

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RESULTS

Effect of serum deprival on in vivo protein synthesis Serum starvation of growing cultures of Vero Ma cells results in an immediate decrease in the intracellular rate of protein synthesis (Fig. 1). This rate is depressed to 35 % that of the control rate by 10 h. We were unable to detect any differences in the acid-soluble pool of amino acids between control and serum-deprived cells after a 1-h pulse (Hassell, J. A. unpublished). The acid-soluble pool was determined by subtracting the acid-precipitable from the total cell bound counts. tO0

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Fig. l. T h e effect o f s e r u m deprivation on the intracellular rate o f protein synthesis. To determine the intracellular rate o f protein synthesis, the cells were labelled for 1 h with 2.5/~Ci/ml o f t-[14C]leucine (312 Ci/mole) at intervals after s e r u m deprivation as described in Materials a n d M e t h o d s . T h e results are expressed as the percentage o f a n o n - s e r u m - d e p r i v e d control culture. Every determination was carried o u t in triplicate.

There is an apparent decrease in the rate of cell growth when Vero M3 cells are deprived of serum, and cell growth ceases completely three days later (Hassell, J. A., unpublished). The viability of the cells in unaffected by the absence of serum in the culture media. This is indicated by the ability of the cells to exclude trypan blue and to resume growth when supplemented with serum (Hassell, J. A., unpublished). Results similar to these are obtained when primary cultures of chick embryo cells, or mouse 3T3 cells are deprived of serum, but not when chemically or virally transformed cells are serum deprived 1'5'6 (Hassell, J. A., unpublished). Protein synthesis in cytoplasmic extracts Upon removing serum from the media of animal cells in culture a decrease in the overall rate of intracellular protein synthesis takes place. By 10 h a lower steadystate rate of protein synthesis has been established (Fig. 1). To further characterize this step-down phenomenon we have prepared extracts from cells deprived of serum for 20 h and assayed their protein synthetic capacity. Fig. 2A shows that extracts prepared from serum-deprived cells are only 35 % as active in endogenous protein synthesis when compared to non-serum-deprived controls. Fig. 2B shows that the capacity of these same extracts to synthesize polyphenylalanine is also depressed to 35 % of the control extract. This difference in translational capacity for both endogenous m R N A and polyuridylate-mediated translation is observed at all extract concentrations assayed (see Figs 2A and 2B).

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Fig. 2. (A) The capacity of cytoplasmic extracts to synthesize proteins mediated by endogenous mRNA. (B) The capacity of cytoplasmic extracts to synthesize polyphenylalanine mediated by polyuridylate. Extracts prepared from cells which had been deprived of serum for 20 h ( O - O ) . Extracts prepared from control cells ( 0 - 0 ) . Each value is the average of two determinations.

The time-course of the in vitro protein synthesis reaction is shown for endogenous mRNA-mediated translation with control extracts and extracts prepared from serum-deprived ceils in Fig. 3A. The initial rate of endogenous mRNA-mediated pro20

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Fig. 3. (A) The time-courses o f protein synthesis in cytoplasmic extracts mediated by endogenous mRNA. (B) The time-courses of polyphenylalanine synthesis in cytoplasmic extracts mediated by polyuridylate. For this determination a 2.0-ml reaction was initiated, and at the time intervals indicated 0.1 ml was withdrawn and processed as indicated in Materials and Methods. In each case the final extract concentration was 250/~g/ml o f protein. Extracts prepared from control cells ( O - O ) . Extracts prepared from cells which had been deprived o f serum for 20 h ( O - O ) . Each value is the average of two determinations.

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J . A . HASSELL, D. k. ENGELHARDT

tein synthesis in extracts prepared from serum-deprived cells is 30 ~o that of the control Similarly the initial rate of polyuridylate-mediated translation in extracts prepared from serumdeprived cells is 15 % that of the control (Fig. 3B). Hence, our results show that the rate as well as the extent of in vitro translation is affected by depriving Vero M 3 cells of serum for 20 h. To determine if the decrease in the rate of protein synthesis observed in t,ivo correlated in time with that assayed in vitro, we prepared extracts from cells at various times after serum deprivation and assayed their capacity to translate endogenous m R N A and polyuridylate. The results are depicted in Fig. 4. They show that the extracts lose capacity for both endogenous m R N A and polyuridylate-mediated protein synthesis at the same rate as the observed loss of net intracellular protein synthesis (compare Fig. 1 and Fig. 4). A temporal correlation therefore can be shown to exist between the decreased rate of protein synthesis assayed in vivo and in vitro. 1OO, c-

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Fig. 4. The translational activity of cytoplasmic extracts prepared from cells at intervals following serum deprivation. Cytoplasmic extracts were assayed in duplicate at three different concentrations (50, 150 and 250 pg/ml of protein). Incorporation of amino acids into protein was linear over the range of 10-300 #g/ml of extract protein. This is the case for both endogenous m R N A and polyuridylate-mediated translation. The activity of the extracts was calculated per mg of protein and was then expressed as the percentage of a control extract which was prepared from cells treated identically but from which serum had not been removed. Polyuridylate translation ( O - G ) ; endogenous m R N A translation ( O - O ) .

Effects o f serum-deprivation on polysomes Serum starvation of animal cells could cause the disaggregation of polysomes, and this could account for the decreased rate of endogenous mRNA-directed protein synthesis observed in extracts prepared from serum-deprived cells. Therefore we analyzed the ribosomal sedimentation profile of extracts derived from serum-deprived and non-serum-deprived control cells prepared at intervals after serum deprivation using linear 15--50 % sucrose gradients (Figs 5A--5C). These profiles reveal no differences in the distribution of ribosomes at 3, 10 or 20 h after serum deprivation. By summing the total amount of absorbance at 260 nm in the polysome region we conclude that there are as many polysomes per mg of cell extract protein (i.e. per cell) in extracts prepared from serum-starved cells as there are in extracts of control cells, at all time periods examined. Similarly no difference in the amounts of subunits (40 S and 60 S) or monosomes (80 S) can be discerned using this measurement. The maintenance of the polysomes in serum-starved cells, which must be less active, as

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Fig. 5. Sucrose gradient sedimentation analysis of ribosomes prepared from cells at intervals following serum deprivation. Each value is expressed as the percentage of the total absorbance at 260 nm layered onto the gradient. (A) 3 h, (B) l0 h, (C) 20 h after serum deprivation. Ribosomes from control cells (O-O). Ribosomes from cells deprived of serum for the time intervals indicated (O-O).

well as the reduced capacity of extracts prepared from serum-starved cells to translate polyuridylate argues against the contention that a limitation of m R N A is responsible for the observed effects. Therefore, our experiments allow us to conclude that the overall efficiency of the protein synthetic machinery is decreased in cells deprived of serum, and that this is not a consequence of either a limitation in m R N A for translation or due to a decreased number of ribosomes. Hence we postulate a translational control mechanism operative in animal cells deprived of serum.

Partial restoration of extract activity To determine if extracts prepared from serum-deprived cells were limited in factors other than ribosomes necessary for the translation of polyuridylate, we supplemented extracts derived from control and serum-deprived cells with an ammonium sulfate fraction of a post-ribosomal supernatant purified from growing HeLa cells (see Materials and Methods). This fraction will be referred to as crude supernatant factors. The results of such an experiment are shown in Fig. 6. At saturating concentrations of crude supernatant factors polyuridylate-mediated polyphenylalanine synthesis is stimulated two-fold in control extracts, but more than ten-fold in extracts prepared from serum-deprived cells. In the absence of added supernatant factors the experimental extracts were less than 10 % as active as controls, however, at saturating concentrations of crude supernatant factors this activity was increased to 55 % of the control. From this we conclude that an activity is present in the crude supernatant fraction which either replaces an inactive protein synthesis factor or partially negates the effects of an inhibitor of protein synthesis in extracts prepared from serumdeprived cells.

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Fig. 6. Stimulation of polyuridylate-mediated translation by crude supernatant factors. Increasing concentrations of crude supernatant factors were added to a constant concentration (250/~g/ml of extract protein) of extract and the results recorded. Extracts prepared from control cells ( Q - O ) . Extracts prepared from cells which had been deprived of serum for 20 h ( O - O ) . Each value is the average of two determinations. DISCUSSION

Extracts prepared from cells deprived of serum have a decreased translational activity when compared to their non-serum-deprived counterparts. This is true both for polyphenylalanine synthesis as well as for endogenous protein synthesis. Our data indicates however that neither the total amount of polysomes or the number of ribosomes available for translational activities changes significantly when cells are deprived of serum for 20 h or less. Thus the serum-deprived induced loss in translational activity is the result of an impairment in the efficiency of translation. The translational impairment must be brought about either through the elimination of a factor required for translation of polyuridylate or by the appearance of an inhibitor, or both after serum starvation. This is indicated since crude supernatant factors when added exogenously will partially restore polyphenylalanine synthetic capacity to extracts prepared from serum-starved cells. Experiments to determine the mechanism by which protein synthesis is controlled in serum-deprived cells are currently underway. Elongation factor I, the factor which mediates the binding of aminoacyl-tRNA to the template--ribosome complex, has been postulated to have a regulatory function 7,s. Specifically recent reports indicate that a correlation can be shown to exist between the extent of cell proliferation in a particular tissue, and the activity of EF I (refs 9--13). In each system examined an increase in the activity of EF I has been

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shown to occur at the onset o f cellular proliferation. W e have shown t h a t s e r u m depr i v a t i o n o f g r o w i n g V e r o M 3 cells slows d o w n the rate o f cell division, a n d extracts p r e p a r e d f r o m such cells have r e d u c e d activity for p r o t e i n synthesis, W e are investigating the possibility t h a t s e r u m s t a r v a t i o n o f Vero M 3 cells causes a decrease in the activity o f E F I. ACKNOWLEDGEMENTS This investigation was s u p p o r t e d by grants f r o m the N a t i o n a l Science F o u n d a tion (GB-37863) a n d the D a m o n R u n y o n M e m o r i a l F u n d for C a n c e r Research, Inc. ( D R G - 1 2 1 2 ) . J o h n A. Hassell is an N . I . H . p r e d o c t o r a l fellow ( G , M . 00317). REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13

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