Effects of serum deprivation, insulin and dexamethasone on polysome percentages in C2C12 myoblasts and differentiating myoblasts.

An increase in the rate of protein synthesis in living cells can be achieved by regulating the quantity of mRNA, ribosomes, and enzymes available for translation or by regulating the efficiency at which existing components are used. Efficiency can be measured by comparing the number of ribosomes actively engaged in the synthesis of protein (polysomes) to the pool of free ribosomes. The objective of this study was to determine the percentage of ribosomes found as polysomes in C2C12 cells deprived of serum or exposed to insulin or dexamethasone 24 h before and after being stimulated to differentiate. Individual 60 mm culture dishes were exposed to serum-free control medium, medium containing serum, insulin, or dexamethasone for a period of 1 h or 2 h and then quickly frozen. The ribosomes and polysomes from these cells were separated by ultracentrifugation on 15 to 60% sucrose gradients and the absorbance across the gradient at 254 nm was recorded. Polysome percentages were determined as the area under the polysome peak divided by the total area under the curve. Serum deprivation caused a 12% decline in the percentage of ribosomes found as polysomes (P < 0.01). Dexamethasone caused a quadratic decline (P < 0.05) in polysome percentage, while insulin yielded a quadratic increase (P < 0.05). Protein synthesis assays measuring 3H-tyrosine uptake showed similar responses. These changes occurred in the absence of any differences in total RNA concentration. It was concluded that differentiation and the absence of serum in the media reduced the rate of recruitment of ribosomes for protein synthesis. Insulin increased ribosome recruitment which was also observed by a similar increase in incorporation of radio-labeled tyrosine.     

Identification of the fibroblast growth factor receptor of Swiss 3T3 cells and mouse skeletal muscle myoblasts

Two distinct fibroblast growth factors (FGF) were purified to homogeneity from bovine brain on the basis of their ability to stimulate skeletal muscle myoblast proliferation. These growth factors are also mitogenic for Swiss 3T3 cells and appear to be closely related to or identical with previously isolated anionic and cationic fibroblast growth factors. The half-maximum concentrations (EC50) for stimulation of myoblast DNA synthesis by the anionic and cationic growth factors were 30pM and 1pM, respectively. In contrast, an EC50 of 45 pM was observed for stimulation of 3T3 cell DNA synthesis by both growth factors. Binding of 125I-labeled anionic FGF was saturable with apparent Kd values of 45 pM and 11 pM and approximately 60 000 and 2000 receptor sites per cell for 3T3 cells and MM14 murine myoblasts, respectively. Unlabeled anionic and cationic FGF equally displaced 125I-labeled anionic FGF from 3T3 cells while cationic FGF was more potent than anionic FGF for displacement from skeletal muscle myoblasts, demonstrating that a single receptor binds the two distinct growth factors. Binding was specific for these factors since platelet-derived growth factor, insulin, insulin-like growth factor 1, epidermal growth factor, and nerve growth factor were unable to displace bound 125I-labeled anionic FGF from Swiss 3T3 cells. Chemical cross-linking of specifically bound 125I-labeled anionic FGF to 3T3 cells and MM14 myoblasts identified a single detergent-soluble FGF receptor with an apparent molecular weight of 165 000.     

Fibroblast growth factor inhibits insulin-like growth factor-II (IGF-II) gene expression and increases IGF-I receptor abundance in BC3H-1 muscle cells.

Muscle is an important target tissue for insulin-like growth factor (IGF) action. We have previously reported that muscle cell differentiation is associated with down-regulation of the IGF-I receptor at the level of gene expression that is concomitant with an increase in the expression and secretion of IGF-II. Furthermore, treatment of myoblasts with IGF-II resulted in a similar decrease in IGF-I receptor mRNA abundance, suggesting an autocrine role of IGF-II in IGF-I receptor regulation. To explore further the role of IGF-II in IGF-I receptor regulation, BC3H-1 mouse muscle cells were exposed to differentiation medium in the presence of basic fibroblast growth factor (FGF), a known inhibitor of myogenic differentiation. FGF treatment of cells resulted in a 50% inhibition of IGF-II gene expression compared to that in control myoblasts and markedly inhibited IGF-II secretion. Concomitantly, FGF resulted in a 60-70% increase in IGF-I binding compared to that in control myoblasts. Scatchard analyses and studies of gene expression demonstrated that the increased IGF-I binding induced by FGF reflected parallel increases in IGF-I receptor content and mRNA abundance. These studies indicate that FGF may up-regulate IGF-I receptor expression in muscle cells through inhibition of IGF-II peptide expression and further support the concept of an autocrine role of IGF-II in IGF-I receptor regulation. In addition, these studies suggest that one mechanism by which FGF inhibits muscle cell differentiation is through inhibition of IGF-II expression.     

Insulin-like growth factor-I-coupled mitogenic signaling in primary cultured human skeletal muscle cells and in C2C12 myoblasts. A central role of protein kinase Cdelta

In this study, we have investigated the effects of insulin-like growth factor-I (IGF-I) on cellular responses of primary human skeletal muscle cells and mouse C2C12 myoblasts. In human muscle, IGF-I stimulated proliferation and fusion of the cells and the expression of the differentiation marker desmin. These effects were completely inhibited by Rottlerin, the inhibitor of the protein kinase C (PKC)delta, but were not affected by the inhibition of the mitogen-activated protein kinase (MAPK) or the phosphatidylinositide 3-kinase (PI-3K) pathways. Furthermore, IGF-I initiated the selective translocation of PKCdelta to the nucleus. In C2C12 myoblasts, the growth-promoting effects of IGF-I were abrogated by inhibition of PKCdelta, but not by the inhibition of the PI-3K system. However, in contrast to the human data, the MAPK inhibitor PD098059 partially (yet significantly) also inhibited the action of IGF-I and, furthermore, IGF-I induced phosphorylation of the MAPK Erk-1/2. In addition, overexpression of constitutively active form of PKCdelta in C2C12 cells fully mimicked, whereas overexpression of kinase inactive mutant of the isoform prevented the action of IGF-I. Finally, the inhibition of PKCdelta suspended the IGF-I-induced phosphorylation of Erk-1/2 and, moreover, the inhibition of the MAPK pathway partially (yet significantly) inhibited the accelerated growth of C2C12 cells overexpressing PKCdelta. Taken together, these results demonstrate a novel, central and exclusive involvement of PKCdelta in mediating the action of IGF-I on human skeletal muscle cells, with an additional yet PKCdelta-dependent contribution of the MAPK pathway on C2C12 myoblasts.     

Isolation of myosin-synthesizing polysomes from cultures of embryonic chicken myoblasts before fusion.

Mononucleated myoblasts and multinucleated myotubes were obtained by culturing embryonic chicken skeletal muscle cells. Comparison of total polysomes isolated from these mononucleated and multinucleated cell cultures by density gradient centrifugation and electron microscopy revealed that mononucleated myoblasts contain polysomes similar to those contained by multinucleated myotubes and large enough to synthesize the 200,000-dalton subunit of myosin. When placed in an in vitro protein-synthesizing assay containing [³H]leucine, total polysomes from both mononucleated and multinucleated myogenic cultures were active in synthesizing polypeptides indistinguishable from myosin heavy chains as detected by measurement of radioactivity in slices through the myosin band on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Fractionation of total polysomes on sucrose density gradients showed that myosin-synthesizing polysomes from mononucleated myoblasts may be slightly smaller than myosin-synthesizing polysomes from myotubes. Multinucleated myotubes contain approximately two times more myosin-synthesizing polysomes per unit of DNA than mononucleated myoblasts, and the proportion of total polysomes constituted by myosin polysomes is only 1.2 times higher in multinucleated myotubes than it is in mononucleated myoblasts. The results of this study suggest that mononucleated myoblasts contain significant amounts of myosin messenger RNA before the burst of myosin synthesis that accompanies muscle differentiation and that a portion of this messenger RNA is associated with ribosomes to form polysomes that will actively translate myosin heavy chains in an in vitro protein-synthesizing assay.     

Effects of IGF-I, EGF, and FGF on proteoglycans synthesized by fractionated chondrocytes of rat rib growth plate

The effects of insulin-like growth factor (IGF-I), epidermal growth factor (EGF), fibroblast growth factor (FGF), or 10% newborn calf serum (NCS) on the amount and structure of the proteoglycans synthesized by fractionated chondrocytes from rat growth plate were investigated. Proliferative cells (fraction II) or resting cells (fraction III) synthesized more proteoglycans than hypertrophic cells (fraction I). Addition of IGF-I to the cultures increased proteoglycan synthesis more than addition of EGF or FGF. EGF and FGF induced synthesis of proteoglycans of smaller molecular size with a lower proportion of aggregates. The size of the constituent glycosaminoglycan chains did not differ between control and growth factor-treated cells. The present study demonstrates that proteoglycan structure and synthesis are modified by growth factors to different extents, depending on the maturation stage of the target cells.     

Insulin-like growth factors (IGF) in muscle development. Expression of IGF-I, the IGF-I receptor, and an IGF binding protein during myoblast differentiation

The insulin-like growth factors (IGFs) I and II exert pleiotropic effects on diverse cell types through interaction with specific high affinity cell surface receptors and with locally produced binding proteins. In skeletal muscle and in myoblast cell lines, the functions of IGF-I and -II are complex. Both growth factors appear capable of stimulating cellular proliferation and differentiation, as well as exerting insulin-like effects on intermediary metabolism. We have demonstrated recently that the expression of IGF-II and its receptor is induced during the terminal differentiation of the myoblast cell line, C2, and have suggested that IGF-II may be an autocrine growth factor in these cells (Tollefsen, S.E., Sadow, J.L., and Rotwein, P. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 1543-1547). We now have examined this cell line for expression of other components involved in IGF signaling. The synthesis of IGF-I is low during myoblast proliferation; IGF-I mRNA can be detected only through use of a sensitive solution hybridization assay. Typical IGF-I receptors can be measured in myoblasts, whereas IGF binding proteins cannot be detected in proliferating cells or in conditioned culture medium. During myogenic differentiation, IGF-I mRNA levels increase transiently by 6-10-fold within 48-72 h. The expression of IGF-I mRNA is accompanied by a 2.5-fold accumulation of IGF-I in the culture medium. IGF-I receptors also increase transiently, doubling by 48 h after the onset of differentiation. By contrast, secretion of a Mr 29,000 IGF binding protein is induced 30-fold to 100 ng/ml within 16 h and continues to increase throughout differentiation. These studies demonstrate that several components critical to IGF action are produced in a fusing skeletal muscle cell line in a differentiation-dependent manner and suggest that both IGF-I and IGF-II may be autocrine factors for muscle.     

Role of the p66Shc isoform in insulin-like growth factor I receptor signaling through MEK/Erk and regulation of actin cytoskeleton in rat myoblasts

To investigate the role of Shc in IGF action and signaling in skeletal muscle cells, Shc protein levels were reduced in rat L6 myoblasts by stably overexpressing a Shc cDNA fragment in antisense orientation (L6/Shcas). L6/Shcas myoblasts showed marked reduction of the p66Shc protein isoform and no change in p52Shc or p46Shc proteins compared with control myoblasts transfected with the empty vector (L6/Neo). When compared with control, L6/Shcas myoblasts demonstrated 3-fold increase in Erk-1/2 phosphorylation under basal conditions and blunted Erk-1/2 stimulation by insulin-like growth factor I (IGF-I), in the absence of changes in total Erk-1/2 protein levels. Increased basal Erk-1/2 activation was paralleled by a greater proportion of phosphorylated Erk-1/2 in the nucleus of L6/Shcas myoblasts in the absence of IGF-I stimulation. The reduction of p66Shc in L6/Shcas myoblasts resulted in marked phenotypic abnormalities, such as rounded cell shape and clustering in islets or finger-like structures, and was associated with impaired DNA synthesis in response to IGF-I and lack of terminal differentiation into myotubes. In addition, L6/Shcas myoblasts were characterized by complete disruption of actin filaments and cell cytoskeleton. Treatment of L6/Shcas myoblasts with the MEK inhibitor PD98059 reduced the abnormal increase in Erk-1/2 activation to control levels and restored the actin cytoskeleton, re-establishing the normal cell morphology. Thus, the p66Shc isoform exerts an inhibitory effect on the mitogen-activated protein kinase signaling pathway in rodent myoblasts, which is necessary for maintenance of IGF responsiveness of the MEK/Erk pathway and normal cell phenotype.     

EGF responsiveness and receptor regulation in normal and differentiation-defective mouse myoblasts

The interrelationship between cell proliferation and terminal myogenic differentiation has been analyzed by studying a differentiation-defective subclone (DD-1) of the permanent mouse myoblast line MM14. Parental MM14 myoblasts withdraw irreversibly from the cell cycle and initiate terminal differentiation when they are deprived of certain mitogens. In contrast, DD-1 cells become quiescent in a mitogen-depleted environment and less than 0.4% of the cells differentiate. When refed with mitogen-rich medium quiescent DD-1 cells resume proliferation. Expression of this differentiation-defective phenotype is apparently coupled to an alteration in mitogen sensitivity: MM14 myoblasts require horse serum plus either chick embryo extract or fibroblast growth factor (FGF) to sustain cell growth: DD-1 variants are responsive to FGF, but also proliferate in response to serum alone or to reduced serum plus epidermal growth factor (EGF). Interestingly, EGF also appears to retard DD-1 cell differentiation in a manner similar to the FGF repression of differentiation in normal myoblasts. Normal and differentiation-defective myoblasts which have been maintained under growth-promoting conditions exhibit similar EGF binding, internalization, and degradation. However, whereas the EGF binding capacity of MM14 myoblasts declines to less than 5% of its initial level within 24 hr of FGF removal, DD-1 variants exhibit an increase in EGF binding when switched to an FGF-depleted medium. The relationship of altered EGF receptor regulation to changes in mitogen sensitivity and differentiation capacity of the DD-1 variant is discussed, and implications for general in vivo processes governing cell proliferation and differentiation are considered.     

Effect of fibroblast growth factor on the division and fusion of bovine myoblasts

The effect of fibroblast growth factor (FGF) on the rate of proliferation and fusion of bovine myoblast has been examined. Addition to the cultures of 0.1 mug-1 mug/ml of FGF stimulates the rate of proliferation and delays the fusion of primary cultures of bovine myoblasts cultured in 10% serum. Final cell densities reached in the presence of 0.1 mug/ml of FGF were fivefold higher than in controls; with 1 mug/ml, they were 10-fold higher. Increases in cell density were paralleled by increases in acetylcholine receptor sites as measured by the binding of 125I-alpha-bungarotoxin. Both fusion and the appearance of acetylcholine receptor sites were delayed in the presence of FGF. Growth hormone, insulin and testosterone, which have been reported to be mitogenic for rat and chick embryo myoblasts, did not have significant effects on DNA synthesis in bovine myoblasts when compared to the FGF. Conversely, FGF did not stimulate the proliferation of chick embryo myoblasts, indicating that it is not active in all vertebrate species.     

Sphingosine kinase activity is required for myogenic differentiation of C2C12 myoblasts

Sphingosine kinase (SphK) is a conserved lipid kinase that catalyzes the formation of sphingosine 1-phosphate (S1P), an important lipid mediator, which regulates fundamental biological processes. Here, we provide evidence that SphK is required for the achievement of cell growth arrest as well as myogenic differentiation of C2C12 myoblasts. Indeed, SphK activity, SphK1 protein content and S1P formation were found to be enhanced in myoblasts that became confluent as well as in differentiating cells. Enforced expression of SphK1 reduced the myoblast proliferation rate, enhanced the expression of myogenic differentiation markers and anticipated the onset of differentiated muscle phenotype. Conversely, down-regulation of SphK1 by specific silencing by RNA interference or overexpression of the catalytically inactive SphK1, significantly increased cell growth and delayed the beginning of myogenesis; noticeably, exogenous addition of S1P rescued the biological processes. Importantly, stimulation of myogenesis in SphK1-overexpressing myoblasts was abrogated by treatment with short interfering RNA specific for S1P(2) receptor. This is the first report of the role of endogenous SphK1 in myoblast growth arrest and stimulation of myogenesis through the formation of S1P that acts as morphogenic factor via the engagement of S1P(2).     

Stimulation of mechano-growth factor expression by myofibrillar proteins in murine myoblasts and myotubes

Mechano-growth factor (MGF) is a product of alternative splicing of the insulin-like growth factor 1 (IGF-1) mRNA. MGF is known to stimulate myoblast proliferation and to protect neurons and cardiomyocytes from apoptosis. MGF expression is dramatically increased in response to mechanical stimuli and tissue damage. The mechanisms of induction of MGF expression are as yet imperfectly understood. There is certain evidence that some protein factors able to stimulate MGF synthesis in normal myoblasts are released from damaged muscle. This study was undertaken to explore the nature of these protein inductors of MGF expression and to investigate the mechanism of their action. We report here that myofibrillar fraction of skeletal muscle homogenate activated MGF expression in murine myoblasts and myotubes in culture. The expression of another splice form of IGF-1 gene, IGF-1Ea, was also stimulated by myofibrils. Three myofibrillar proteins able to stimulate MGF synthesis were isolated. These proteins were identified by MALDI and immunoblotting as myomesin, myosin-binding protein C, and titin. The activation of MGF expression was associated with the increase of cAMP level in the cells. Inhibitor of adenylyl cyclase dideoxyadenosine arrested stimulation of MGF synthesis by all three myofibrillar proteins.     

Transforming growth factor-beta,basic fibroblast growth factor,and platelet-derived growth factor-BB interact to affect proliferation of clonally derived porcine satellite cells

Transforming growth factor beta-1 (1GF-β) stimulated porcine satellite cell proliferation in basal serum-free medium by 25%, but inhibited growth in serumcontaining medium by 58%. The effect of TGF-β on cell proliferation in serumfree medium was examined in combination with the following human recombinant growth factors: platelet-derived growth factor-BB (PDGF), basic fibroblast growth factor (FGF), insulin-like growth factor I (IGF-I), and epidermal growth factor (EGF). TGF-β inhibited PDGF-stimulated proliferation, enhanced FGF-stimulated proliferation, and had no effect on proliferation stimulated by IGF-I. The response of satellite cells to EGF and TGF-β in serum-free medium was not different than TGF-β alone. TGF-β depressed proliferation stimulated by the following combinations of two growth factors: PDGF and IGF-I, PDGF and EGF, PDGF and FGF, and IGF-I and EGF. In combination with IGF-I and FGF, TGF-β did not affect proliferation. TGF-β inhibited proliferation stimulated by the combination of PDGF, EGF, and IGF-I, but had no effect on proliferation stimulated by combinations of three growth factors that included FGF. FGF stimulated proliferation in Minimum Essential Medium containing 10% porcine serum (MEM-10% PS) by 13% above control. When the combination of TGF-β and FGF was added to MEM-10% PS, a 78% increase in proliferation was observed. Polyclonal antihuman PDGF-AB (this form neutralizes PDGF-AA, AB, and BB) reduced proliferation in MEM-10% PS by 44%. The combination of TGF-β and anti-PDGF-AB reduced proliferation by 59%, indicating the effects were not additive. These data indicate that: (1) FGF and TGF-β interact to increase proliferation of clonally derived porcine satellite cells, and (2) the inhibitory effect of TGF-β on proliferation of clonally derived porcine satelite cells can be primarily attributed to a reduction in the mitogenic effects of PDGF. © 1993 Wiley-Liss, Inc.     

Free ribosomes and growth stimulation in human peripheral lymphocytes: Activation of free ribosomes as an essential event in growth induction

During the initial ten hours of growth in lymphocytes stimulated by phytohemagglutinin, the cells are converted from a state in which over 70% of all ribosomes are inactive free ribosomes, to one in which over 80% of ribosomes are in polysomes or in native ribosomal subunits. In this initial period, there was a neglible increase in total ribosomal RNA due to increased RNA synthesis, and abolition of ribosomal RNA synthesis with low concentrations of actinomycin D did not interfere with polysome formation. Therefore, the conversion is accomplished by the activation of existing free ribosomes rather than by accumulation of newly synthesized particles. The large free ribosome pool of resting lymphocytes is thus an essential source of components for accelerated protein synthesis early in lymphocyte activation, before increased synthesis can provide a sufficient number of new ribosomes. Free ribosomes accumulate once more after 24 to 48 hours of growth, when RNA and DNA synthetic activity are maximal. This reaccumulation of inactive ribosomes at the peak of growth activity may represent preparation for a return to the resting state where cells are again susceptible to stimulation. Activation of free ribosomes to form polysomes appears to involve modification of at least two steps: (a) dissociation of free ribosomes with stabilization as native subunits, and (b) adjustment of a rate-limiting step at initiation.     

Post-transcriptional control of protein synthesis in Balb/c-3T3 cells by platelet-derived growth factor and platelet-poor plasma

Platelet-derived growth factor (PDGF) and platelet-poor plasma, which lacks PDGF, both induce a rapid increase in the rate of total protein synthesis within quiescent, density-arrested Balb/c-3T3 cells. This stimulation of protein synthesis is associated with an increased aggregation of ribosomes into polyribosomes. Nuclear functions are not required for this response, as demonstrated by the observation that this stimulation of protein synthesis occurs in cells pretreated with actinomycin D and in enucleated cells (cytoplasts). The response to PDGF persists even after PDGF has been removed from the culture medium, but in contrast, when plasma is removed from the medium, polysomes disaggregate and protein synthesis declines. PDGF and plasma do not function synergistically to increase protein synthesis, whereas they do to induce optimum DNA synthesis. Thus stimulation of the translational apparatus may be necessary for the mitogenic response of Balb/c-3T3 cells to growth factors, but it is not by itself sufficient.     

Insulin signaling leading to proliferation, survival, and membrane ruffling in C2C12 myoblasts

We have recently shown that insulin induced myogenesis in the mouse C2C12 skeletal muscle cell line by activation of phosphatidylinositol (PI) 3-kinase/p70S6-kinase and p38-mitogen-activated protein kinase (MAPK) and downregulation of p42/p44-MAPK. This study investigated the insulin-signaling pathways involved in mitogenesis, survival, and membrane ruffling in C2C12 myoblasts, a cellular system that besides IGF-I receptors, expressed a high number of functional insulin receptors. Insulin (10 nM) rapidly stimulated beta-chain insulin receptor and IRS-1 tyrosine phosphorylation, IRS-2 being poorly and SHC not phosphorylated at all. However, an association of SHC with IRS-1 was found under insulin stimulation. Insulin stimulated IRS-1 association with p85alpha leading to the activation of PI3-kinase, and, subsequently AKT and p70S6-kinases. Moreover, both p42/p44- and p38-MAPKs resulted in phosphorylation after insulin stimulation. Insulin treatment for 24 h produced mitogenesis, as demonstrated by the increase in ((3)H)-thymidine incorporation, DNA content, the expression of PCNA and cyclin D1 proteins, and the proportion of cells in S + G2/M phases of the cell cycle. This mitogenic effect of insulin was precluded by inhibition of p70S6-kinase (either by rapamycin or by the PI3-kinase inhibitor LY294002) as well as by inhibition of p44/p42-MAPK with PD098059, but was not affected by inhibition of p38-MAPK. Serum deprivation of C2C12 myoblasts resulted in growth arrest at the GO/G1 phases of the cell cycle and apoptosis, as detected either by DNA laddering or by increase in the percentage of hypodiploid cells. Insulin rescued serum-deprived cells from apoptosis in an AKT-dependent manner, as demonstrated by the inhibition of AKT-activity by the use of LY294002 and ML-9, meanwhile neither inhibition of p70S6-kinase, nor MAPK affected insulin-induced survival. Finally, we evaluated the capacity of insulin to modulate actin cytoskeleton rearrangement. Insulin stimulation of myoblasts produced membrane ruffling and decreased actin stress fibers; this biological response being dependent of p38-MAPK, as demonstrated by the use of the p38-MAPK inhibitors SB203580 or PD169316, but independent of PI3-kinase and p42/p44-MAPK.     

Regulation of initiation factors during translational repression caused by serum depletion. Abundance, synthesis, and turnover rates

During growth in unreplenished medium, the fraction of active, polysomal ribosomes progressively decreases about 3-fold from 80-90% to only 20-40% due to a reduced rate of initiation. To assess whether the abundance of initiation factors could be involved in this repression of translational activity. HeLa cell cytoplasmic lysates were resolved by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and spots corresponding to the initiation factor proteins were quantitated. The relative abundance of most of the initiation factor proteins only decreases by 10-40% and roughly parallels that of the ribosomes. Measurement of the rates of synthesis and turnover of the initiation factor proteins establishes that during periods of active growth, synthesis and degradation occur coordinately with total cell protein. As growth rate decreases, the synthesis of some initiation factor proteins, particularly eukaryotic initiation factor (eIF)-3 subunits, becomes depressed. Serum stimulation of serum-depleted cells recruits most inactive ribosomes and mRNAs into polysomes, but most initiation factor mRNAs are not selectively recruited. The principal exceptions are eIF-3p24 which exhibits 4-5 fold enhanced synthesis and eIF-3p44 and eIF-4A whose syntheses are moderately stimulated.