Inactivation of protein synthesis stimulating activity in serum by cells

When Ehrlich ascites cells were cultivated in serum-free media their cellular protein synthetic rate declined to a new steady-state level and the cells stopped multiplying. On addition of serum the cellular protein synthetic rate increased to the level before serum starvation and cells resumed multiplication. The activity in serum stimulating protein synthesis was inactivated on incubation with cells. At cell concentrations of the usual culture conditions this inactivation took several hours; at very high cell concentrations it was complete in ten minutes. Serum-starved cells inactivated low serum (2%–6%) media in the same length of time. Studies of inactivation of high serum media demonstrated that cells had a limted capacity to inactivate. Cells grown in 10% serum were unable to inactivate. Inactivation was not due to accumulation in the medium of either low molecular or macromolecular cell products. Inactivation was strongly inhibited at 4° or by treatment of cells with fluoride or cycloheximide (long exposure): less inhibited by treatment with 2-deoxyglucose or glutaraldehyde; and slightly inhibited by treatment with cyanide or cycloheximide (short exposure). Inactivating ability was unaffected by trypsinization. These findings are best explained by the hypothesis that cells take up the serum activity by endocytosis.     

Cell-serum factor interaction. Characteristics of stimulation of protein synthesis in Ehrlich ascites cells by factors in serum and in cell extracts.

Stimulation of protein synthesis induced by serum in serum-starved Ehrlich ascites tumor cells was directly proportional to the concentration of added serum, inversely proportional to the concentration of cells, in the culture, and dependent on the length of exposure of cells to serum. Stimulation was markedly decreased in cells incubated with serum at temperatures lower than 37 °C. During the exposure of cells to serum, active protein synthesis was not required in order for subsequent stimulation of protein synthesis to take place. These characteristics were consistent with the possibility that stimulation of protein synthesis followed uptake of serum factors by cells. Extracts of cells stimulated protein synthesis in a similar fashion to serum. Stimulations by extracts and by serum were additive. The factors in cell extracts were macromolecular, associated with articulate fractions, and inactivated by trypsin, but not by RNAase, DNAase, ether or chloroform. Extracts of serum-grown cells were more stimulatory than extracts of serum-starved cells. When serum-starved cells were incubated with serum, stimulatory activities of their extracts increased as a function of time of incubation with serum.     

Requirement for protein synthesis in the regulation of protein breakdown in cultured hepatoma cells.

The modes of action of insulin and of inhibitors of protein synthesis on the degradation of labeled cellular proteins have been studied in cultured hepatoma (HTC) cells. Protein breakdown is accelerated upon the deprivation of serum (normally present in the culture medium), and this enhancement is inhibited by either insulin or cycloheximide. An exception is a limited class of rapidly turning over cellular proteins, the degradation of which is not influenced by insulin or cycloheximide. Alternative hypotheses to explain the relationship of protein synthesis to the regulation of protein breakdown, viz., control by the levels of precursors of protein synthesis, regulation by the state of the ribosome cycle, or requirement for a product of protein synthesis, have been examined. Protein breakdown was not influenced by amino acid deprivation, and measurements of valyl-tRNA levels in HTC cells subjected to various experimental conditions showed no correlation between the levels of charged tRNAVal and the rates of protein degradation. Three different inhibitors of protein synthesis (puromycin, pactamycin, and cycloheximide) suppressed enhanced protein breakdown in a similar fashion. A direct relationship was found between the respective potencies of these drugs to inhibit protein synthesis and to block enhanced protein breakdown. When cycloheximide and insulin were added following a prior incubation of HTC cells in a serum-free medium, protein breakdown was maximally suppressed within 15-30 min. Actinomycin D inhibited protein breakdown only after a time lag of about 90 min. It is suggested that the regulation of protein breakdown in hepatoma cells requires the continuous formation of a product of protein synthesis, in a manner analogous to the mode of the control of this process in bacteria.     

The regulation of protein synthesis by translation control RNA

The mechanism by which translational control RNA (tcRNA) inhibits protein synthesis was investigated. In the presence of heme the inhibitory role of muscle tcRNA on hemoglobin synthesis was confirmed. Upon the addition of muscle tcRNA to a rabbit reticulocyte cell-free system the binding of [³²P]-globin mRNA to 40S ribosomal subunits and its subsequent incorporation into polysomes was inhibited. Furthermore, muscle tcRNA inhibits met-tRNA binding to polysomes and yet stimulates the formation of methionine-puromycin. These results suggest that muscle tcRNA blocks the binding of globin mRNA to ribosomes resulting in an abortive initiation complex that is, however, still capable of the methionine-puromycin reaction.     

The control of serum protein synthesis in hepatoma-fibroblast hybrids.

Hybrids between mouse hepatoma cells (which secrete several serum proteins) and mouse or rat fibroblasts (which do not secrete these proteins) produce transferrin and the third component of complement (C3) like the parental hepatoma cells, while they do not secrete either albumin or alpha-fetoprotein (AFP). This lack of albumin and AFP secretion is probably due to a lack of synthesis, rather than to a simple defect in secretion. The cessation of albumin and AFP production is not dependent upon the parental fibroblast nor upon the selection conditions; it is best explained by a shut-off synthesis and could thus reflect the existence of a regulatory mechanism. This would imply a difference between the control of albumin and AFP synthesis and that of transferrin and C3 synthesis. On the other hand, in agreement with Peterson and Weiss (1972), hybrids between rat hepatoma cells and mouse fibroblasts continue to product rat albumin. This suggests that the mouse hepatoma cells differ from the rat hepatoma cells in the way they control albumin production.     

The regulation of protein synthesis in the liver of rats. Mechanisms of dietary amino acid control in the immature animal

Weanling (23-day-old) rats were fed either on an amino acid-deficient diet (6% of casein, which in effect represents an `amino acid-deficient' diet) or on a diet containing an adequate amount of protein (18% of casein) for 28 days. The hepatic cells from the animals fed on the low-protein diet were characterized by low amino acid content, almost complete inhibition of cell proliferation and a marked decrease in cell volume, protein content and concentration of cytoplasmic RNA compared with cells from control rats. The lower concentration of cytoplasmic RNA was correlated with a decreased ribosomal-RNA content, of which a larger proportion was in the form of free ribosomes. The protein-synthetic competence and messenger-RNA content of isolated ribosomes from liver cells of protein-deprived animals were 40–50% of those noted in controls. At 1hr. after an injection of radioactive uridine, the specific radioactivity of liver total RNA was greater in the group fed on the low-protein diet, but the amount of label that was associated with cytoplasmic RNA or ribosomes was significantly less than that noted in control animals. From these data it was concluded that dietary amino acids regulate hepatic protein synthesis (1) by affecting the ability of polyribosomes to synthesize protein and (2) by influencing the concentration of cytoplasmic ribosomes. It is also tentatively hypothesized that the former process may be directly related to the concentration of cellular free amino acids, whereas the latter could be correlated with the ability of newly synthesized ribosomal sub-units to leave the nucleus.     

Nonspecific and specific interactions of Y-box-binding protein 1 (YB-1) with mRNA and posttranscriptional regulation of protein synthesis in animal cells

Y-box-binding protein 1 (YB-1) is an animal multifunctional DNA/RNA-binding protein that is involved in reproduction, storing, and expression of genetic information. YB-1 accompanies mRNA throughout its life, from synthesis to degradation, and has a high specific and nonspecific affinity for RNA. In the nucleus YB-1 regulates mRNA processing. In the cytoplasm YB-1 is responsible for global and selective regulation of protein synthesis, as well as the mRNA life. This review focuses on the role of YB-1 in regulating translation. The possible mechanisms of the positive and negative effects of YB-1 on this process are considered. The recent original data are described, supporting the role of YB-1 as a major structural component of mRNP. Data about specific interactions of YB-1 with RNA are summarized for the first time.     

Control of protein synthesis in extracts from poliovirus-infected cells. I. mRNA discrimination by crude initiation factors.

By using cell-free systems prepared from uninfected and poliovirus-infected cells, we have been able to demonstrate that crude preparations of initiation factors from infected cells do not stimulate the initiation of translation by polyribosomes containing endogenous host cell mRNA. When tested with polysomes containing endogenous viral mRNA, however, they were able to stimulate initiation of translation nearly as well as uninfected cell initiation factors. The uninfected cell initiation factor preparations were able to stimulate initiation of translation of both cell and viral mRNA. The results indicate an mRNA-specific activity present in crude initiation factor preparations from infected cells. Furthermore, the ability of eIF2 from infected cells to form a ternary complex with GTP and formyl [35S]methionine-tRNAfmet, an mRNA-independent step in initiation, was found not to be deficient. Implications of these data for proposed mechanisms of poliovirus-induced host cell shutoff are discussed.     

Induction of the nuclear protein ''cyclin'' in quiescent mouse 3T3 cells stimulated by serum and growth factors. Correlation with DNA synthesis

The effect of serum and growth factors [platelet-derived growth factor (PDGF), fibroblast growth factor (FGF)] on the synthesis of the nuclear protein cyclin and its correlation with DNA synthesis has been studied in quiescent mouse 3T3 cells by means of quantitative two-dimensional gel electrophoresis. Serum must be present in the medium for at least 8-12 h to induce maximal synthesis of cyclin (6- to 7-fold increase compared with quiescent cells). The stimulation of cyclin synthesis is dose-dependent and correlates directly with DNA synthesis. In addition, partially purified PDGF and FGF also induce cyclin and DNA synthesis in a coordinate way. Both growth factors, like serum, exhibit a similar lag phase to induce maximal cyclin (6- to 7-fold) and DNA synthesis (90% of the cells). Pure PDGF at a concentration as low as 10 ng/ml has the same effect as 10% serum. The coordinate induction of cyclin and DNA synthesis can only be observed with growth factors that induce DNA synthesis. These results strengthen the notion that cyclin is an essential component of the events leading to DNA replication.     

On the regulation of DNA synthesis in a line of adrenocortical tumor cells: effect of serum, adrenocorticotropin and pituitary factors.

Y-1 adrenal cells responded to serum step down by a several fold decrease in DNA synthesis. Serum starved cells resumed DNA synthesis upon serum step up. ACTH and cAMP inhibited DNA synthesis both at low and high serum concentrations, a fact previously known. Pituitary, brain and liver crude extracts stimulated DNA synthesis in serum starved cells. Purified pituitary factors preparations contained two activities: one specific for Y-1 cells and another active with both fibroblasts and Y-1 cells. The kinetics of restimulation of DNA synthesis by serum and pituitary factors was studied. DNA synthesis restimulation occurred after a lag of 11 hours. This lag did not vary irrespective of the type of stimulator or its concentration. Cells entered S phase continuously at a rate which increased with increasing concentrations of the stimulator. Cells became refractory to the inhibitory action of ACTH five hours before entering S phase. The implications of these data to the understanding of cell growth control are considered.     

Osmotic regulation of aldose reductase protein synthesis in renal medullary cells

Renal medullary cells are normally exposed to high extracellular NaCl as part of the urinary concentrating mechanism. They react to this stress by accumulating sorbitol and other organic osmolytes. PAP-HT25, a line of epithelial cells derived from rabbit renal inner medulla, expresses this response. In hypertonic medium, these cells accumulate large amounts of sorbitol. There is a large increase in the amount of aldose reductase, which catalyzes production of sorbitol from glucose. The purpose of the present study was to investigate whether the aldose reductase protein increases because of faster synthesis or slower degradation. We measured the rate of synthesis and degradation of aldose reductase protein by pulse-chase with [35S]methionine, followed by immunoprecipitation with specific antiserum and autoradiography. The protein synthesis rate was 6 times greater in cells grown in hypertonic (500 mosmol/kg) medium, than in those grown in normal (300 mosmol/kg) medium. When control cells were switched to hypertonic medium, the synthesis rate increased 15-fold by 24 h, then decreased to 11-fold after 48 h. In contrast, synthesis rate continued to increase past 24 h when accumulation of sorbitol was prevented by inhibiting aldose reductase activity with Tolrestat. Thus, there is a feedback mechanism by which cellular sorbitol accumulation inhibits aldose reductase protein synthesis. Degradation of aldose reductase protein was slow (only about 25% in 3 days) and was not affected by osmolality. Thus, the osmoregulatory increase in aldose reductase protein is due to an increase in its synthesis rate and not to any change in its degradation.     

Effect of secretagogues on chromogranin A synthesis in bovine cultured chromaffin cells. Possible regulation by protein kinase C.

Chromogranin A is a major component of storage granules in many different secretory cell types. After [35S]methionine labelling of proteins from cultured bovine chromaffin cells, chromogranin A was immunoprecipitated with specific antibodies, and the radioactivity incorporated into chromogranin A was determined and used as an index of its synthesis rate. Depolarization of cells with nicotine or high K+ evoked a Ca2+-dependent increase in chromogranin A synthesis, whereas muscarine, which does not evoke significant Ca2+ influx from bovine chromaffin cells, had no effect on chromogranin A synthesis. Forskolin, an activator of adenylate cyclase, affected neither the basal nor the nicotine-stimulated rate of chromogranin A synthesis. In contrast, 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C, significantly enhanced the incorporation of radioactivity into chromogranin A. Sphingosine, an inhibitor of protein kinase C, abolished both nicotine-stimulated and TPA-induced chromogranin A synthesis. In addition, long-term treatment of chromaffin cells with TPA decreased protein kinase C activity and inhibited the nicotine-stimulated chromogranin A synthesis. These results suggest that protein kinase C may play an important role in the control of chromogranin A synthesis.     

Inhibition of RNA synthesis in animal cells by sodium selenite

The effect of selenium-containing compounds on RNA synthesis in rat liver cells was studied in vivo. All the selenium derivatives under study inhibited the rRNA synthesis in liver cells. The most potent inhibiting effect was exerted by sodium selenite. It was shown that in a cell-free system of RNA biosynthesis sodium selenite selectively inhibited the activity of RNA-polymerase I in isolated nuclei and purified enzyme preparations of normal and tumour cells. A feasible mechanism of inhibition of the RNA-polymerase I activity by selenium and a hypothesis on the anticarcinogenic effect of selenium are postulated.