Mechanism of inhibition of polypeptide chain initiation in calcium-depleted Ehrlich ascites tumor cells.

Protein synthesis in Ehrlich ascites tumor cells is inhibited when cellular calcium is depleted by the addition of EGTA to the growth medium. This inhibition is at the level of polypeptide chain initiation as evidenced by a disaggregation of polyribosomes accompanied by a significant elevation in 80-S monomers. To identify direct effects of calcium on the protein synthesis apparatus we have developed a calcium-dependent, cell-free protein-synthesizing system from the Ehrlich cells by using 1,2-bis(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA), a recently developed chelator with a high (greater than 10(5)) selectivity for calcium (pKa = 6.97) over magnesium (pKa = 1.77). BAPTA inhibits protein synthesis by 70% at 1 mM and 90% at 2 mM. This effect was reversed by calcium but not by other cations tested. The levels of 43-S complexes (i.e., 40-S subunits containing bound methionyl-tRNAf.eIF-2.GTP) were significantly lower in the calcium-deprived incubations, indicating either inhibition of the rate of formation or decreased stability of 43-S complexes. Analysis of 43-S complexes on CsCl gradients showed that in BAPTA-treated lysates, 40-S subunits containing eIF-3, completely disappeared and the residual methionyl-tRNA-containing complexes were bound to 40-S subunits lacking eIF-3. Our results demonstrate a direct involvement of Ca2+ in protein synthesis and we have localized the effect of calcium deprivation to decreased binding of eIF-2 and eIF-3 to 40-S subunits.

Antitumor immunity induced by hybrid murine tumor cells: requirements for optimal immunization.

Hybrid tumor cells have been evaluated for their ability to induce specific antitumor immunity in inbred female C3H/He mice challenged with the syngeneic BA tumor. Hybrid cells were produced by fusion of BA cells with a BALB/c renal adenocarcinoma, which is hypoxanthine phosphoribosyl transferase-deficient and grows well in culture. Corynebacterium parvum was evaluated as an adjuvant for BA and hybrid cells. The BA tumor was shown to be poorly immunogenic, and four weekly injections of BA cells alone or C. parvum alone did not confer significant immunity. When BA cells and C. parvum were mixed, survival time was prolonged and most mice remained tumor-free. Hybrid cell lines derived from the BA tumor were produced in culture in unlimited quantities and were successfully used as immunogens. The addition of C. parvum to hybrids gave a significant incremental increase in survival when compared to the survival resulting from immunization by hybrids without adjuvant. When hybrids without adjuvant were used, several weekly injections were required for effective immunization. Irradiated and unirradiated hybrids were compared, and it was found that irradiation did not diminish hybrid immunogenicity. The potential problems and advantages of this concept of therapy are discussed.

Antitumor immunity induced by hybrid tumor cells: comparison between hybrids and the parental tumor.

The ability of hybrid tumor cells to induce antitumor immunity has been evaluated in the line 1 alveolar cell carcinoma (L1) model of BALB/c mice. Hybrid tumor cells were produced by fusing freshly dissociated L1 cells isolated from in vivo tumors with the hypoxanthine:aminopterin:thymidine-sensitive cell line, GM 347, derived from C3H mice. Each hybrid was characterized by DNA content and expression of H-2 antigens using a fluorescence-activated cell sorter. Irradiated L1 cells in the presence or absence of Corynebacterium parvum were capable of immunizing BALB/c mice against a challenge of live L1 cells, provided the challenge dose was small (50% lethal dose was between 6 X 10(4) and 1.2 X 10(5) L1 cells). Testing of five hybrid clones and 1 uncloned hybrid line for their immunizing ability demonstrated a range in immunizing ability with none showing a statistically significant improvement in survival (p less than 0.0018) when compared to untreated controls. However, one hybrid clone, MoHb-L1-C2, was selected in which the survival of mice immunized with it compared to controls had a p value of 0.0255. A tumor (labeled L1/A) which grew in one of the mice immunized with this clone was removed and hybridized with GM 347 to yield a second set of hybrids. Both this variant of L1 cells and a hybrid clone made from it (MoHb-L1A-C18) were capable of immunizing mice against a challenge of live L1/A (p values of 0.0000 and 0.0028, respectively, when compared to controls). However, L1 cells were not able to immunize effectively against L1/A, and MoHb-L1A-C18 did not immunize against L1. This suggests that L1/A is a subpopulation of L1 cells with a different antigenic composition. The limited success of MoHb-L1A-C18 against L1/A is thought to be due to the narrower range of antigenic specificities in L1/A and the ability of MoHb-L1A-C18 to represent an important antigenic subpopulation of L1/A.

Variations in the protein pattern of Ehrlich ascites tumor cell polyribosomes.

The two-dimensional gel electrophoretic protein pattern of polyribosomes from Ehrlich ascites tumor cells which have been fasted in a nutritionally deprived medium was compared to the corresponding pattern derived from unstarved cells. The proteins were labeled with 3H-labeled amino acids and their counts determined to make the comparison quantitative. Three proteins were found in the "fasted" polyribosomes in 4--6-fold greater amounts than in the fed control.

Serum growth factors cause rapid stimulation of protein synthesis and dephosphorylation of eIF-2 in serum deprived Ehrlich cells.

In Ehrlich ascites tumor cells maintained in serum-free medium for 16 h the rate of protein synthesis was about 50% of the rate in control (well-fed) cells. The addition of 10% calf serum led to a 1.5- to 2-fold stimulation of protein synthesis within 10 min. Stimulation was effected through a non-transcriptional mechanism which operated at the level of polypeptide chain initiation. The effect was due to non-dialyzable serum growth factors which were sensitive to treatment with dithiothreitol and iodoacetamide. Replacing the 16-h-conditioned serum-free medium with fresh serum-free medium stimulated protein synthesis about 30% in serum-deprived cells, and the effect of these low molecular weight nutrients was additive with the effect of serum factors. Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) inhibits protein synthesis by competitively inhibiting the guanine nucleotide exchange factor (GEF), and modulation of the extent of phosphorylation of eIF-2 alpha has been suggested as a probable regulatory mechanism in serum-deprived mammalian cells. We measured the ratio of phosphorylated to total eIF-2 alpha in serum-deprived cells. The ratio was elevated in serum-deprived cells compared to control (serum-fed) cells. eIF-2 was rapidly dephosphorylated in response to serum refeeding and returned to near control levels after 10 min. The rapidity of this response and the close temporal correlation between eIF-2 dephosphorylation and increased rate of protein synthesis provide evidence that eIF-2 plays an important role in the regulation of protein synthesis by serum growth factors.

Cyclic AMP and growth of Ehrlich ascites tumor cells. Lack of cyclic AMP elevation in nutritionally deprived cells and mechanism of retardation of growth by dibutryl cyclic AMP.

Cyclic AMP levels in Ehrlich ascites tumor cells changed little after deprivation of cells of essential nutrients, serum, glucose and amino acids, deprival of each of which leads to marked inhibition of growth and protein synthesis. Cyclic AMP levels also changed little after the addition of these nutrients to deprived cells. Thus cyclic AMP is not likely to be the intracellular mediator for growth regulation by these three nutrients. Elevation of cyclic AMP levels for short periods by exposure of cells to choleratoxin or theophylline produced only slight changes in parameters of protein synthesis (polyribosome pattern and rate of [3H]leucine incorporation). An exposure for 1 day to dibutyryl cyclic AMP did not inhibit cell growth. However, prolonged exposure to dibutyryl cyclic AMP inhibited the multiplication of Ehrlich ascites cells both in suspension and in stationary cultures. No morphological effects were evident in the former; in the latter, cells attached firmly to the substratum and formed elongated cytoplasmic processes. Inhibition of cell multiplication by dibutyryl cyclic AMP was related to cell density and to serum concentration. Cells in dibutyryl cyclic AMP-containing media plated at low cell densities multiplied as rapidly as control cells. The final densities cells reached were determined by the serum concentration; in dibutyryl cyclic AMP-containing media these densities were about one-half those of respective control cells. Limitation of cell multiplication by dibutyryl cyclic AMP was reversed by the addition of serum, by resuspending cells at lower densities, or by resuspending cells in media without dibutyryl cyclic AMP. These findings suggested that dibutyryl cyclic AMP may affect the utilization of serum factors by cells. Dibutyryl cyclic AMP did not inactivate serum factors and did not change the rate at which cells depleted the growth medium of serum factors. Dibutyryl cyclic AMP may limit cell multiplication by increasing the cellular requirement for serum factors.

The isolation of large polysomes in high yield from unfractionated tissue homogenates.

It was found that if large quantities of both exogenous RNA and Mg-2+ were present during gentle tissue homogenization, the subsequent addition of deoxycholate to the whole homogenate produced a viscous mass from which polysomes could be isolated in large yields. These polysomes were substantially less degraded than those isolated by previous methods. In the case of rat liver, 15 ribosomes per mRNA was the species present in highest concentration. The parameters of this method were investigated and optimized. About 80 percent of the rRNA in the homogenates was recovered in the polysomes. Omission of deoxycholate permitted the isolation of less-degraded free polysomes as well. In the liver of fed rats these represented one-fourth of the total polysomes, in good agreement with results obtained by an independent approach. Using the method to isolate polysomes from the liver of starving rats, it was found that only about one percent of the large amount of monomers and dimers present resulted from polysome breakdown during isolation. It was further shown that random RNAase hydrolysis of polysomes could not produce the patterns of liver polysomes seen during starvation. Polysomes isolated by this procedure were quite stable in solution and were very active in cell-free protein synthesis. Application of this method without adaptation to eight other tissues also permitted the isolation of large polysomes in high yields.

Binding of Met-tRNA-f to native and derived 40S ribosomal subunits.

Our previous work has shown that the native 40S ribosomal subunits (those found free in the cell sap) but not polyribosomal 40S subunits have additional associated proteins that are removed by 0.5 M KCl. In this communication we present evidence that in the Ehrlich cell one of the native subunit associated proteins is the mammalian initiation factor that forms a Met-tRNA-f-factor-GTP complex, and is required for the binding of Met-tRNA-f to the 40S subunit. Initial examination of the KCl wash of the Ehrlich cell total ribosomal pellet revealed a factor which (1) shifted the elution of Met-tRNA-f and of GTP from the included to the excluded volume on Sephadex G-100 chromatography, (2) stimulated the binding of Met-tRNA-f to Millipore filters, and (3) stimulated the binding of Met-tRNA-f to salt-washed 40S subunits. These activities were dependent upon or enhanced by GTP; were inhibited by GDP; were much greater for Met-tRNA-f than for Met-tRNA-m or for lysyl-tRNA; and were concentrated in the KCl ribosomal wash and were not detected in the cell soluble fraction. Met-tRNA-f bound in conjunction with a specific amount of KCl wash protein, to form a distinctive particle of bouyant density 1.40 g cm minus 3 in CsCl, identical in density to one form of the native 40S subunit. Native 40S subunits, but no other subunits, contained a factor which was eluted by 0.5 M KCl and which (1) stimulated the binding of Met-tRNA-f to Millipore filters, and (2) stimulated the binding of Met-tRNA-f to salt-washed 40S subunits. The factor appeared to be localized on the native 40S subunit of density 1.40 g cm minus 3.

Binding of Met-tRNAf to native 40 S ribosomal subunits in Ehrlich ascites tumor cells.

Two forms of native 40 S ribosomal subunits, distinguishable by their buoyant densities, are recovered from Ehrlich ascites cells. In this communication, we describe experiments designed to test whether Met-tRNAf is associated with either form. Our results indicate that (a) in the cell, Met-tRNAf is bound to the native 40 S subunit, and in particular, to the subunit of density 1.40 g x cm-3; (b) under the growth conditions used, less than 10% of the low density native subunits have bound Met-tRNAf; (c) the majority of the Met-tRNAf containing native 40 S subunits, isolated from sucrose gradient analyses of cell extracts, join with 60 S subunits in vitro to form 80 S monosomes only if additional ribosomal wash factors are provided; and (d) the 80 S monosomes so formed are completed initiation complexes that can form peptide bonds. These results support the hypothesis that various forms of the native 40 S subunit represent different stages in the formation of a complete initiation complex.

A GDP/GTP exchange factor essential for eukaryotic initiation factor 2 cycling in Ehrlich ascites tumor cells and its regulation by eukaryotic initiation factor 2 phosphorylation.

Formation of the ternary complex Met-tRNAi X eukaryotic initiation factor (eIF) 2 X GTP from eIF-2 X GDP requires exchange of GDP for GTP. However, at physiological Mg2+ concentrations, GDP is released from eIF-2 exceedingly slowly (Clemens, M.J., Pain, V.M., Wong, S.T., and Henshaw, E.C. (1982) Nature (Lond.) 296, 93-95). However, GDP is released rapidly from impure eIF-2 preparations, indicating the presence of a GDP/GTP exchange factor. We have now purified this factor from Ehrlich cells and refer to it as GEF. CM-Sephadex chromatography of ribosomal salt wash separated two peaks of eIF-2 activity. GEF was found in association with eIF-2 in the first peak and co-purified with eIF-2 under low salt conditions. It was separated from eIF-2 in high salt buffers and further purified on hydroxylapatite and phosphocellulose. Gel electrophoresis of our purest preparations showed major bands at 85, 67, 52, 37, 27, and 21 kDa. Purified GEF increased the rate of exchange of [32P] GDP for unlabeled GDP 25-fold but did not function with phosphorylated eIF-2 (alpha subunit). The factor also stimulated markedly the rate of ternary complex formation using eIF-2 X GDP as substrate with GTP and Met-tRNAi but not using phosphorylated eIF-2 X GDP as substrate. eIF-2 is released from the 80 S initiation complex with hydrolysis of GTP. If eIF-2 X GDP is actually the complex released, then GEF is absolutely required for eIF-2 to cycle and it is therefore a new eukaryotic initiation factor. Furthermore, the inability of GEF to utilize eIF-2 (alpha P) X GDP explains how phosphorylation of eIF-2 can inhibit polypeptide chain initiation.

Initiation of protein synthesis in Ehrlich ascites tumour cells. Evidence for physiological variation in the association of methionyl-tRNAf with native 40-S ribosomal subunits in vivo.

Binding of methionyl-tRNAf to native 40-S ribosomal subunits is thought to be an early stage in the process of polypeptide chain initiation, and [35S]Met-tRNAf - 40-S-subunit complexes can be isolated from Ehrlich ascites tumour cells following a brief incubation with [35S]methionine. To determine whether this step is subject to modulation by physiological conditions, we have estimated the extent of binding of Met-tRNAf to native- 40S ribosomal subunits in Ehrlich cells under nutritional conditions known to affect the rate of protein synthesis in these cells. Deprivation of either an essential amino acid, lysine, or of glucose, results in a substantial reduction in the proportion of native 40-S subunits which have Met-tRNAf associated with them, and refeeding of lysine to cells deprived of this amino acid partially reverses this effect within 10 min. These effects on the concentration of Met-tRNA - 40-S-subunit complexes are paralleled by changes of similar magnitude in the rate of protein synthesis and in polyribosome profiles. Native 40-S subunits can be spearated by equilibrium density gradient analysis on caesium chloride into two species, with buoyant densities approximately 1.40 and 1.49 g X cm-3. In cells deprived of either lysine or glucose, the radioactivity from [35S]methionine is bound exclusively to the particle of buoyant density 1.40 g X cm-3. In well-fed cells, or in starved cells shortly after refeeding, a significant proportion of the label is associated with a region of the CsCl gradient corresponding to a particle of higher density. The results suggest that the binding of Met-tRNAf to native 40-S ribosomal subunits can be greatly affected by physiological conditions which alter the rate of protein synthesis. This is consistent with a regulatory role for this step in the sequence of reactions involved in initiation of translation.

TPA stimulates S6 phosphorylation but not protein synthesis in Ehrlich cells.

Increased phosphorylation of ribosomal protein S6 has been extensively correlated with an increased rate of protein synthesis. We report here that under two separate conditions in Ehrlich cells an increase in the level of S6 phosphorylation does not result in any increase in the rate of protein synthesis. 1) In glutamine-deprived cells TPA stimulates S6 phosphorylation but has no effect on the rate of protein synthesis, 2) In cells deprived of serum growth factors, addition of serum stimulates both S6 phosphorylation and protein synthesis while TPA stimulates only S6 phosphorylation. These results show that increased phosphorylation of S6 is not sufficient to cause increased rates of protein synthesis, and suggest that additional factors may play a more direct role.

Mechanism of inhibition of polypeptide chain initiation in heat-shocked Ehrlich ascites tumour cells.

The rate of polypeptide synthesis is inhibited by 80% in Ehrlich cells incubated at 43 degrees C compared to those at 37 degrees C. The regulatory site of translation resides at polypeptide chain initiation. Polypeptide synthesis does not recover at the higher temperature; however, the inhibition is reversed by returning the cells to 37 degrees C. Neither new RNA synthesis or protein synthesis is required for recovery at 37 degrees C, eliminating degradation of mRNA and irreversible denaturation of a protein essential for polypeptide chain initiation. The concentration of 40-S initiation complexes was found to be reduced markedly in heat-shocked cells compared to controls. This was confirmed in the cell-free protein-synthesizing systems prepared from heat-shocked and control cells. Reversible alteration in the activity of components affecting eIF2 function is, therefore, a likely mechanism of regulation in heat-shocked Ehrlich cells. In extracts from heat-shocked cells, Met-tRNA synthetase activity was unaltered compared to control extracts.