Effect of serum on heat response of synchronized mouse neuroblastoma cells: protection of cell cycle progression, protein synthesis and survival

The effect of serum and temperature elevation on proliferation has been studied in synchronized mouse neuroblastoma (Neuro-2A) cells. The effects of serum were studied on the induction of (a) mitotic delay due to a non-lethal heat treatment (30 min at 42.7 degrees C) and (b) the loss of colony-forming capacity after a more extensive heat treatment (45 min at 44 degrees C or a continuous 42.7 degrees C heat treatment). The following results were obtained. Under conditions of serum depletion, cell cycle extension of heated G1 phase cells was more than that of heated G2 phase cells. Serum protected against heat-induced alterations of cell cycle progression in G1- but not in G2 phase cells. This effect of serum could be mimicked by a supplement to the medium of human transferrin, bovine pancreas insulin and selenium, and was correlated with protection of protein synthesis. Serum also affected heat-induced cell killing. Under conditions of serum depletion, G1 phase cells were more resistant to heat compared to G2 cells. The presence of serum during heat treatment further increased the thermoresistance of G1 phase cells, but did not affect sensitivity of G2 phase cells. This effect of serum could not be mimicked by a supplement of transferrin, insulin and selenium. These results indicate that serum protects G1 phase cells for heat-induced changes of cell cycle progression as well as on cell survival, but the mechanisms involved in both phenomena seem to be different.     

The effect of serum deprivation on the initiation of protein synthesis in mouse neuroblastoma cells

Growth of mouse neuroblastoma cells becomes stationary when cultured in serum-free medium. Within 60 h, the protein-synthesizing capacity of the cells declines to 25% as compared to that of exponentially growing cells. The transitional activity of the crude ribosomal salt washes from serum-deprived and control cells was compared in in vitro protein-synthesizing pH 5 systems. It appears that the ribosomal salt wash from serum-deprived cells has significantly (70%) lost its ability to support the translation of neuroblastoma poly(A)+ RNA. This activity of the ribosomal wash from serum-deprived cells can be restored to control level with rabbit reticulocyte initiation factor eIF-4B only. The ability of the ribosomal wash from serum-deprived cells to support the translation of encephalomyocarditis virus (EMC) and Semliki Forest virus (SFV) 42 S mRNA was tested. We found that EMC-mRNA is efficiently translated with the ribosomal salt wash from serum-deprived cells, whereas on the other hand the translation of SFV 42 S mRNA is severely impaired. Therefore, we conclude that in serum-deprived neuroblastoma cells protein synthesis is regulated in both a quantitative and a qualitative way. Modulation of the activity of initiation factor of protein synthesis eIF-4B is at least partly responsible for the observed (selective) blockade of protein synthesis in serum-deprived cells.     

Effects of p-chlorophenylalanine and alpha-methylphenylalanine on amino acid uptake and protein synthesis in mouse neuroblastoma cells.

The phenylalanine analogues p-chlorophenylalanine and alpha-methylphenylalanine were used to inhibit phenylalanine hydroxylase in animal models for phenylketonuria. The present report examines the affects of these analogues on the metabolism of neuroblastoma cells. p-Chlorophenylalanine inhibited growth and was toxic to neuroblastoma cells. Although in vivo this analogue increased cell monoribosomes by 42%, it did not significantly affect poly(U)-directed protein synthesis in vitro. P-Chlorophenylalanine did not compete with phenylalanine or tyrosine for aminoacylation of tRNA and was therefore not substituted for those amino acids in nascent polypeptides. The initial cellular uptake of various large neutral amino acids was inhibited by this analogue but did not affect the flux of amino acids already in the cell; this suggested that an alteration of the cell's amino acid pools was not responsible for the cytotoxicity of the analogues. In contrast with p-chlorophenylalanine, alpha-methylphenylalanine did not exert these direct toxic effects because the administration of alpha-methylphenylalanine in vivo did not affect brain polyribosomes and a comparable concentration of this analogue was neither growth inhibitory nor cytotoxic to neuroblastoma cells in culture. The suitability of each analogue as an inhibitor of phenylalanine hydroxylase in animal models for phenylketonuria is discussed.     

Synthesis of immunoglobulin and nuclear protein in synchronized mouse myeloma cells

The synthesis of immunoglobulin and of nuclear proteins has been studied in synchronized mouse myeloma cells of the C1 line. Synchronization has been obtained by a double thymidine block. C1 cells synthesize immunoglobulin at a relatively constant rate throughout the cell cycle except for mitosis, when a decrease in the rate of synthesis of total protein and of immunoglobulin is observed. Cell synchrony around mitosis is not sufficiently good to determine whether immunoglobulin is synthesized at all. Nuclear protein and in particular histones appear to be synthesized synchronously with DNA during the S phase of the cell cycle.     

Effect of colchamine on Ehrlich ascitic carcinoma cells showing synchronized cell division]

The author studied the 24-hour changes in the number of normal and colchamine mitoses in the cells of Ehrlich's ascites carcinoma in mice after the injection of colchamine argainst the background of partial synchronization of cell division, obtained as a result of preliminary injection of dibutyryl cyclic 3',5'-AMP, and also in mice after the injection of colchamine alone or dibutyryl cyclic 3',5'-AMP. As shown, synchronization of cell division in the tumour led to the 2,6-fold increase in the number of tumour cells blocked by colchamine and also to the accelerated arrest of colchamine mitoses.     

The relationship of protein synthesis to cell division and oral development in synchronized Tetrahymena pyriformis GL-C: An analysis employing cycloheximide

The effects of cycloheximide on synchronized Tetrahymena pyriformis strain GL-C were investigated at concentrations ranging from 0.01 to 10 μg/ml. The initial inhibition of protein synthesis was nearly total (>85%) at 1 μg/ml and above, partial (50–80%) at 0.2 to 0.05 μg/ml, and slight (<30%) at 0.02 μg/ml. Eventual recovery of protein synthesis to a rate approaching that of the controls took place at concentrations of 1 μg/ml and less. When the drug was added before a “transition point” at 55 minutes after the end of the synchronizing treatment (EST), cell division was blocked by 10 μg/ml, and delayed at concentrations of 1 μg/ml or less. The duration of delay was related to the degree of initial inhibition, and to the time required for recovery of protein synthesis; it also depended on the time after EST at which the drug was added. At a given concentration, maximum division delay was observed just prior to the “transition point;” this maximum delay was correlated with resorption of differentiating oral primordia, followed by the appearance of new primordia. The lesser delays observed at earlier times were correlated with temporary blockage of development of primordia in the “stomatogenic field” stage. Resumption of oral primordium development was, in both cases, temporally correlated with a substantial recovery of protein synthesis. After the “transition point,” cell division, and completion of oral development, was delayed slightly at the lower concentrations, and more substantially at 1 and 10 μg/ml, with some division-arrest at the latter concentration. Except for the recovery phenomenon, the developmental responses elicited by cycloheximide were similar to those observed earlier with puromycin. The bearing of these findings on the mechanism of synchronization in Tetrahymena is considered in the Discussion.     

Heat shock protein synthesis and thermotolerance in Salmonella typhimurium

The resistance of stationary phase Salmonella typhimurium to heating at 55°C was greater in cells grown in nutritionally rich than in minimal media, but in all media tested resistance was enhanced by exposing cells to a primary heat shock at 48°C. Chloramphenicol reduced the acquisition of thermotolerance in all media but did not completely prevent it in any.
The onset of thermotolerance was accompanied by increased synthesis of major heat shock proteins of molecular weight about 83, 72, 64 and 25 kDa. When cells were shifted from 48°C to 37°C, however, thermotolerance was rapidly lost with no corresponding decrease in the levels of these proteins. There is thus no direct relationship between thermotolerance and the cellular content of the major heat shock proteins. One minor protein of molecular weight about 34 kDa disappeared rapidly following a temperature down-shift. Its presence in the cell was thus correlated with the thermotolerant state.     

Dibutyryl cAMP-induced protein changes in differentiating mouse neuroblastoma cells.

Proteins from mouse neuroblastoma cells treated with dibutyryl adenosine-3',5'-monophosphate (B2cAMP) were analyzed by high resolution, two-dimensional gel electrophoresis. Quantitative changes in proteins and charge modifications of proteins apparently induced B2cAMP were detected by isoelectric focusing. Some proteins appeared to be modified and one protein was increased 7- to 8-fold in cells treated with B2cAMP. Since neuroblastoma cells differentiate when treated with B2cAMP, understanding the protein changes induced by B2cAMP may help to understand cellular differentiation in neural tissue.     

Heat shock protein synthesis and thermotolerance in Salmonella typhimurium

The resistance of stationary phase Salmonella typhimurium to heating at 55 degrees C was greater in cells grown in nutritionally rich than in minimal media, but in all media tested resistance was enhanced by exposing cells to a primary heat shock at 48 degrees C. Chloramphenicol reduced the acquisition of thermotolerance in all media but did not completely prevent it in any. The onset of thermotolerance was accompanied by increased synthesis of major heat shock proteins of molecular weight about 83, 72, 64 and 25 kDa. When cells were shifted from 48 degrees C to 37 degrees C, however, thermotolerance was rapidly lost with no corresponding decrease in the levels of these proteins. There is thus no direct relationship between thermotolerance and the cellular content of the major heat shock proteins. One minor protein of molecular weight about 34 kDa disappeared rapidly following a temperature down-shift. Its presence in the cell was thus correlated with the thermotolerant state.     

Evidence for a role of heat-shock proteins in proliferation after heat treatment of synchronized mouse neuroblastoma cells

A role for heat-shock proteins (HSPs) in proliferation after heat treatment was considered in synchronized mouse neuroblastoma cells. For this purpose enhancement of HSP synthesis after heat treatment was inhibited by actinomycin D and the effect of this on cell cycle progression into mitosis and on cell survival was studied both in thermoresistant G1- and in thermosensitive late S/G2-phase cells. In G1-phase cells expression of basal and heat-induced HSP synthesis was the same as that in late S/G2-phase cells, which suggests that regulation of thermoresistance throughout the cell cycle is not directly linked with HSP synthesis. The synthesis of HSP36, HSP68, and HSP70 was enhanced after a 30-min treatment at 41-43 degrees C. Increase of HSP synthesis after heat shock was partly suppressed by the presence of 0.1 microgram/ml actinomycin D during heat treatment, while 0.2 micrograms/ml prevented enhancement of HSP synthesis completely. Suppression of heat-induced HSP synthesis by actinomycin D had the same concentration dependency in G1- and late S/G2-phase cells. Actinomycin D potentiated induction of mitotic delay by heat treatment (30 min, 42.5 degrees C) but only under conditions where it actually inhibited heat-induced enhancement of HSP synthesis. Heat-induced cell killing was also potentiated by actinomycin D. The potentiating effect of actinomycin D on heat-induced mitotic delay and on heat-induced cell killing was more pronounced in G1-phase cells than in late S/G2-phase cells. These results give evidence for a role of HSPs in the resumption of proliferation after heat treatment and suggest that heated G1-phase cells are more dependent on HSP synthesis for recovery of proliferation after heat treatment than heated late S/G2-phase cells.