Incubation Of HeLa Cells Research Articles

Morphological alterations and ganglioside sialyltransferase activity induced by small fatty acids in HeLa cells.

Incubation of HeLa cells in the presence of millimolar concentrations of propionate, butyrate, or pentanoate increases the specific activity of CMP-sialic acid:lactosylceramide sialyltransferase 7-20-fold within 24 h. Longer-chain saturated fatty acids or acetate are much less effective, decanoate showing no induction. Unsaturated fatty acid analogs of butyrate and other compounds are ineffective. Only the three most effective compounds also produce characteristic smooth extended cell processes in HeLa cells. Butyrate (5 mM) induces the sialyltransferase after a 4-h lag, producing maximum specific activity by 24 h. The amount of sialyl-lactosylceramide, the glycolipid product of the enzyme, increases during that time 3.5 times more than in control cultures. No other glycosphingolipid enzyme is significantly altered by butyrate exposure. The cellular shape changes occur 2-3 h later than the increase of sialyltransferase activity, and both processes require the continuous presence of inducer and the synthesis of RNA and protein but not the synthesis of DNA or the presence of serum.

Sensitization of HeLa cells for viral RNA infection by cytochalasin B

Incubation of HeLa cells in medium containing 5 μg cytochalasin B/ml for 5–10 min at 37° results in: (a) 30- to 80-fold increase in the sensitivity of cells for infection by isolated poliovirus RNA, (b) reduced viral RNA adsorption and penetration, and (c) immediate inhibition of incorporation of labeled amino acids into proteins. Protein synthesis in poliovirus-infected cells, however, continues unabated for 15 min in the presence of a ten-fold higher concentration of cytochalasin B (50 μg/ml). Apparently, viral RNA can efficiently direct protein synthesis under conditions where host RNA translation is inhibited.

Rapid labeling of DNA-C during incubation of HeLa cells with (5-T) uridine

(5-T) uridine is a widely used radioactive precursor for the analysis of RNA metabolism. It has been proposed as a specific marker for RNA formation, cf. [ 1 ] because its label is lost during conversion from U to dT. As this may be valid for very short incubation times, it certainly introduces serious errors in studies exceeding l/ 10 of the cell doubling time. As shown in this paper, the radioactivity very soon enters DNA via [“HI UTP+[3H]CTP+[3H] dCTP conversion, thus labeling DNA exclusively in C bases. The U to C conversion was analysed in HeLa suspension cultures labeled with (5-T) uridine at different concentrations. C bases in RNA became rapidly labeled, though with some delay to U bases. The time needed to reach equilibrium depended on the concentration of the exogenous uridine indicating profound differences of UTP and CTP pools under these conditions. Base analysis of DNA showed a time-dependent increase in radioactivity which was exclusively located in the cytosine residues.

Reversible Inhibition of Protein Synthesis in HeLa Cells by Dimethylsulfoxide

Abstract Incubation of HeLa cells with dimethylsulfoxide results in a partial or complete inhibition of protein synthesis. At a concentration of 12% dimethylsulfoxide, a complete inhibition of protein synthesis occurs within 3 to 4 min. This inhibition is reversible; on removal of dimethylsulfoxide, cells resume their normal rate of protein synthesis. Inhibition of protein synthesis by dimethylsulfoxide is accompanied by a complete breakdown of polyribosomes and release of nascent polypeptides. The breakdown of polyribosomes can be prevented by preincubating cells either with cycloheximide or anisomycin. These studies suggest that dimethylsulfoxide may be a useful tool to synchronize chain initiation in cultured cells.

RNA metabolism in HeLa cells at reduced temperature. II. Steps in the processing of transfer RNA.

Incubation of HeLa cells at 24 degrees C results in the modification of the processing of pre-tRNA to tRNA. Both methylation and size reduction were shown to take place in vitro when purified pre-tRNA was subjected to processing in a cytoplasmic extract of HeLa cells The migration of pre-tRNA from the nucleus to the cytoplasm was not significantly altered at 24 degrees C

A Study of Mitochondrial Protein Synthesis in Intact HeLa Cells

Mitochondrial protein synthesis has been studied in intact HeLa cells by utilizing specific inhibitors. We have observed that in the presence of pederine, an inhibitor of cytoplasmic protein synthesis in mammalian cells, protein synthesis continues for at least 1 h at approximately 0.5% of the rate of untreated cells. When control cells and cells treated with pederine are fractionated after an incubation with labeled amino acids into a mitochondrial and a supernatant fraction, it is found that incorporation into the mitochondrial fraction is higher in the presence of the inhibitor. Mitochondrial protein synthesis has been shown to take place on aggregates of mitochondrial ribosomes.The synthesis of mitochondrial proteins has been studied by incubating HeLa cells with labeled amino acids and either pederine or chloramphenicol, an inhibitor of mitochondrial protein synthesis. We have been able to establish that the structural protein of mitochondrial ribosomes is synthesized on cytoplasmic ribosomes and its synthesis is thus inhibited by pederine, but not by chloramphenicol. Mitochondrial proteins have been analyzed by acrylamide gel electrophoresis after fractionation of the mitochondria into inner and outer membranes. Proteins synthesized by mitochondria in the presence of pederine are located predominantly on the inner mitochondrial membranes. Only few protein species synthesized by mitochondria are resolved by gel electrophoresis. This finding is discussed in relationship to the information content of mitochondrial DNA.

RNA metabolism in HeLa cells at reduced temperature. I. Modified processing of 45S RNA.

Incubation of HeLa cells at suboptimal temperature has been used to study the synthesis of 45S ribosomal RNA precursor and the individual steps of the subsequent processing to 28S RNA. Below 20 degrees C no detectable 45S RNA is formed. The processing of 45S RNA to 32S RNA ceases around 15 degrees C, and the processing of 32S RNA to 28S RNA is inhibited near 25 degrees C. Prolonged incubation at reduced temperature results in further modification of the processing, resulting in the apparent accumulation of 41S RNA. The products of these reactions at reduced temperature appear normal in that the ribosomal RNA made at 27 degrees C can be isolated from functional polyribosomes in the cytoplasm after a short incubation at 37 degrees C.

Effect of sodium fluoride on protein synthesis in HeLa cells: Inhibition of ribosome dissociation

Incubation of HeLa cells with sodium fluoride results in inhibition of protein synthesis, disaggregation of polyribosomes, accumulation of 80 s ribosomes and decrease of free ribosomal subunits. Part of the messenger released from polyribosomes by fluoride is recovered in a particle sedimenting at 95 s. The decrease of ribosomal subunits occurs only in cells which are active in protein synthesis and appears to be due to the block of ribosome dissociation which in turn prevents ribosome recycling. The analysis of small ribosomal subunits “chasing” into ribosomes, when the free ribosomal subunit pool is depleted in the presence of sodium fluoride, shows no evidence of selectivity between relatively new and old particles in this conversion. After the removal of sodium fluoride, the normal level of free ribosomal subunits is restored at the expense of a random dissociation of ribosomes. The results are consistent with a model of ribosome function in which cyclic dissociation into free ribosomal subunits takes place.

Ultraviolet motion picture studies of effects of ribonuclease and deoxyribonuclease on living HeLa cells.

Summary1) Living HeLa cells were incubated in various concentrations of ribonu-clease and deoxyribonuclease and their ultraviolet absorption characteristics were followed for 6 hours by means of time lapse motion pictures obtained with the ultra-violet flying spot television microscope. 2) No change in ultra-violet absorption images of cells was detectable as long as 2 hours after the time that replicate cultures demonstrated a loss of affinity of cytoplasm for basic dyes and the nuclei were negative with Fuelgen stain. 3) Living HeLa cells react to incubation in ribonuclease by first shrinking slightly and then expanding their cytoplasm markedly. 4) Prolonged incubation of HeLa cells in either enzyme eventually produces collapse and pyknosis of all of the cells in the culture.