Protein Synthesis In HeLa Cells Research Articles

Structural difference between the 5' termini of viral and cellular mRNA in poliovirus-infected cells: possible basis for the inhibition of host protein synthesis

Host protein synthesis in poliovirus-infected HeLa cells is interrupted, but the host mRNA appears to remain completely intact and unmodified. The average size and poly (A) content of host mRNA was previously known to be unchanged (Koschel, 1974; Leibowitz and Penman, 1971), and this was confirmed. In addition, the 5' terminal methylated "cap" structures remained intact, and no further base modifications at the level of 1 base in 1,000 could be detected. Poliovirus RNA from viruses was previously shown not to have "caps" (Wimmer, 1972), and in this work poliovirus RNA from polyribosomes was found to have pUp at its 5' end. Since, initiation of protein synthesis is probably the basis for the inhibition of cellular protein synthesis in infected cells, the difference in the 5' ends of the host cell and viral RNA could be the basis of selective translation of viral RNA during infection.

Inhibition of protein and RNA synthesis by 5-mercaptopyridoxine

The effect of 5-mercaptopyridoxine (vitamin B 6SH) on nucleic acid and protein synthesis in HeLa cells was studied with radioactive precursors. The compound inhibited both RNA and protein synthesis, independently and simultaneously. In these studies, vitamin B 6SH did not interfere with amino acid transport or the uptake of nucleic acid precursors and vitamin B 6. The addition of pyridoxine or pyridoxal phosphate did not prevent the inhibitory effect of the compound on protein synthesis. The previously reported reduction in antibody titers in patients with auto-immune disease treated with this compound might be a reflection of its inhibitory effect on RNA and protein synthesis, as demonstrated in these studies.

Inhibition of protein synthesis in intact HeLa cells.

Polysome analysis has proved to be a sensitive probe for the mode of action of inhibitors of protein synthesis in intact HeLa cells. To classify the active compounds as inhibitors of initiation, elongation, or termination, their effects on the cellular polyribosome pattern were compared under three conditions. These conditions tested (i) their direct effect on the polyribosome profile; (ii) their effect on ribosome run-off produced by hypertonicity; and (iii) their effects on recovery from hypertonicity. Using this technique, diacetoxyscirpenol, 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide, and three alkaloids, harringtonine, isoharringtonine, and homoharringtonine, were found to be inhibitors of initiation. Polysome analysis indicated that in HeLa cells 7.8 x 10(-7) M pactamycin, which inhibited protein synthesis 94%, interfered with elongation as well as initiation under these conditions. Emetine, anisomycin, cycloheximide, and trichodermin each gave polysome patterns consistent with inhibition of elongation. Fusidic acid and aurintricarboxylic acid inhibited incorporation of [(14)C]leucine into intact HeLa cells, but polysome analysis did not localize any specific inhibitory effects to the initiation, elongation, or termination steps of protein synthesis. The use of specific inhibitors of initiation of protein synthesis has indicated that most, if not all, mammalian messenger ribonucleic acids contain a single initiation site.

Identification of discrete electrophoretic components among the products of mitochondrial protein synthesis in HeLa cells.

Abstract Proteins synthesized in vivo by HeLa cell mitochondria in the presence of emetine, an inhibitor of cytoplasmic protein synthesis, have been characterized as to their electrophoretic mobility, solubility properties in organic solvents, and kinetics of labeling with [3H]isoleucine. Ten distinct electrophoretic components in the molecular weight range from about 11,000 to 42,000 have been identified with high reproducibility among the products of mitochondrial protein synthesis. Control experiments involving direct sodium dodecyl sulfate lysis of whole cells, or the use of the protease inhibitor phenylmethylsulfonylfluoride during cell homogenization and fractionation, have excluded a significant role of enzymic degradation during extraction in producing the observed electrophoretic pattern of mitochondrial protein products. A selective solubilization of mitochondrial protein products, representing a 20 to 30-fold purification with respect to the cytoplasmically synthesized proteins, has been obtained by using a neutral chloroform/methanol mixture. At least six of the electrophoretic components with a molecular weight greater than 20,000 were found to be extracted to an appreciable extent with neutral chloroform/methanol. A fairly co-ordinate labeling of the discrete electrophoretic components has been observed during a one-hour exposure period of the cells to [3H]isoleucine in the presence of emetine, without any clear evidence of intorconversion.

Inhibition of host protein synthesis by vaccinia virus: fate of cell mRNA and synthesis of small poly (A)-rich polyribonucleotides in the presence of actinomycin D.

Purified vaccinia virus rapidly inhibited HeLa cell protein synthesis in the presence of actinomycin D. Under these conditions host polyribosomes were extensively degraded but the mRNA was stable as indicated by a greater than 90% recovery of prelabeled polyadenylylated RNA. Although actinomycin D prevented the synthesis of host mRNA and poly(A) in uninfected cells, incorporation of adenosine into poly(A) was inhibited by less than 50% in infected cells. Further analysis indicated that there was little or no normal size viral mRNA but that a unique class of small poly(A)-rich RNA was made in the presence of actinomycin D. From measurements of the RNase resistance and base composition of the RNA, approximately 40% of the nucleotide sequence was estimated to be poly(A). The poly(A)-rich RNA was found associated with small polyribosomes and monoribosomes that were inactive in protein synthesis. It was suggested that the poly(A) segment of the RNA is formed by the poly(A) polymerase previously found in vaccinia virus cores and that the inactive RNA, by competing with host mRNA, may contribute to the virus-mediated inhibition of host protein synthesis observed in the presence of actinomycin D.

Conformation-Dependent Antigenic Determinants in the Toxic Lectin Ricin

The major part of the ricin-precipitable antibodies in sera produced by immunizing rabbits with formaldehyde-treated ricin is precipitated also by the isolated ricin A and B chains. In contrast, in antisera produced by immunizing with formaldehyde-treated ricinus agglutinin only a small part of the antibodies cross-reacting with ricin can be precipitated by the isolated A and B chains, or bound to immunoabsorbents containing the isolated ricin chains. In immunodiffusion studies with anti-ricinus agglutinin sera, a star-shaped precipitate was formed when isolated A and B chains recombined to form intact ricin. Both anti-ricin and anti-ricinus agglutinin sera neutralized effectively the ability of ricin to inhibit protein synthesis in HeLa cells. Anti-ricin serum also neutralized the inhibitory effect of the isolated A chain on protein synthesis in a cell-free system and the ability of the isolated B chain to induce indirect hemagglutination. In contrast, antiricinus agglutinin serum did not neutralize the biologic activities of the isolated ricin A and B chains. Anti-ricinus agglutinin serum formed a precipitate with the hybrid ricin A chain/abrin B chain, and protected against the toxic effect on HeLa cells of this hybrid, indicating conformational changes of ricin A chain upon binding to the B chain. It is concluded that the anti-ricinus agglutinin serum contains antibodies directed against conformational determinants present on intact ricin, but not present or exposed in the isolated A and B chains. At least part of these conformational determinants appears to be carried by the A chain.

Effects of cycloheximide on chromatin biosynthesis

In the presence of sufficient cycloheximide, puromycin or NaCl to quantitatively inhibit protein synthesis in HeLa cells, thymidine incorporation continues at 20% of control rates for 60 to 90 minutes, after which incorporation gradually ceases. Both DNA and protein synthesis revert to control rates in about five minutes after removal of cycloheximide. DNA synthesis in the presence of cycloheximide appears to be a continuation of the replicative process by several criteria. The persistent DNA synthesis in the presence of cycloheximide is abolished by hydroxyurea, which does not inhibit repair synthesis, while ethidium bromide, an inhibitor of mitochondrial DNA synthesis, is without effect. Nuclear DNA is not nicked during incubation in cycloheximide. Low molecular weight Okazaki fragments (4 to 5 S) are both synthesized and processed to high molecular weight DNA in cells treated with cycloheximide. Replication forks, identified in alkaline CsCl gradients by incorporation of bromodeoxyuridine as a density marker just before the addition of cycloheximide, are selectively labeled with radioactive thymidine during DNA synthesis. In the presence of cycloheximide the maturation of DNA intermediates into high molecular weight DNA is defective. All size classes of DNA fragments, normally present during progression of low to high molecular weight DNA, are demonstrable in cells preincubated in cycloheximide for prolonged periods. However, 21 S fragments, intermediate in size between Okazaki pieces and mature, high molecular weight DNA, accumulate in cells treated with cycloheximide, demonstrating a defect in maturation of the 21 S intermediates into high molecular weight DNA. After removal of the cycloheximide, the 21 S DNA fragments are processed to high molecular weight DNA at a significantly impaired rate, requiring about three hours for completion of chain growth as compared to 40 to 60 minutes in controls. The slowed growth of DNA fragments synthesized in the presence of cycloheximide following drug removal is not due to persisting effects of cyeloheximide since DNA synthesis immediately following removal of the drug has chain growth rates similar to that of controls. Pools of chromatin proteins exist in HeLa cells, as demonstrated by a brief, labeled amino acid pulse followed by a chase with cycloheximide. The specific activity of chromatin proteins increases significantly during 60 minutes of cycloheximide inhibition. Histone f2a1 accumulates preferentially during this chase period, suggesting that a supply of this highly conserved histone might be requisite to continued replication. Comparison of chromatin synthesized during cycloheximide treatment with pulse-labeled control chromatin has provided insight into the mechanism of assembly of proteins and DNA into the nucleoprotein complex. The DNA of ch-chromatin † † Abbreviations used: ch-chromatin, chromatin synthesized during exposure of cells to cycloheximide; chDNA, nuclear DNA synthesized during exposure of cells to 200 μg cycloheximide/ml. is more susceptible to nuclease digestion than control chromatin, suggesting that it is deficient in protein content. Upon reversal of cycloheximide inhibition, the recovery of nuclease digestibility of ch-chromatin to control values takes two to three hours, a time similar to that required for conversion of the corresponding 21 S chDNA fragments to high molecular weight DNA. Briefly pulse-labeled (30 to 60 s) DNA in control chromatin also has an enhanced susceptibility to nuclease digestion of the same degree as found in ch-ehromatin. The time of recovery of increased nuclease susceptibility of newly made chromatin DNA ( via protein addition) to control levels is about 10 to 15 minutes and corresponds to the time required for synthesis of replicon-sized units of DNA. In addition to being nuclease-sensitive, both cycloheximide and newly synthesized (30 to 60 s) chromatin have lighter buoyant densities in CsCl gradients than bulk chromatin. This property exists for only one to two minutes in controls and is probably due to structural properties distinct from those rendering nuclease sensitivity. Limit digests of chromatin by micrococcal nuclease yield a characteristic pattern of polynucleotides when resolved in polyacrylamide gels. The radioactivity profiles of limit digest polynucleotides from control and ch-chromatin are identical, indicating that pre-existing chromatin proteins remain in place on newly replicated DNA in the same fashion as in mature chromatin.

Effect of DEAE-dextran on protein synthesis in HeLa cells.

Concentrations of DEAE-dextran which are currently used to stimulate the interaction between animal cells and natural or synthetic polynucleotides, inhibit protein synthesis in HeLa cells. This inhibition results from the blocking of more than one of the steps in protein synthesis, including transport of amino acids. The results support the hypothesis that enhancement of cellular competence for infection by viral RNA involves interference with initiation of cellular protein synthesis.

Source of amino acids used for protein synthesis in HeLa cells.

HeLa cells, prelabeled with [3H]leucine for several generations, were pulse‐labeled with [14C]‐leucine. The 14C/3H ratio of the leucine in the medium, in the total amino acid pool and bound to transfer RNA was determined.It was found that the 14C/3H ratio of the leucyl‐tRNA was identical with that of the extracellular medium while that of the amino acid pool was much lower. These results confirm previous reports that selection of amino acids for protein synthesis takes place before the amino acids are released intracellularly.Further, it was concluded that amino acids resulting from intracellular proteolysis are not reutilized directly under the conditions of our experiments.A possible model of intracellular amino acid pathways is discussed.

Reversible inhibition of protein synthesis in HeLa cells by exposure to proteolytic enzymes

Protein synthesis in suspended HeLa S 3 cells is inhibited by more than 50% immediately after addition of 100 μg pronase/ml or 500 μg trypsin/ml. Polyribosome profiles are not altered by exposure of cells to 1 or 2 mg trypsin/ml suggesting that the inhibition affects peptide chain elongation. Protein synthesis resumes after removal of proteases by sedimentation and resuspension of the cells.

Lectins from Abrus Precatorius and Ricinus Communis

Abstract The quantitative precipitation reactions of rabbit anti-abrin and anti-ricin sera have been studied with the purified toxins, abrin, and ricin, their isolated A- and B-chains and with the corresponding nontoxic Abrus and Ricinus agglutinins. In the case of ricin, the results are consistent with a model in which the agglutinin contains two lactose-binding B-chains, immunologically indistinguishable from the single B-chain of ricin and two A-chains which lack certain antigenic determinants present on the ricin A-chain. The relation between abrin and its agglutinin appears basically similar, although somewhat more complex. Both Abrus and Ricinus lectins react with lactose and with glycoproteins containing terminal nonreducing galactose residues that are present on certain proteins in the normal serum of many animal species and on the membranes of animal cells. Therefore precipitation reactions must be carried out in the presence of lactose or galactose. Dose-response curves of the effect of abrin and ricin on protein synthesis in HeLa cells are shown in the absence and presence of lactose. A method for estimation of potency of ricin antitoxin using the HeLa cell system is described.

Selective and reversible inhibition of initiation of protein synthesis in mammalian cells.

Abstract Hypertonicity effected by elevation of NaCl concentration in the growth medium results in a rapid cessation of protein synthesis in HeLa cells accompanied by a complete breakdown of polyribosomes. Since elongation and termination of polypeptide synthesis proceeds normally, it is concluded that elevated NaCl concentration in the medium selectively inhibits the initiation of peptide chain formation. Pulse-labeling of poliovirus-infected cells at different times after incubation of the cells in hypertonic medium can be used to map the poliovirus genome. Upon restoration of isotonicity re-initiation of protein synthesis occurs only at the proper initiation site and synthesis proceeds at a normal rate. This synchronized re-initiation of protein synthesis observed upon restoration of isotonicity, in turn, allowed us to determine the gene sequence of poliovirus RNA in a novel way.

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.

The mechanism of inhibition of protein synthesis by 5-azacytidine in HeLa cells

The base analogue 5-azacytidine is shown to inhibit protein synthesis in HeLa cells with a half-life of 90 min. Inhibition apparently occurs at the level of initiation of translation. The analogue must be incorporated into RNA, since its effect is suppressed by actinomycin. The defective RNA which causes inhibition is probably neither messenger nor ribosomal RNA. 5-Azacytidine may interfere with the actinomycin-sensitive metabolism involved in initiating translation in HeLa cells.

Regulation of protein synthesis in mammalian cells: V. Further studies on the effect of actinomycin D on translation control in Hela cells

The rate of protein synthesis in HeLa cells appears to be regulated, in part, by a factor which promotes the association of ribosomes with messenger RNA and whose production is inhibited by actinomycin. The decline in protein synthesis after the administration of actinomycin is not primarily due to a decay of available messenger RNA but, rather, is a result of a decrease in the rate of ribosomal association with message. The decay of protein synthesis in actinomycin can be varied over a wide range by altering the temperature of cell incubation. Thus the half-life of protein synthesis decay ranges from eight hours at 34 °C to two hours at 41°C. The rapid decline of protein synthesis at 41 °C is not accompanied by a corresponding decay of the messenger RNA. Polyribosomes decrease in size, but they can be restored to normal sedimentation distributions by low levels of cycloheximide, suggesting that messenger RNA remains functional. The translation rate at 41 °C is unaltered. The dose-response of protein synthesis inhibition by actinomycin was measured and a half-maximum inhibition was found to be effected by 0·1 μg of the drug/ml. Another important aspect of the regulation of translation in HeLa cells is the response of cells to depressed rates of protein synthesis. At 42 °C, protein synthesis is severely inhibited, due to a failure in the association of ribosomes with messenger RNA. Prolonged incubation at the elevated temperature results in a significant repair of the lesion. This repair is inhibited by actinomycin. The half-maximum inhibition is achieved at levels of from 0·05 to 0·1 μg of the drug/ml. The cell response to depressed rates of protein synthesis can also be demonstrated using the drug cycloheximide. Prolonged incubation in the drug results in a response which then can promote protein synthesis at 42 °C. Here again, the half-maximum inhibition of the response to cyclohemixide is achieved by 0·1 μg of actinomycin/ml. These experiments suggest, but do not prove, that the cellular response may be mediated through the synthesis of RNA that promotes the initiation of translation and does not involve the subsequent production of protein.

Intracellular amino acids and protein synthesis in Hela cells

(1) When the intracellular amino acid pool is prelabelled and subsequently chased in non-radioactive medium, the radioactivity of the amino acid pool is not found to have been incorporated into protein. (2) Leucine transport into Hela cells is reduced in the presence of 10 mM valine in the medium. This results in a lower specific radioactivity of leucine in the intracellular amino acid pool. However, neither the overall rate of protein synthesis nor the incorporation of radioactive leucine into protein is affected. From these experiments it is concluded that incoming amino acids entering the intracellular amino acid pool are not used for de novo synthesis of protein.

Atypical pattern of utilization of amino acids for mitochondrial protein synthesis in HeLa cells.

The capacity of HeLa cell mitochondria, either isolated or in intact cells, to incorporate different labeled amino acids into proteins was investigated. Eight amino acids (alanine, arginine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, and lysine), which include most of the charged polar ones, showed a very low amount, if any at all, of chloramphenicol-sensitive incorporation, relative to that expected for an "average" HeLa-cell protein. By contrast, the most hydrophobic amino acids (leucine, isoleucine, valine, phenylalanine, and methionine) were the most actively incorporated by HeLa mitochondria. The available evidence suggests that pool effects cannot account for this general pattern of utilization of amino acids; furthermore, this pattern is in good agreement with the known hydrophobic properties of proteins synthesized in mitochondria.

A screen for pesticide toxicity to protein and RNA synthesis in HeLa cells.

A screen for pesticide toxicity to protein and RNA synthesis in HeLa cells.

XXIV. Effect of mikamycin, carbomycin, spiramycin, erythromycin, and paromomycin on growth and respiration of HeLa cells

HeLa cells have been grown in tissue culture for a period corresponding to three control cell generations in the presence of the antibiotics mikamycin, carbomycin, spiramycin, erythromycin, and paromomycin. All of these antibiotics inhibit yeast mitochondrial and Bacteriol protein synthesis. It has been found that at low concentrations mikamycin and carbomycin selectively inhibit mitochondrial protein synthesis in HeLa cells, with reduction in cytochrome a and a 3 levels and consequent growth inhibition after two cell divisions. Erythromycin and paromomycin do not show this effect at all, while spiramycin has some minor effects on protein synthesis and cytochrome formation. At high concentrations all five antibiotics show a second effect, namely a direct and immediate slowing or cessation of growth. In this instance mikamycin appears to act by means of its direct inhibitory effect on electron transport between cytochromes b and c 1; no inhibition of respiratory processes by the other antibiotics was demonstrated, and the cause of their growth inhibitory effects remains to be established. The results are discussed in terms of antibiotic effects on an intact cell system compared to isolated organelle studies; the usefulness of the HeLa cell system as a model to explain effects in the intact animal is emphasized.

Initiation of protein synthesis in HeLa cells.

Initiation of protein synthesis in HeLa cells has been synchronized by exposure of the cells to fluoride. Double-labeling of such cells for short pulses with [(35)S]methionine and a tritiated amino acid, followed by Edman degradation of the puromycin-released nascent peptides, has shown that the percent of N-terminal methionine incorporated compared to total incorporation is significantly higher than the value obtained with any of the other amino acids tested. The results suggest that the bulk of the nascent proteins synthesized in vivo by HeLa cells are initiated with methionine.