Initial inhibition and recovery of protein synthesis in cycloheximide-treated hepatocytes

Previous studies conducted with intact rats had demonstrated that protein synthesis was reversibly inhibited by cycloheximide. Polysome aggregation occurred during inhibition with a return to normal during recovery, suggesting that the block of translational activity involved termination and release of polypeptides. This study involving freshly isolated hepatocytes was undertaken to clarify the mechanism of the biphasic response to cycloheximide. Cycloheximide at 1 μM inhibited [³H]leucine incorporation into both cellular and secreted proteins by at least 86%, without having deleterious effects on membrane integrity as indicated by trypan blue uptake and lactate dehydrogenase (LDH) (EC 1.1.1.27) release. After removal of cycloheximide, incorporation of labeled amino acids into cellular protein and protein secreted into the medium returned to control levels. Kinetically, incorporation into secreted protein exhibited a lag of 30–45 min, indicating that a longer recovery period for restoration of proteosynthetic ability is required for membrane-bound polysomes. During the first 100 min of the recovery period, 30% of the cellular protein, which had been prelabeled during cycloheximide inhibition, was secreted into the medium; treated cells, however, secreted prelabeled protein at a lower initial rate. To elucidate the mechanism of action of cycloheximide, the content of the cytoplasmic ribo-nucleoprotein complexes (RFC), polysome size classes, and the distribution of radioactivity among the various ribosome classes were determined during inhibition and recovery. Larger size class polysomes (7+) were increased by cycloheximide treatment and remained increased during recovery. During inhibition, there was enhanced [³H]leucine labeling with increasing polysome size, implicating termination as the rate-limiting step, whereas during the recovery phase the labeled nascent polypeptides were removed from the ribonucleoprotein complex at a 3- to 4-fold greater rate than control, indicating an accelerated release of completed proteins.     

Aflatoxin induced inhibition of protein synthesis

The effect of aflatoxins B₁, G₁ and M₁ on protein synthesis in rat liver was studied. Wistar rats (80–100 g body weight) were injected i.p. with aflatoxins B₁, G₁ and M₁ (6, 12 and 6 mg/kg) respectively dissolved in dimethyl sulfoxide and were sacrificed 24 hr after dosing. Aflatoxins B₁ and M₁ but not G₁, inhibited in vivo protein synthesis. Aflatoxins M₁ G₁, but not B₁ inhibited in vitro mitochondrial protein synthesis, whereas both B₁ and M₁ inhibited cytoplasmic protein synthesis.     

DNA and protein synthesis inhibition in Chinese hamster ovary cells by dichlorophenoxyacetic acid

The effects of the herbicide dichlorophenoxyacetic acid (2,4-D) on DNA and protein synthesis were investigated in chinese hamster ovary cells (CHO) employing two different methods. The results showed that the herbicide affects DNA and protein synthesis depending on the stage of growth and method of treatment. 2,4-D action appears to concentrate the cells mainly in the G1/S boundary of the cell cycle. The effect is expressed as an inhibition of DNA and protein synthesis. This effect was revealed not only by the chemical determination of DNA and protein synthesis but also by experiments using autoradiography, using the labelling index to detect the incorporation of [3H]thymidine into the cells. Labelling of the cell nucleus was reduced markedly when cells treated with 2,4-D were in confluency for 4 days after reaching plateau growth.     

Reversible inhibition of DNA, RNA and protein synthesis in human cells by lead chloride.

An acute intoxication by lead chloride (conc. 2.5 - 10(-4)M) produces a temporary reduction of macromolecular syntheses in HeLa cells growing asynchronously. The reduction is similar for DNA, RNA and proteins and differs only in the intensity. After a one-day intoxication, if the cells are put back in a fresh medium, the syntheses return to normal within 10 h. The histochemical sulphide-silver method shows that lead is present in the cells during the inhibition.     

Role of protein kinase C in the phosphatidylserine-induced inhibition of DNA synthesis in blood mononuclear cells.

The mechanism of immunosuppressant activity of phosphatidylserine has been studied in peripheral blood mononuclear cells depleted or not of monocytes. After the addition of phosphatidylserine, mass determinations and uptake of labeled compound demonstrate its transfer into the cells. Phosphatidylserine incorporation causes a 2.5-fold increase of membrane-bound protein kinase C activity. The activation of translocated enzyme is indicated by the inhibition of phosphoinositide hydrolysis, and early feedback effect induced by activated protein kinase C. This action of phosphatidylserine is reproduced by tetradecanoylphorbolacetate and is prevented by the protein kinase C inhibitor, staurosporine. Consistently, phosphatidylserine (8 nmol/10(6) cells) decreases by 46% the production of inositol phosphates in cells responding to phytohemagglutinin. The decrease of phosphoinositide signal pathway as well as the inhibition of mitogen-induced DNA synthesis are produced at the same phosphatidylserine concentration and are equally manifest in total mononuclear cells or in preparations depleted of monocytes. However, only in the presence of monocytes does tetradecanoylphorbolacetate enhance the action of phospholipid, decreasing its IC50 from 13-15 microM to 7 microM. Thus, the data suggest that a reaction driven by protein kinase-C and a factor released by activated monocytes are involved in the phosphatidylserine-induced inhibition of lymphocyte DNA synthesis.     

Rapid recovery of in vivo prostacyclin formation after inhibition by aspirin

Summary The effect of aspirin on the in vivo formation of prostacyclin and thromboxane A₂ in normal healthy individuals was studied by measuring the urinary excretion of 2,3-dinor-6-keto-PGF₁ and 2,3-dinor-TxB₂ by gas chromatography-mass spectrometry. Administration of 500 mg aspirin twice daily caused a sustained reduction in the excretion of 2,3-dinor-TxB₂ to 10–15% of the predose value, while the excretion of 2,3-dinor-6-keto-PGF₁ was reduced for only about 3 hours after the aspirin dose. The data demonstrate a considerable difference in the inhibitory effect of aspirin on the in vivo synthesis of thromboxane A₂ and prostacyclin.     

Acetaldehyde inhibition of protein synthesis in isolated rat pancreatic acini

Exposure of isolated dispersed pancreatic acini to increasing concentrations of ethanol (5 to 500 mM) or acetaldehyde (0.5 to 100 mM) produced a progressive inhibition of [3H]leucine incorporation into both "cellular" (those remaining in the cell) and "secretory" (those released into the medium) proteins. Whereas 500 mM ethanol caused 90-95% inhibition in the synthesis of "cellular" and "secretory" proteins, the concentration of acetaldehyde needed to produce a similar inhibition was found to be 50 mM. All subsequent experiments were performed with 12.5 mM acetaldehyde, a concentration that consistently inhibited acinar protein synthesis by about 50%. The acetaldehyde-mediated inhibition of acinar protein synthesis was partially normalized when this metabolite was removed after 30 min during a 90-min incubation period. In the presence of acetaldehyde, the secretion of 3H-pulse-labeled proteins, but not amylase, trypsinogen, or chymotrypsinogen, was greatly depressed. Acetaldehyde also caused a marked reduction in [3H]uridine incorporation into acinar RNA. The entry of [3H]uridine, [3H]leucine, and [3H]aminoisobutyric acid into isolated acini was found to be slightly (15-25%) decreased by acetaldehyde. It is concluded that acetaldehyde exerts a direct toxic effect on isolated dispersed pancreatic acini as evidenced by diminution of both protein and RNA synthesis and decreased secretion of the newly synthesized proteins. This inhibitory effect of acetaldehyde could be partially reversed.     

Carbocyclic puromycin: synthesis and inhibition of protein biosynthesis

The carbocyclic analogue of puromycin was prepared by the coupling of N-(benzyloxycarbonyl)-p-methoxy-L-phenylalanine to the racemic aminonucleoside (+/-)-9-[3 beta-amino-2 beta-hydroxy-4 alpha-(hydroxymethyl)cyclopent-1 alpha-yl]-6-(dimethylamino)purine, followed by separation of the diastereomers and subsequent removal of the Cbz blocking group. Kinetic studies indicate that carbocyclic puromycin is an excellent substrate for the peptidyltransferase reaction with both prokaryotic and eukaryotic ribosomes. A comparison of carbocyclic puromycin with previously synthesized analogues indicate that the furanosyl ring oxygen and the hydroxymethyl group of puromycin do not contribute to ribosomal binding, but both moieties contribute to the rate of product formation from the enzyme-substrate complex. Carbocyclic puromycin was equal to puromycin when evaluated for cytotoxicity using P-388 mouse lymphoid leukemia cells in culture.     

Barbiturate inhibition of growth and of synthesis of deoxyribonucleic acid, ribonucleic acid and protein in cultured mammalian cells

Pentobarbital (PB) inhibited growth and the synthesis of nucleic acids and protein in murine, mastocytoma cells (P815Y) grown in culture. The inhibition increased with an increase in the concentration of drug and was also time-dependent with a high level of drug. For example, 0.5 mM PB (id₅₀) reduced both the rate of division of the cells and the synthesis of DNA, RNA and protein by about one-half, compared with the control, over a 12-hr period. In contrast, treatment with a 2-fold higher concentration of PB (1 mM; id₁₀₀) blocked both cell division and the synthesis of protein promptly. It also reduced the synthesis of DNA and RNA by about one-half, compared with the control, during the first 4 hr of treatment. After this time, however, the synthesis of both DNA and RNA stopped abruptly. It is concluded that the inhibition of DNA synthesis caused by barbiturate in the latter case may have been secondary to the inhibition of protein synthesis. Transfer of the inhibited (1 mM PB; 12 hr) cells to drugfree medium caused the synthesis of protein and RNA to begin without apparent delay. In contrast, the synthesis of DNA proceeded slowly for about 8 hr; then the amount of DNA increased parasynchronously. Cell division, which also proceeded slowly during the first 10 hr, occurred as a parasynchronous wave some 2 hr after DNA synthesis began. The inhibitory effects of PB were also studied in murine, lymphoblastic cells (L5178Y) synchronized by sequential treatment with thymidine (5 hr) and deoxycytidine plus Colcemid (5 hr). When the mitotically inhibited cells were transferred to normal medium containing PB (1.5 mM; id₁₀₀), slightly more than one-half of the cells failed to complete mitosis and the synthesis of DNA, RNA and protein was blocked by the drug. The synthesis of DNA, RNA and protein was also blocked by PB addition in the middle of the S-phase. Its addition at the onset of the second wave of mitosis prevented mitosis as before and blocked the initiation of DNA synthesis.     

Role of protein phosphorylation in the inhibition of protein synthesis caused by hypoxia in rat hepatocytes

Hypoxia causes a rapid and reversible inhibition of translation in freshly isolated rat hepatocytes. This inhibition is neither due to an ATP loss nor to an increase in cell death. Because protein synthesis is mainly regulated by reversible phosphorylation of initiation and/or elongation factors, we investigated whether translation inhibition by hypoxia may be related to changes in the phosphorylation status of proteins. Whatever the incubation conditions, three phosphoreactive bands (molecular weights 220, 129, and 83 kDa) were detected by antiphosphotyrosine antibodies. The phosphorylation in the 129- and 83-kDa bands, however, was significantly and progressively decreased under hypoxia. Although this time-dependent decrease was sensitive to changes in oxygen tension, it occurred after the early protein synthesis inhibition caused by hypoxia. Moreover, sodium orthovanadate prevented tyrosine dephosphorylation in hypoxic cells, but did not restore the depressed protein synthesis caused by hypoxia. Under aerobic conditions, orthovanadate inhibited the synthesis of proteins, confirming that protein phosphorylation is a major mechanism involved in translational regulation. Once again, this inhibitory effect occurred only after 90 minutes of incubation whereas hypoxia inhibits the protein synthesis at the beginning of the incubation. Labeling cells with [33-32P]-ortho-phosphoric acid allowed detection of several phosphorylated proteins that appeared under hypoxia. Because they were not recognized by the phosphotyrosine antibodies, we suggest that serine/threonine residues of key proteins may be the putative hypoxic targets.     

Fluoride inhibition of DNA synthesis in isolated nuclei from cultured cells

DNA synthesis in isolated nuclei from both fluoride sensitive and resistant LS cells was inhibited by fluoride at and above 3 mM. These fluoride concentrations also had an inhibitory effect on DNA synthesis in intact sensitive cells, but not in resistant cells. Due to previous findings that the intracellular fluoride concentration in the sensitive cells is only 30-40% of the extracellular, it is suggested that inhibition of DNA synthesis in intact cells is secondary to the inhibitory effect on protein synthesis.