DNA and protein synthesis in cultured lens epithelial cells. I. Test system]

An in vitro test system for measuring DNA and protein synthesis in cultivated lens epithelium cells was developed. The method is suited also for other monolayer cultures; it has the following advantages: a) Cultivation of cells, incubation with radionuclides, preparation of the samples and measurement of radioactivity are carried out in the same vessel (scintillation vial); b) The use of 3H-thymidine and 14C-phenylalanine allows simultaneous measurement of DNA and protein synthesis; c) Only small amounts of cells (10(4) to 10(5) cells) are required to measure DNA and protein synthesis. The test system is highly sensitive to synthetic effectors (cytosone arabinoside, actinomycin D, puromycin), and is thus appropriate for the detection of inhibitors of DNA and protein synthesis and for testing the toxicity of drugs.     

In vitro DNA and RNA synthesis by human platelets.

In vitro incorporation of [Me-3H] thymidine and [5-3H] uridine into human platelets was demonstrated. Thymidine incorporation was inhibited by three specific inhibitors of DNA synthesis: hydroxyurea, cytosine arabinoside and daunomycin. The effect was dose-dependent. Uridine uptake by platelets was found to be inhibited by specific inhibitors of RNA synthesis such as actinomycin D, rifampicin and vincristine, the effect of actinomycin D being dose dependent. The drug also led to a time-dependent inhibition of protein synthesis when preincubated with platelets. The platelet RNA profile on polyacrylamide gel was demonstrated to be similar to that of embryonic mouse erythroblast RNA. Synthesis of all three fractions, 28 S, 18 S and 4 S, was inhibited by actinomycin D. These findings show that human platelets are capable of DNA and RNA synthesis, and that these activities play a role in controlling protein synthesis in these cells. Detectable amounts of DNA have been found in whole human platelets, and in isolated mitochondria derived from these cells. Isolated platelet mitochondria incorporated [3H] thymidine and [3H] uridine into their macromolecules. These activities were inhibited by daunomycin and by both rifampicin and actinomycin D, respectively. These results support the assumption that DNA and RNA synthesis found in intact cell preparations takes place most probably in platelet mitochondria.     

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.     

Synthesis of protein and mRNA is necessary for transition of suspension-cultured Catharanthus roseus cells from the G1 to the S phase of the cell cycle

The effects of inhibition of the synthesis of protein, mRNA or rRNA on the progression of the cell cycle have been analyzed in cultures of Catharanthus roseus in which cells were induced to divide in synchrony by the double phosphate starvation method. The partial inhibition of protein synthesis at the G₁ phase by anisoniycio or cycloheximide caused the arrest of cells in the G₁ phase or delayed the entry of cells into the S phase. When protein synthesis was partially inhibited at the S phase, cell division occurred to about the same extent as in the control. When asynchronously dividing cells were treated with cycloheximide, cells accumulated in the G₁ phase, as shown by flow-cytometric analysis. The partial inhibition of mRNA synthesis by α-amanitin at the G₁ phase caused the arrest of cells in the G₁ phase, although partial inhibition of mRNA synthesis at the S phase had little effect on cell division. In the case of inhibition of synthesis of rRNA by actinomycin D at the G₁ phase, initiation of DNA synthesis was observed, but no subsequent DNA synthesis or the division of cells occurred. However, the addition of actinomycin D during the S phase had no effect on cell division. These results suggest that specific protein(s), required for the progression of the cell cycle, are synthesized in the G₁ phase, and that the mRNA(s) that encode these proteins are also synthesized at the G₁ phase.     

Contribution of a change in mRNA half-life to the accumulation of the tissue-specific S-100 protein during postnatal development of the mouse brain

The S-100 protein accumulates rapidly in the mouse brain between 15 and 21 days of postnatal development. The accumulation of this protein is brought about mainly by an increased rate of its synthesis. The present study focuses on attempts to determine if a change in the half-life of messenger RNA (mRNA) is involved in bringing about the increased rate of synthesis of the S-100 protein. Utilizing the inhibition of RNA synthesis by actinomycin D, we were able to show that the halflife of mRNA increases concurrently with an increase in the rate of synthesis of the S-100 protein. Utilization of actinomycin D does present hazards in interpretation of results on mRNA stability; experiments were performed to determine if the results obtained were due to side effects of the drug. As far as could be determined, the possible side effects of actinomycin D did not affect our results.     

Treatment of Chinese hamster ovary cells with the transcriptional inhibitor actinomycin D inhibits binding of messenger RNA to ribosomes

Inhibitors of RNA synthesis such as actinomycin D, MPB, and cordycepin progressively inhibit the initiation of protein synthesis in intact, nucleated mammalian cells. This inhibition is not dependent on the levels of mRNA, ribosomes, or tRNA. Lysates prepared from CHO cells treated with actinomycin D do not incorporate labeled globin mRNA or ovalbumin mRNA into 80S initiation complexes at the rates of untreated control extract. The ability of the extracts to produce and accumulate 48S preinitiation complexes was assessed using the 60S subunit joining inhibitors edeine and 5'-guanylyl imidodiphosphate. Control extracts were able to accumulate both the 48S preinitiation complexes and the migration-related intermediates in the presence of both inhibitors. However, lysates derived from CHO cells treated with actinomycin D were unable to produce these complexes. This was also true at low temperature, a condition that does not inhibit mRNA binding but prevents migration of the 43S complex along the mRNA. Mixing experiments with extracts from untreated control or AMD-treated CHO cells provided no evidence for a translational inhibitor. Thus, our data are consistent with the hypothesis that treatment of whole cells with actinomycin D inhibits protein synthesis initiation at the level of mRNA binding and not at migration or 60S subunit joining.     

Effects of actinomycin D on ribosomal RNA and protein synthesis as revealed by high resolution autoradiography.

Incorporation of tritiated amino acids and uridine was studied in untreated and actinomycin D treated HeLa cells by high resolution autoradiography. Results showed a non-selective inhibition of protein synthesis by actinomycin, as measured by the decrease in radioactive amino acid uptake. When cells pretreated with actinomycin D were incubated with radioactive amino acids and uridine, amino acid uptake in the nucleolus still occurred, while uridine uptake was almost completely eliminated. These findings suggest that in the absence of ribosomal RNA precursor synthesis, nucleolar protein synthesis continues to some extent, and that this protein is transported to the nucleolus.     

Gene induction by gamma-irradiation leads to DNA fragmentation in lymphocytes

An early event in death of interphase lymphocytes exposed in vivo or in vitro to low doses of gamma-irradiation is the degradation of DNA into nucleosome-sized fragments. Induction of fragmentation required RNA and protein synthesis because actinomycin D and cycloheximide, respectively, are able to inhibit DNA fragmentation in irradiated lymphocytes. Studies adding cycloheximide and actinomycin D at various times postirradiation suggest that once the metabolic process is initiated within an individual cell it proceeds to completion. The reversible RNA synthesis inhibitor, 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole inhibits DNA fragmentation in irradiated thymocytes. When this drug is removed after 6 hr, irradiated thymocytes proceed to fragment their DNA; this suggests that an inducing "signal" that is not simply mRNA persists within the irradiated cell for at least 6 hr after irradiation. In contrast to mitogen-activated T and B lymphoblasts, resting T and B cells show significant DNA fragmentation after exposure to 100 to 500 rad. At 2000 rad, all of the splenic subpopulations die rapidly via a different mechanism. By studying the mechanism of DNA fragmentation induced during the interphase death of lymphocytes, we hope to understand better the extreme sensitivity of resting lymphocytes to radiation and what may be the common final pathway of programmed cell death.     

Delayed inhibition of DNA synthesis in mouse jejunum by low doses of actinomycin D

Actinomycin D (0.008 μg/gm of body weight) injected intraperitoneally every two hours, produced a prompt 50% inhibition of RNA synthesis in the jejunum of mice, and a delayed inhibition of DNA synthesis, that reached its maximum inhibition (68% of control values) 4.5 hours after the first injection of actinomycin D. Autoradiographic studies indicated that this low level of actinomycin D inhibited a step in the G₁ phase of the cell cycle, preventing the initiation, but not affecting the continuation, of DNA biosynthesis. The activity of DNA polymerase was not affected under these conditions. The results are substantially similar to those previously obtained with Ehrlich ascites cells growing in the peritoneal cavity of mice and can be interpreted as indicating that in the G₁ phase of dividing cells there is an actinomycin sensitive step whose inhibition prevents the entrance of cells into the DNA-synthesis phase.     

Inhibition of cell-substratum attachment of cultured rat heart cells by protein synthesis inhibitors.

Addition of cycloheximide to growth medium of neonatal rat heart cell cultures prevented cell-substratum attachment. Even concentrations of cycloheximide which inhibited only 50% of normal protein synthesis prevented some cells from attaching. Cells which required the longest time to attach were not dependent on protein synthesis. The kinetics of cell-substratum adhesion in the presence of various concentrations of cycloheximide supported the hypothesis that repair of damaged cell membranes was required prior to attachment. An alternate hypothesis that protein synthesis was required for substratum attachment either to synthesize new unique proteins or higher concentrations of existing proteins not damaged by enzymes was not supported by experimentally obtained data. If the second hypothesis were true, no cells would have attached when protein synthesis was completely inhibited (greater than 95%) and all cells should have been equally affected by protein synthesis inhibition; such was not the case. Inhibition of mRNA formation by actinomycin D also should have inhibited attachment completely and this was not observed. Since attachment was minimally affected by actinomycin D, protein synthesis on long-lived mRNA was apparently sufficient for cell-substratum adhesion.     

Early changes in the synthesis of acidic nuclear proteins in human diploid fibroblasts stimulated to synthesize DNA by changing the medium

When resting WI-38 cells in a confluent monolayer were stimulated to proliferate by changing the medium, the incorporation of leucine-³H into nuclear acidic proteins was promptly stimulated, although its incorporation into total cellular proteins was unchanged or even decreased. Three fractions, all acidic by aminoacid analysis, were extracted from the nuclei: (1) ribonucleoproteins (RNP); (2) a fraction extractable with 0.15 M NaC1; and (3) a fraction tenaciously bound to the insoluble residue (residual fraction). A first increase occurred between one and three hours after stimulation in all three fractions. The synthesis of NaCl-soluble proteins then returned to control levels, while the synthesis of residual and RNP proteins remained high between 6 and 12 hours and increased even further at 18 hours, the peak of DNA synthesis. Pulse chase experiments indicated that the proteins synthesized in the first hour after stimulation have a turnover time of less than four hours, while the same fractions in non-proliferating cells were stable for at least 12 hours. 2-mercapto-1-(β-4-pyridethyl) benzimidazole, when added at the same time as the fresh medium, produced an inhibition of the increase in nuclear protein synthesis at one hour, but, if added at five hours after stimulation, it did not inhibit the increase in nuclear protein synthesis occurring at six hours. Actinomycin D (0.01 μg/ml) inhibited both the stimulation of DNA synthesis and the increases in nuclear acidic protein synthesis occurring at one and six hours after stimulation. These results seem to indicate that the serum factors responsible for the stimulation of WI-38 cells, after binding to cells, induce an early synthesis of acidic nuclear proteins which is sensitive to very low doses of actinomycin D. In turn, the newly synthesized proteins could in some way activate in the nuclei the genes that control DNA synthesis and cell division.     

Regulation of Herpesvirus Macromolecular Synthesis I. Cascade Regulation of the Synthesis of Three Groups of Viral Proteins

Based on evidence that 50% of herpes simplex 1 DNA is transcribed in HEp-2 cells in the absence of protein synthesis we examined the order and rates of synthesis of viral polypeptides in infected cells after reversal of cycloheximide- or puromycin-mediated inhibition of protein synthesis. These experiments showed that viral polypeptides formed three sequentially synthesized, coordinately regulated groups designated alpha, beta, and gamma. Specifically: (i) The alpha group, containing one minor structural and several nonstructural polypeptides, was synthesized at highest rates from 3 to 4 h postinfection in untreated cells and at diminishing rates thereafter. The beta group, also containing minor structural and nonstructural polypeptides, was synthesized at highest rates from 5 to 7 h and at decreasing rates thereafter. The gamma group containing major structural polypeptides was synthesized at increasing rates until at least 12 h postinfection. (ii) The synthesis of alpha polypeptides did not require prior infected cell protein synthesis. In contrast, the synthesis of beta polypeptides required both prior alpha polypeptide synthesis as well as new RNA synthesis, since the addition of actinomycin D immediately after removal of cycloheximide precluded beta polypeptide synthesis. The function supplied by the alpha polypeptides was stable since interruption of protein synthesis after alpha polypeptide synthesis began and before beta polypeptides were made did not prevent the immediate synthesis of beta polypeptides once the drug was withdrawn. The requirement of gamma polypeptide synthesis for prior synthesis of beta polypeptides seemed to be similar to that of beta polypeptides for prior synthesis of the alpha group. (iii) The rates of synthesis of alpha polypeptides were highest immediately after removal of cycloheximide and declined thereafter concomitant with the initiation of beta polypeptide synthesis; this decline in alpha polypeptide synthesis was less rapid in the presence of actinomycin D which prevented the appearance of beta and gamma polypeptides. The decrease in rates of synthesis of beta polypeptides normally occurring after 7 h postinfection was also less rapid in the presence of actinomycin D than in its absence, whereas ongoing synthesis of gamma polypeptides at this time was rapidly reduced by actinomycin D. (iv) Inhibitors of DNA synthesis (cytosine arabinoside or hydroxyurea) did not prevent the synthesis of alpha, beta, or gamma polypeptides, but did reduce the amounts of gamma polypeptides made.     

Regulation of synthesis and turnover of an interferon-inducible mRNA.

Regulation of synthesis and turnover of an interferon (IFN)-inducible mRNA, mRNA 561, in HeLa monolayer cells was studied. Cytoplasmic levels of this mRNA were estimated by hybridization analyses with a cDNA clone that we have isolated as a probe. IFN-alpha A induced a high level of this mRNA in a transient fashion, whereas no induction was observed in response to IFN-gamma. Surprisingly little mRNA 561 was induced in cells treated simultaneously with IFN-alpha A and an inhibitor of protein synthesis, suggesting that in addition to IFN-alpha A, an interferon-inducible protein was needed for induction of this mRNA. Apparently this putative protein could be induced by IFN-gamma as well. Thus, although little mRNA 561 was synthesized in cells treated either with IFN-gamma alone or with IFN-alpha A and cycloheximide, a large quantity of this mRNA was induced in cells which had been pretreated with IFN-gamma and then treated with IFN-alpha A and cycloheximide. Once mRNA 561 was induced by IFN-alpha A, it turned over rapidly. This rapid turnover could be blocked by actinomycin D or cycloheximide indicating that another IFN-inducible protein may mediate this process.