Expression of oncogenes in the rat liver under conditions of template biosynthesis uncoupling by sublethal doses of cycloheximide

Injection of sublethal doses of cycloheximide (CHI) to rats allowed to reveal three stages in the dynamics of protein synthesis: 1) suppression stage (0-6 hrs), 2) regeneration stage (6-12 hrs), 3) stimulation stage (6-12 hrs). RNA-polymerases are activated when protein synthesis is inhibited. The stimulation stage precedes the activation of DNA replication. This model of DNA replication induced by CHI is specified by the expression of various cell oncogenes (c-fos, c-mys, p53, c-Ha-ras, c-sis, c-src). The investigated oncogenes may be divided into 4 groups according to the character of their expression. 1. Oncogenes (c-fos, c-myc) are switched on step-by-step 1 hour after CHI injection, the superexpression of the oncogenes being comparatively short. Maximum expression of c-fos and c-myc oncogenes is registered after 2-3 hours, respectively. 2./p53 oncogene expression increases within a few hours' after CHI injection and manifests itself at all three stages of protein synthesis till DNA replication. 3. c-Ha-ras oncogene is expressed at a high level in control and experimental animals. 4. Expression of c-sis and c-src oncogenes are absent both before and after CHI injection. Sublethal doses of CHI have the same effect on oncogene expression as the lethal ones.     

Dynamics of the biosynthesis of components of the protein synthesizing apparatus of the rat liver at the stage of restoration of translation, inhibited by cycloheximide

The biosynthesis of proteins, ribosomal RNA and other components of the rat liver protein-synthesizing system during the reparation and subsequent activation of translation inhibited by a sublethal dose cycloheximide (CHI, 3 mg/kg) was studied. It was found that the incorporation of labeled precursors into proteins and ribosomal rRNA isolated from free and membrane-bound polysomes is repaired already 3 hours after CHI injection. 6-9 hours thereafter, the level of component labeling reaches control values, whereas the total protein biosynthesis is retarded. After 12-24 hours, marked stimulation of ribosome biosynthesis and the integration of ribosomes into polysomes are observed together with an asymmetric accumulation of excessive amounts of newly synthesized 40S subunits into polysomes 12 hours after CHI infection. The putative mechanisms of the activation of expression of the part of the genome responsible for protein and ribosomal rRNA synthesis as well as for the synthesis of other components of the protein-synthesizing system are discussed.     

Adaptation to cycloheximide of macromolecular synthesis in Tetrahymena

Cycloheximide (CHI) at 10 ng/ml partially inhibited protein synthesis in exponential cultures of Tetrahymena Sp. At 20 ng/ml or greater, inhibition was complete. When protein synthesis was inhibited to any extent, cell division ceased immediately. In all instances where measured, synthesis of RNA and DNA also ceased. After a period of delay, cellular functions reinitiated in the order: (i) protein synthesis, (ii) DNA synthesis and, (iii) RNA synthesis and cell division. The delay in cell division was divided into three phases of: I, zero; II, low; and, III, fully recovered rates of exponential protein synthesis. The length of the three phases increased with increasing concentration of CHI Prior growth of cells for one generation in the presence of 7.5 ng/ml CHI (facilitation) eliminated phase I and slightly decreased phases II and III following subsequent challenge with an inhibitory concentration of CHI. Facilitation for six generations further decreased phases II and III. Protein synthesis and cell division were not inhibited during facilitation In the culture, succinate dehydrogenase activity did not increase during the delay but increased normally at the onset of division. In contrast, NADPH-cytochrome c reductase activity continued to increase for an hour after inhibition of protein synthesis, was constant for a period and did not increase again until an hour after reinitiatoin of cell division and RNA synthesis Inhibition of division of all cells was immediate and reinitiation of synthesis and cell division was non-synchronous.     

RNA biosynthesis in mitochondria under conditions of prolonged inhibition of protein biosynthesis in rat liver cytoplasm]

When cytoplasmic protein synthesis is inhibited by cycloheximide (CHI) in vivo synthesis of water-soluble mitochondrial proteins and of mitochondrial RNA is decreased. These changes measured in isolated rat liver mitochondria are similar to those observed in vivo and correlate with the changes the synthesis of water-soluble proteins in mitochondria. When the cytoplasmic fraction (30,000 g-supernatant) had been added to the mitochondria showing decreased RNA synthesis, the RNA synthesis increased to the control level (the incubation conditions were favourable for the protein transport from microsomes to mitochondria). RNA synthesis in mitochondria was not stimulated by cytoplasmic fractions from the CHI-pretreated rats. After prolonged dialysis these fraction stimulated RNA synthesis even to a greater extent than cytoplasmic fractions from the untreated animals. Mitochondrial RNA polymerase activity (measured in mitochondrial extracts supplemented with exogenous DNA) was higher in extracts of mitochondria from livers of normal rats than in extracts of mitochondria from livers of animals injected with CHI.     

Biogenesis of chromatin in animal cells. Stimulation of nuclear protein and DNA synthesis in hepatocytes after brief inhibition of translation by cycloheximide]

A drastic and brief inhibition of protein synthesis (up to 95% for 3--6 hrs) by cycloheximide in the liver of rats starved for 24 hrs results in a recovery and subsequent marked stimulation of non-histone proteins, histone chromosomal proteins and DNA. The stimulation of non-histone protein synthesis was observed after 1 hr (inhibition) 12--24 hrs (recovery and stimulation of protein synthesis) and 48--60 hrs (stimulation of DNA synthesis) following the administration of cycloheximide. Two periods of histone biosynthesis were observed. The first one (24--36 hrs) was not coupled and the second one (48--60 hrs) was coupled with DNA replication. During the recovery and stimulation of protein synthesis acetylation of the histone and non-histone proteins proceeds at an increased rate. Possible applicability of the model in question for investigations of chromatin biogenesis is discussed.     

Characteristics of [3H]cycloheximide distribution, protein and DNA biosynthesis in rat organs after sublethal blocking of translation

The kinetics of accumulation and release of [3H]cycloheximide (CHI) as well as protein and DNA biosyntheses in some organs of the rats injected with sublethal doses of CHI were studied. It was shown that in the majority of organs under study (especially in the liver, kidneys and adrenals) the inhibition is completed within 12 hours after CHI injection followed by the resumption of protein and DNA syntheses. In the thymus and pancreas the levels of these biosyntheses remain below control values up to the 72nd hour. A positive correlation was observed between the decrease of CHI (or its metabolites) concentration and the beginning of protein and DNA syntheses in different organs. However, there was a reverse correlation between the high values of squares below the kinetic curves of CHI release from the liver, kidneys and adrenals and the intensive resumption of protein and DNA biosyntheses in these organs. It was thus assumed that in these particular organs CHI is subjected to intensive biotransformations. The contribution of the endocrine system to the induction of intensive compensatory protein and DNA syntheses in the liver were estimated from the viewpoint of the nature of reconstructive processes occurring in the appropriate organs.     

Study of the ultrastructural and functional expression of the hepatocyte genome under conditions of prolonged depression of protein biosynthesis by cycloheximide. III]

Dynamics of changes in mtRNA synthesis and mitochondria ultrastructure is strictly dependent on the level of inhibition of biosynthesis of cytoplasm proteins and "soluble" proteins of mitochondria by cycloheximide in hepatocytes: 1-6 hrs later a progressive weakening of protein synthesis is accompanied by a drop in mtRNA synthesis and essential destruction of mitochondria; from 12 to 24 hrs a partial restoration of protein biosynthesis induces the processes of the above-mentioned indexes normalization.     

Ultrastructural and functional expression of the hepatocyte genome in conditions of prolonged suppression by cycloheximide of protein synthesis. IV]

RNA synthesis was intensified in isolated nuclei of the rat liver an hour after the animal treatment with cycloheximide (CHI). Incubation of the nuclei with a cytoplasmic fraction 160000 g obtained from normal rats decreases RNA synthesis, and that obtained from CHI-treated animals-increases it. Mechanisms of this effects are discussed.     

Regulation of de novo purine biosynthesis in Chinese hamster cells

Regulation of de novo purine biosynthesis was examined in two Chinese hamster cell lines, CHO and V79. De novo purine biosynthesis is inhibited at low concentrations of adenine. The mechanism of inhibition was studied using the RNA and protein synthesis inhibitors actinomycin D, cycloheximide, and azacytidine. Although all three inhibitors rapidly inhibited de novo purine biosynthesis in vivo, neither adenine nor the RNA and protein synthesis inhibitors could be found to have an effect in vitro on either phosphoribosylpyrophosphate (PRPP) synthetase or amido phosphoribosyltransferase, the first enzymes of the de novo pathway. However, in the presence of actinomycin D, cycloheximide, and azacytidine, there was a 50% or greater reduction in PRPP concentrations. This reduction in PRPP levels is correlated with a 2-fold increase in purine nucleotides in the acid-soluble pool. It is proposed that in the presence of the metabolic inhibitors there is an increase in nucleotide pools due to degradation of RNA, with a resulting feedback inhibition on de novo purine biosynthesis. In contrast to a previous report (Martin, D. W., Jr., and Owen, N. T. (1972) J. Biol. Chem. 247, 5477-5485), we could find no evidence for a repressor type mechanism in these cells.     

Relation of the biosynthesis of intracellular and export proteins in rat liver cells during induction of proliferation by cycloheximide

After a single injection of a sublethal dose of cycloheximide (CHI) the biosynthesis of extracellular proteins in rat hepatocytes was rapidly suppressed, the reconstitution being very slow. On the contrast, the biosynthesis of intracellular proteins (e.g., histones, and other acid-soluble liver proteins) was more resistant to CHI. The activation of biosynthesis of acid-soluble and acid-insoluble proteins was found to occur stepwise. It was assumed that the activation of synthesis and accumulation of intracellular proteins after CHI release accompanied by a decreased synthesis of extracellular proteins is one of possible causes of stimulation of DNA synthesis in the hepatocytes following a single injection of CHI.     

Effects of abscisic Acid on growth, RNA metabolism, and respiration in germinating bean axes

The effect of abscisic acid on growth, respiration, the ATP pool, and rate and amount of RNA synthesis in aseptically cultured axes of Phaseolus vulgaris during the first 24 hours of germination has been measured in experiments where the duration of abscisic acid application and its concentration have been varied. At concentrations from 10(-7) to 10(-4)m, abscisic acid inhibits synthesis of RNA with maximal inhibition (80%) at 10(-5)m. RNA synthesis is inhibited by abscisic acid at all times examined (12, 18, and 24 hours), but the extent of inhibition is maximal at 18 hours. In 18-hour axes RNA synthesis is inhibited 42%, ATP pool size is reduced 3%, and O(2) consumption is decreased by 6% after 75 minutes of abscisic acid treatment. Inhibition of RNA synthesis is complete by 2 hours of treatment with abscisic acid, and recovery to near control levels occurs by the 3rd hour after removal from abscisic acid.     

Macromolecular syntheses during biosynthesis of prodigiosin by Serratia marcescens.

Amino acids that were utilized as sole sources of carbon and nitrogen for growth of Serratia marcescens Nima resulted in biosynthesis of prodigiosin in non-proliferating bacteria. Addition of alanine, proline, or histidine to non-proliferating cells incubated at 27 C increased the rate of protein synthesis and also caused biosynthesis of prodigiosin. No increase in the rate of protein synthesis was observed upon the addition of amino acids that did not stimulate prodigiosin biosynthesis. Increased rates of synthesis of ribonucleic acid (RNA) and of deoxyribonucleic acid (DNA) (a small amount) also occurred after addition of amino acids that resulted in biosynthesis of prodigiosin. After incubation of 24 h, the total amount of protein in suspensions of bacteria to which alanine or proline was added increased 67 and 98%, respectively. Total amounts of DNA and of RNA also increased before synthesis of prodigiosin. The amounts of these macromolecules did not increase after addition of amino acids that did not induce biosynthesis of progidiosin. However, macromolecular synthesis was not related only to prodigiosin biosynthesis because the rates of DNA, RNA, and protein synthesis also increased in suspensions of bacteria incubated with proline at 39 C, at which temperature no prodigiosin was synthesized. The quantities of DNA, RNA, and protein synthesized were lower in non-proliferating cells than in growing cells. The data indicated that amino acids causing biosynthesis of prodigiosin in non-proliferating cells must be metabolized and serve as sources of carbon and of nitrogen for synthesis of macromolecules and intermediates. Prodigiosin was synthesized secondarily to these primary metabolic events.     

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.     

Glycoprotein biosynthesis in B lymphocytes: induction of protein N-glycosylation, RNA synthesis, and DNA synthesis by phorbol ester plus ionomycin is blocked by protein kinase inhibitors

The combination of phorbol 12-myristate 13-acetate (PMA) and ionomycin produces a dramatic increase in the incorporation of [2-3H]mannose into Glc3Man9GlcNAc2-P-P-dolichol and glycoprotein, and the induction of RNA and DNA synthesis in murine splenic B lymphocytes (B cells). The kinetics of the induction processes and the concentrations of PMA and ionomycin required for the optimal response have been defined. While the levels of induction of RNA and DNA synthesis by PMA + ionomycin were similar to the mitogenic response to bacterial lipopolysaccharide, activation by PMA and the calcium ionophore resulted in a threefold higher stimulation in dolichol-linked oligosaccharide biosynthesis and protein N-glycosylation. These results indicate that all signalling mechanisms that trigger RNA and DNA synthesis may not be sufficient to produce maximal induction of the N-glycosylation apparatus. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7), a potent protein kinase C inhibitor, prevented the induction of protein N-glycosylation activity (IC50 = 11 microM), as well as RNA (IC50 = 18 microM) and DNA synthesis (IC50 = 12 microM), two common indices of B cell activation. N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide (H-8) also inhibited the induction of oligosaccharide-lipid intermediate, glycoprotein, RNA, and DNA synthesis, but required higher concentrations than H-7 for 50% inhibition. N-(2-Guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), a potent inhibitor of cyclic nucleotide-dependent protein kinases, had little effect on the activation of the B cell metabolic processes. The H-7-sensitive reactions involved in the induction of RNA and DNA synthesis occurred within 4 h, but induction of lipid intermediate and glycoprotein biosynthesis remained sensitive to H-7 for 10 h after exposure to PMA and ionomycin. Direct in vitro assays in the presence of 0.6% Brij 58 reveal that a cytosolic, phospholipid-dependent protein kinase activity is translocated to a membrane site(s) after treatment with PMA and ionomycin, and the translocated protein kinase is sensitive to H-7. The relative order of potency of the protein kinase inhibitors on the metabolic processes strongly supports the hypothesis that protein kinase C, acting synergistically with Ca2+ mobilization, plays a key regulatory role in the early stages of B cell activation. The synthesis of oligosaccharide-lipid intermediates and protein N-glycosylation are also shown to be induced in B cells activated by PMA + ionomycin.