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Effect of cycloheximide on the synthesis and processing of 5S RNA in HeLa cells.

The synthesis and incorporation into ribosomes of 5S RNA were investigated under conditions of protein synthesis inhibition by cycloheximide. Within 1 h after addition of cycloheximide to the culture medium, the synthesis of 5S RNA in HeLa cells, as measured by the pulse labeling of total 5S RNA, was inhibited by 25-30 per cent, but the degree of inhibition did not further increase for several hours. The synthesis of 4S RNA was less markedly inhibited, especially at earlier time periods. Although the appearance of labeled 5S RNA in cytoplasmic ribosomoes after chase was inhibited by approximately 60% or more this presumably reflects the inhibition of 60S ribosomal subunit formation by cycloheximide. Some utilization of preformed 5S RNA for new ribosomes was suggested after resumption of protein synthesis. In confirmation of the results of Kumar and Wu (1), a low dose of actinomycin D inhibited the appearance of 28S RNA in the cytoplasm rather specifically, whereas cycloheximide showed an apparently opposite differential effect.

Cycloheximide↗

Two different genes from Schwanniomyces occidentalis determine ribosomal resistance to cycloheximide.

Two genes (SCR1 and SCR2) encoding natural cycloheximide resistance in the budding yeast Schwanniomyces occidentalis have been cloned by expression in Saccharomyces cerevisiae. Both genes determine resistance to the inhibitory action of cycloheximide on the ribosome, SCR1 and SCR2 are present as single copies in Schwanniomyces occidentalis, where they map on chromosomes II and V, respectively. The nucleotide sequence of SCR2 contains an open reading frame of 321 nucleotides which is interrupted by an intron of 452 nucleotides. It encodes a polypeptide of 106 amino acids of molecular mass 12.25 kDa and pI 11.19. The deduced amino acid sequence shows a high degree of similarity to the L41 protein of the 60S ribosomal subunit from several eukaryotic organisms. The intron and the 5' non-coding region of SCR2 possess conserved elements which are typical of yeast ribosomal protein genes. A single amino acid change determines the resistance or sensitive phenotype to cycloheximide of the 80S ribosome since replacement of Gln56 in L41 from Schwanniomyces with Pro, by site-directed mutagenesis, confers cycloheximide sensitivity. SCR2 may serve as a practical yeast cloning marker if integrated in a multicopy plasmid.

Amino Acid Sequence↗

The effect of cycloheximide and ganglioside GM1 on the viability of retinotectally projecting ganglion cells following ablation of the superior colliculus in neonatal rats.

The time-course and extent of death of retinal ganglion cells (RGCs) following ablation of the superior colliculus (SC) in neonatal Wistar rats has recently been described [Harvey, A. R. and Robertson, D. (1992) J. Comp. Neurol., 325, 83-94]. Normal and pyknotic nuclei of retinotectally projecting ganglion cells were visualized using the fluorescent retrograde tracer diamidino yellow (DY), which had been injected into the SC at P2 (day of birth = P0), 2 days prior to tectal removal. The present report sets out to determine whether cycloheximide, an inhibitor of protein synthesis, or ganglioside GM1 reduced this lesion-induced RGC death. All surgery was carried out under ether anaesthesia; DY was injected into the left SC at P2 and the injected area was removed at P4. Cycloheximide (20-500 ng) was injected into the vitreous chamber of the right eye immediately after the lesion and again 11-12 h later. In some rats, cycloheximide administration was delayed until 12 h after the SC ablation. Control rats received SC lesions alone or lesions plus sham eye injections of saline. Different doses of GM1 were applied i.p. or intraocularly. Rats were perfused 24 h after the SC lesion, at the time of peak RGC death. Retinae of lesion only or sham eye injected rats contained approximately 11% pyknotic RGCs and the density of normal RGCs was approximately 3400/mm2. The rate of pyknosis in cycloheximide treated retinae was reduced to approximately 3%. Normal RGC density in these retinae was approximately 5500/mm2, similar to that found in retinae of unlesioned animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Identification of the catalase gene promoter region involved in superinduction in Schizosaccharomyces pombe caused by cycloheximide and hydrogen peroxide.

Superinduction of the catalase gene was observed in Schizosaccharomyces pombe cells treated with cycloheximide and hydrogen peroxide. The promoter analysis of the catalase gene revealed that element A (the region from -111 to -90, numbered with the transcription start site as +1), involved in the induction of the gene under oxidative stress, was required for superinduction by hydrogen peroxide and cycloheximide. Although Atf1 is a transcription factor responsible for the induction of the catalase gene by several stresses, a disruptant of atf1 exhibited superinduction. Moreover, in a deletion mutant that lacks element A but has an Atf1 binding site, the cells treated with hydrogen peroxide and cycloheximide expressed as much catalase mRNA as those treated with hydrogen peroxide alone. This suggests that cycloheximide does not stabilize the catalase mRNA but enhances the transcription via element A. Staurosporine, a strong inhibitor of protein phosphorylation, did not inhibit superinduction.

Base Sequence↗

'Superactivation' of alkaline phosphatase activity by cycloheximide in rat hepatoma cell cultures.

We have found that rat hepatoma cells (R-Y121B) retain alkaline phosphatase activity, and that this enzyme activity is increased by cycloheximide. Actinomycin D also increased the enzyme activity. This increase due to actinomycin D was partially inhibited by cycloheximide. The characteristics of alkaline phosphatase of the cells treated or untreated with cycloheximide or actinomycin D were similar to each other; they were heat labile and the enzyme reaction was strongly inhibited by L-homoarginine, but weakly by L-phenylalanine. The increase in alkaline phosphatase activity with cycloheximide has been termed a 'superactivation' of alkaline phosphatase.

Alkaline Phosphatase↗

Cycloheximide: its effects on activity are dissociable from its effects on memory.

Cycloheximide, when injected subcutaneously or intracerebrally, produces changes in the activity level of mice. Isocycloheximide, injected intracerebrally, produces identical effects on activity, but it does not produce inhibition of cerebral protein synthesis or amnesia. Amphetamine, in doses that can antagonize the amnesic action of cycloheximide, does not antagonize the effect of cycloheximide on activity. Effects of cycloheximide on activity do not appear to be responsible for its amnesic action.

Amphetamine↗

Quantitative evaluation of the antifungal properties of cycloheximide.

Although the total growth of both zoopathogenic and saprophytic fungi on cycloheximide media was consistently less than that on the control without the antibiotic, a progressive increase in the growth rate of these organisms occurred during exposure to the drug. The extent of this change depended upon the concentration of cycloheximide, the species and strain of the test organism, and the duration of exposure. Significant alterations were also observed in the macroscopic appearance of the colonies. The results of this investigation agree with those of previous studies regarding the value of cycloheximide in selective isolation media, but there were discrepancies with respect to the degree of sensitivity of several of the organisms studied. The increase in the rate of growth on cycloheximide media may indicate an induced adaptation to the drug.

Antifungal Agents↗

Induction of cycloheximide-resistant mutants in Saccharomyces cerevisiae with N-methyl-N'-nitro-N-nitrosoguanidine and ICR-170.

N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces cycloheximide-resistant mutations in Saccharomyces cerevisiae, but few, if any, resistant mutants are induced by the acridine mustard ICR-170. Cycloheximide sensitivity in yeast is associated with the ribosome, and treatment with the antibiotic at concentrations of 2 mug/ml results in complete inhibition of protein synthesis. Missense mutations induced by MNNG probably lead to the loss of cycloheximide binding sites on the ribosome, resulting in resistance to the antibiotic without altering the activity of the organelle in protein synthesis. ICR-170, however, induced primarily frameshift mutations that would alter ribosome structural integrity, resulting in cell death rather than resistance. ICR-170 and MNNG are both mutagenic in a system in which base-pair substitution and frameshift mutations can be detected. These results indicate that cycloheximide resistance in S. cerevisiae, like streptomycin and spectinomycin resistance in Escherichia coli, can be induced by base-pair substitution mutagens but not by frameshift mutagens such as ICR-170.

Acridines↗

Enhancement of Chlamydia trachomatis infectious progeny by cultivation of HeLa 229 cells treated with DEAE-dextran and cycloheximide.

The effects of DEAE-dextran and cycloheximide on the infection of HeLa 229 cells with Chlamydia trachomatis serotype G were studied in terms of the number of cells infected and the yield of infectious progeny per infected cell. Pretreatment of the host cells with DEAE-dextran resulted in an increase in the number of infected cels but had no significant effect on the yield of infectious progeny per infected cell (burst size). In contrast, the addition of cycloheximide to the medium of infected cells had no significant effect on the number of infected cells but greatly enhanced the burst size. The burst size was calculated to be close to 500. The enhanced burst size was also observed in cells treated with DEAE-dextran and cycloheximide. In addition, there was an increase in the number of cells infected and an augmentation of the infectious progeny yield. Under the conditions of combined treatment, the yield of C. trachomatis serotype G cultivated in HeLa 229 cells was found to be approximately threefold higher than the yield of the organisms cultivated in McCoy cells. The results suggest that HeLa 229 cells treated with DEAE-dextran and cycloheximide offer a most suitable system for the high-yield cultivation of C. trachomatis organisms and possibly also for the diagnosis of infection with these organisms.

Bacteriological Techniques↗

Effects of cycloheximide and puromycin on cytotoxic activity of Escherichia coli verocytotoxin (Shiga-like toxin).

Verocytotoxin (VT)-producing Escherichia coli is closely associated with hemorrhagic colitis and hemolytic uremic syndrome. The diagnosis of this infection requires the demonstration of VT activity in fecal filtrates or the isolation of VT-producing E. coli from stools. To improve the sensitivity of the Vero cell assay for detecting VT, we investigated the interaction between this toxin and cycloheximide and puromycin, agents which, like VT and the related Shiga toxin, are protein synthesis inhibitors. Cycloheximide-treated cells were found to be about eightfold more sensitive to VT, this effect being most pronounced when the drug was added before the toxin. In contrast, puromycin treatment had an antagonistic effect in that it decreased the sensitivity of the cells to VT. In assays of VT in fecal filtrates, the addition of cycloheximide (at 4 to 8 micrograms/ml) increased the sensitivity without affecting the specificity of the assays. Likewise, the use of cycloheximide led to an increase in the sensitivity of the serum VT-neutralizing antibody test by a factor of over eightfold.

Animals↗

RNA synthesis by vesicular stomatitis virus and a small plaque mutant: effects of cycloheximide.

The synthesis of viral RNA by wild-type vesicular stomatitis virus (L(1)VSV) and a small, plaque-size mutant (S(2)VSV) was studied in vitro and in chicken embryo (CE) and mouse L-cell cultures. Virus-specific RNA synthesized in CE or L cells infected with either L(1) or S(2)VSV at low multiplicity was of the same size classes, 12 to 15S, 28S, and 38S. The major differences were in the proportion of RNA produced of each size class. L(1)VSV always synthesized larger proportions of 38S RNA, and S(2)VSV produced larger proportions of 12 to 15S RNA. Both S(2) and L(1)VSV exhibited RNA transcriptase activity in vitro and in cell culture. The products of the in vitro reaction were the same, 12 to 15S for both. The products of the virion-associated transcriptase in CE or L-cell cultures in the presence of cycloheximide were also the same for both viruses but differed from the in vitro products in that 28S and 12 to 15S RNA were made. The effects of addition of cycloheximide at various times after infection demonstrated that new protein synthesis is required early (0-2 h) for both S(2) and L(1)VSV to initiate and maintain the normal rate of viral RNA synthesis. However, the overall rate of RNA synthesis in L(1)VSV infections became independent of protein synthesis after 2 h whereas the rate in S(2)VSV infections did not. With either virus, synthesis of 38S RNA did not occur in the absence of protein synthesis. Moreover, continuous 38S RNA production required continuous protein synthesis. Production of 38S RNA ceased within 30 min after addition of cycloheximide to S(2) (-) or L(1)VSV-infected CE or L cells that had already begun to synthesize the 38S form. The cycloheximide-induced cessation of 38S RNA synthesis was accompanied by a marked increase in production of 12 to 15S and 28S RNA in L(1)VSV-infected cells, but no increase in synthesis of small RNA species occurred in S(2)VSV-infected cells.

Animals↗

Cycloheximide stimulates early adenovirus transcription if early gene expression is allowed before treatment.

The inhibition of protein synthesis by cycloheximide markedly stimulated adenovirus 2 early transcription (assayed in vivo and in vitro in nuclei) when the drug was added 3 h after infection of HeLa cells. The stimulation was not uniform but ranged from three- to eightfold for region 1A to 10- to 30-fold for region 2 of the adenovirus genome. The increase was complete by about 60 min after treatment. The stimulation reversed rapidly after cycloheximide was removed. Treatment with cycloheximide either before infection with wild-type adenovirus type 5 or at 3 h after infection with dl312, a mutant which fails to express early gene products in HeLa cells, both resulted in levels of transcription at or substantially below the control (infection with wild-type adenovirus without treatment). Therefore we conclude that cycloheximide treatment and previous early gene expression interact to yield the maximum levels of transcription.

Adenoviruses, Human↗

Effects of cycloheximide on thermotolerance expression, heat shock protein synthesis, and heat shock protein mRNA accumulation in rat fibroblasts.

A single hyperthermic exposure can render cells transiently resistant to subsequent high temperature stresses. Treatment of rat embryonic fibroblasts with cycloheximide for 6 h after a 20-min interval at 45 degrees C inhibits protein synthesis, including heat shock protein (hsp) synthesis, and results in an accumulation of hsp 70 mRNA, but has no effect on subsequent survival responses to 45 degrees C hyperthermia. hsp 70 mRNA levels decreased within 1 h after removal of cycloheximide but then appeared to stabilize during the next 2 h (3 h after drug removal and 9 h after heat shock). hsp 70 mRNA accumulation could be further increased by a second heat shock at 45 degrees C for 20 min 6 h after the first hyperthermic exposure in cycloheximide-treated cells. Both normal protein and hsp synthesis appeared increased during the 6-h interval after hyperthermia in cultures which received two exposures to 45 degrees C for 20 min compared with those which received only one treatment. No increased hsp synthesis was observed in cultures treated with cycloheximide, even though hsp 70 mRNA levels appeared elevated. These data indicate that, although heat shock induces the accumulation of hsp 70 mRNA in both normal and thermotolerant cells, neither general protein synthesis nor hsp synthesis is required during the interval between two hyperthermic stresses for Rat-1 cells to express either thermotolerance (survival resistance) or resistance to heat shock-induced inhibition of protein synthesis.

Animals↗

Effect of cycloheximide on development of methotrexate resistance of Chinese hamster ovary cells treated with inhibitors of DNA synthesis.

We examined the effects of 18 h of incubation of Chinese hamster ovary (CHO K1) cells with cycloheximide, hydroxyurea, and aphidicolin. Treatment of cells with cycloheximide alone at a concentration adequate to inhibit DNA synthesis to less than 10% of control was significantly less cytotoxic and clastogenic than treatment with hydroxyurea or aphidicolin, did not induce unbalanced cellular growth, and had no effect on the frequency of resistant cells in methotrexate selections compared with control cells. When combined with hydroxyurea or aphidicolin and compared with the effects of either drug alone, cycloheximide blocked the induction of unbalanced growth during drug treatment, reduced the frequency of chromosomal aberrations in recovering cell populations, and decreased cell killing. In addition, the increased frequency of methotrexate-resistant cells observed after treatment with hydroxyurea or aphidicolin was eliminated when cycloheximide was present during drug treatment.

Animals↗

Effects of tamoxifen citrate and cycloheximide on estradiol induction of rat myometrial gap junctions.

Longitudinal muscle of myometrial tissues from immature rats were examined by quantitative thin section electron microscopy for the presence of gap junctions after treatment with estradiol with and without tamoxifen, and cycloheximide for 1-6 days. Gap junctions were present between myometrial cells on days 4, 5, and 6 after treatment with estradiol (500 micrograms/day). Tamoxifen administered concomitantly with estradiol over the 6-day period completely prevented induction of the junctions. Gap junctions were not detected in the myometrium after treatment with tamoxifen alone. Administration of cycloheximide together with estradiol on day 0 of the 6-day period had no effect on gap junction frequency but resulted in a reduction in gap junction size in the myometrium after continued treatment with the hormone. Treatment with cycloheximide on day 1, however, significantly suppressed the effect of further estradiol treatment on the induction of gap junctions in the myometrium. Junctions were not visible in the tissues from animals treated with cycloheximide alone or in the control groups treated with sesame oil. These results indicate that estradiol influences the presence of gap junctions in the myometrium by regulating the synthesis of gap junction proteins through the steroid receptor mechanism.

Animals↗

Cycloheximide blocks insulin-like growth factor I but not somatostatin inhibition of growth hormone secretion.

Growth hormone secretion is controlled by the two hypothalamic hormones, growth hormone releasing factor (GRF) and somatostatin. In addition, the insulin-like growth factors (IGF or somatomedins) which are themselves growth hormone dependent, inhibit growth hormone release in vitro, therefore acting to close the negative feedback loop. The studies reported here examine some of the differences between inhibition of growth hormone secretion by somatostatin and IGF-I in vitro. The major finding is that cycloheximide, a protein synthesis inhibitor, blocks inhibition of GRF-stimulated growth hormone release caused by IGF-I, without changing the inhibition caused by somatostatin. The experiments were done by exposing mixed rat adenohypophysial cells to secretagogues with or without cycloheximide for 24 h in a short term culture. Somatostatin (0.6 nM) totally blocked rat GRF (1 nM) stimulated growth hormone release to values 48% of control (nonstimulated values), while IGF-I (27 nM) only reduced the GRF-stimulated growth hormone release by 27 +/- 3% (N = 5). Cycloheximide (15 micrograms/mL) totally blocked the effect of IGF-I but not somatostatin. A low concentration (0.12 nM) of somatostatin, which only partly inhibited growth hormone release, was also unaffected by cycloheximide. In purified rat somatotrophs, somatostatin (0.1 nM) inhibited GRF-stimulated cAMP levels slightly and reduced growth hormone release while IGF-I (40 nM) had no effect. We suggest that IGF-I inhibits only the secretion of newly synthesized growth hormone, while somatostatin inhibits both stored and newly synthesized growth hormone pools.

Animals↗

Alpha-aminoisobutyric acid transport in human leukemic lymphocytes: in vitro characteristics and inhibition by cortisol and cycloheximide.

We have studied the transport of alpha-aminoisobutyric acid (AIB)-3-(14)C and its response to cortisol and cycloheximide in vitro in blood lymphocytes from untreated patients with chronic lymphocytic leukemia. The accumulation of AIB-3-(14)C increased in a linear fashion for 60 min, and reached an apparent steady state in 120 min. The initial rate of AIB accumulation (V(o)) varied from 1.1 to 10.2 mumoles/kg cell H(2)O per min in cells from 16 different patients; however, V(o) was reproducible in cells from five of six patients which were studied repeatedly over 1-9 months, and correlated positively with the lymphocyte count (r = 0.51, P = < 0.01). Virtually total inhibition of protein synthesis with cycloheximide was found to decrease the accumulation of AIB in cells from four patients which had high rates of AIB transport, but had no effect on transport in cells from four patients which accumulated AIB more slowly. These results indicate that active transport depends, in part, upon the presence of labile protein with a turnover rate which varies among different cell populations. Treatment with 10 muM cortisol for 240 min in vitro reduced the initial rate of AIB-3-(14)C accumulation (V(o)) by 43.4+/-4.1% (SE) (range, 9-66%) in cells from 16 patients. The degree of inhibition did not vary appreciably over a 9 month period in four of five patients. The effect of cortisol was proportional to its starting concentration, and developed at low concentrations (0.1-1.0 muM). Cortisol appears to decrease AIB accumulation by inhibiting active uptake, since it neither enhanced the exodus of AIB, nor inhibited apparently nonsaturable transport. Inhibition was noncompetitive in type, suggesting that cortisol decreases the total capacity of the active transport mechanism.Cortisol inhibited AIB transport indirectly by a process which involved de novo protein synthesis, since inhibition (a) appeared only after 60 min of treatment, (b) was present in treated cells which were subsequently incubated for 60 min in cortisol-free medium, and (c) failed to develop during simultaneous blockade of protein synthesis with cycloheximide, even when cycloheximide itself did not decrease AIB transport.

Aminoisobutyric Acids↗

Cycloheximide inhibits light-induced phase shifting of the circadian clock in Neurospora.

Cycloheximide inhibits light-induced phase shifting of the circadian clock and protein synthesis in Neurospora. Light resetting is not inhibited in mutants whose protein synthesis is resistant to cycloheximide. When light and cycloheximide are presented together at various circadian phases, the final phase shift is always determined by cycloheximide. This dual-treatment phase response curve approach may be useful for other studies using pharmacological treatments to analyze clock pathways. Taken together, the results suggest that synthesis of a protein (or proteins) is involved in the phototransduction pathway of the circadian clock in Neurospora.

Bacterial Proteins↗