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At least 19 recordsLinked to original sources

PubMed1977

DNA replication in Physarum polycephalum: characterization of DNA replication products made in vivo in the presence of cycloheximide in strains sensitive and resistant to cycloheximide.

Synchronous plasmodia of cycloheximide-sensitive and cycloheximide-resistant strains of Physarum polycephalum were labelled with 3[H]-deoxyadenosine in pulse and pulse-chase experiments in presence and absence of cycloheximide. The replication products were studied with alkaline sucrose gradient sedimentation analysis. We show that the action of cycloheximide on DNA replication in Physarum is mediated through the ribosome, since the ribosomally located resistance also makes the plasmodial DNA replication refractile to the action of cycloheximide. Cycloheximide caused inhibition of three stages in DNA replication in the wild type: first, the formation of primary replication units ("Okazaki" size fragments), secondly, the ligation of primary units into secondary ("Replicon" size) units and thirdly, the ligation of secondary units into mature DNA.

Centrifugation, Density Gradient↗

PubMed1975

Cycloheximide production by Streptomyces griseus: control mechanisms of cycloheximide biosynthesis.

Cycloheximide accumulation in a fermentation medium has been shown to be the product of the balance between synthesis and degradation of this antibiotic. Glucose has been shown to prevent cycloheximide degradation. Cycloheximide has been shown to interfere with its own synthesis probably due to feedback inhibition. Approaches for increasing cycloheximide titers in the light of these findings are discussed.

Cycloheximide↗

PubMed1979

Nuclear requirement of post-maturational cortical differentiation of amphibian oocytes: effects of cycloheximide.

Cycloheximide induced a complex series of alterations in the cortical cytoplasm of amphibian (Rana pipiens) oocytes undergoing steroid induced nuclear and cytoplasmic maturation in vitro. The morphological changes were described and the role of nuclear-cytoplasmic interactions in the induction of these changes was investigated in intact, enucleated and enucleated-reinjected oocytes. Three stages of cortical changes were ascertained on the basis of: localized alterations at the animal pole, redistribution of pigment and localized contractility (furrow formation) primarily along the animal:vegetal pole axis. The extent and type of cortical alterations varied depending upon the time at which oocytes were examined following hormonal stimulation and cycloheximide treatment. Cycloheximide did not produce cortical alterations in non-hormone treated oocytes nor in steroid treated oocytes until after germinal vesicle breakdown. Nuclear and cytoplasmic maturation and the appearance of cortical alterations were all inhibited when cycloheximide was added to oocytes at the time of steroid treatment. Cycloheximide induction of cortical alterations occurred only after the inhibitor was no longer effective in preventing germinal vesicle breakdown. Enucleated oocytes underwent cytoplasmic maturation in response to the steroid but exhibited no cortical alterations following the delayed addition of cycloheximide. Simultaneous administration of cycloheximide and steroid to enucleated oocytes inhibited cytoplasmic maturation and all observable cortical alterations. Reinjection of nuclear material into enucleated oocytes restored the ability of cycloheximide to induce cortical alterations following steroid induction of cytoplasmic maturation. Without steroid treatment, such reinjected oocytes did not exhibit cortical changes in response to cycloheximide. The data demonstrate that the nucleus is required for and contains a factor(s) which controls the cycloheximide response and post-maturation differentiation of the oocyte. The maturational changes in the cortical cytoplasm appear to be dependent on the intermixing of the germinal vesicle nucleoplasm materials with mature cytoplasm following germinal vesicle breakdown. The results further suggest that the cortical effects of cycloheximide are dependent upon the initiation of protein synthesis during this period of oocyte development. The significance of these observations and experimental studies are discussed in relation to current understanding of the molecular mechanisms controlling meiosis induction and the composition of the germinal vesicle.

Animals↗

PubMed1975

Synthesis of reovirus-specific polypeptides in cells pretreated with cycloheximide.

When L cells are infected with reovirus in the presence of cycloheximide neither virus-specific polypeptides nor viral double-stranded RNA are synthesized. There is some synthesis of viral single-stranded RNA, transcribed mainly from segments L1, M3, S3, and S4 of the 10 viral genomic segments, and in previous work this has been termed the early mRNA pattern. In an attempt to determine whether these early transcripts are functional mRNA's, the transcripts were allowed to accumulate for a period of 17.5 h at 31 C in cycloheximide-treated cells. The cycloheximide was removed and the cells were exposed for various periods to radioactive amino acids to label any virus-specific polypeptides that might be synthesized. An immunoprecipitation technique was used to separate the viral polypeptides from cellular extracts and this precipitate was then analyzed on sodium dodecyl sulfate-polyacrylamide gels. Within 30 min of cycloheximide removal, four major polypeptides (lambda2, mu0, sigma2a, and sigma3) and two minor polypeptides (lambda1 and mu2) were found. In infected cells without cycloheximide eight viral polypeptides (lambda1, lambda2, mu0, mu2, sigma1, sigma2, sigma2a, sigma3) were found at 17.5 h after infection and the same pattern was found between 3 to 4 h after removal of cycloheximide which had been present for 17.5 h after infection. The latter result shows that the cycloheximide inhibition is reversible and that the cells readily recovered and synthesized the normal complement of viral polypeptides. In one set of experiments cordycepin was added to infected cells immediately after the removal of cycloheximide at 17.5 h to inhibit the synthesis of new viral transcripts. During the succeeding 4 h in the presence of cordycepin, the pattern of protein synthesis was the same as that obtained during the 30 min after cycloheximide removal. It is concluded that the polypeptides formed right after removal of cycloheximide are the translation products of transcripts accumulated during cycloheximide treatment and, therefore, that these transcripts are functional viral mRNA's.

Cycloheximide↗

PubMed1975

Influence of cycloheximide on the lung.

We examined the time course of the influence of cycloheximide on descending pressure-volume curves of excised lungs and on protein and lecithin synthesis and oxygen consumption by lung slices. We also looked at the influence of cycloheximide on granular pneumocyte ultrastructure. Excised lungs from cycloheximide-treated animals are more compliant than controls. After ventilation with air, lungs from control and cycloheximide animals show increased retractive forces and a shift to the right of the deflation P-V curve. Incubation at 38 degrees C for 30 min reverses these changes in control lungs, but not in lungs from cycloheximide-treated rabbits. There is no change in liquid delfation P-V curves after cycloheximide. Cycloheximide causes an immediate decrease of 50% in incorporation of radioactive leucine into protein by lung slices. Incorporation of radioactive palmitate into lecithin and oxygen consumption are also decreased by 50% 6 h after cycloheximide. Lamellar bodies in granular pneumocytes are smaller after cycloheximide. Cycloheximide causes a significant increase in the surface density of the lamellar body envelope. Cytoplasmic area of granular pneumocytes is increased after cycloheximide.

Animals↗

PubMed1989

Protection of doxorubicin cytotoxicity by cycloheximide.

The effect of cycloheximide (an inhibitor of cellular protein synthesis) on doxorubicin-induced cytotoxicity in V79 (rodent fibroblasts) cells was investigated. Cycloheximide is a potent protector of doxorubicin-induced cytotoxicity at concentrations paralleling those required for protein synthesis inhibition. The greatest protective effect was achieved at 10 microM cycloheximide; this concentration correlated with 95% inhibition of protein synthesis. A 15 minute cycloheximide (10 microM) exposure resulted in maximal protein synthesis inhibition; however, 4-6 hr of pretreatment with cycloheximide (10 microM) was required to maximally protect cells from doxorubicin. These results suggest that a time-dependent depletion of a protein is required for cycloheximide's protective effect. Cycloheximide treatments were found to decrease intracellular accumulation of doxorubicin by 35-50% but this decrease accounts for only a small fraction of the total protective effect. When corrections were made for differences in doxorubicin accumulation, cycloheximide had no effect on the formation of DNA-protein crosslinks (DNA-topoisomerase II complexes revealed as single strand DNA breaks in alkaline elution studies). These studies suggest that cycloheximide confers protection from doxorubicin cytotoxicity by a step which occurs following the stabilization of DNA-topoisomerase II complexes.

Animals↗

PubMed1992

Superinduction of c-fos gene expression by estrogen in cultured guinea-pig endometrial cells requires priming by a cycloheximide-dependent mechanism.

The c-fos gene expression is rapidly induced by various mitogenic agents and protein synthesis inhibitors in many cell types. Estradiol-17 beta can induce c-fos gene expression in breast cancer cell lines and in the uterus in vivo, but not in cultured guinea-pig endometrial cells. Using this model, we investigated whether a protein synthesis inhibitor, cycloheximide, could induce the c-fos gene and permit a superinduction by estrogens. In the presence of cycloheximide (10 micrograms/ml), protein synthesis was inhibited at 95% within the first hour. From 190 min after the addition of estradiol-17 beta or diethylstilbestrol (10(-8) M) and cycloheximide (10 micrograms/ml), there was a significant increase (ranging from 3- to 5-fold) of the c-fos messenger RNA level (2.2 kilobase in size), compared with the level in cells treated with cycloheximide alone. Nonestrogenic steroid hormones and estradiol-17 alpha were unable to induce c-fos gene expression in the presence of cycloheximide. The effect of estradiol-17 beta observed in the presence of cycloheximide was completely abolished by 4-hydroxy-tamoxifen or by Ly 156758 or by ICI 164384 (10(-6) M). The c-fos mRNAs were rather stable in cells treated with cycloheximide for 2 h (half-life = 51 +/- 6 min) and there was no further increase in the c-fos messenger RNA stability after the addition of cycloheximide plus estradiol-17 beta (half-life = 40 +/- 3 min). The overall results suggest a response at the transcriptional level. In conclusion, cycloheximide transmits activating signals to the c-fos gene which act as priming elements to allow the estrogen action in cultured guinea-pig endometrial cells.

Androstenedione↗

PubMed1989

Protection by cycloheximide against cytotoxicity induced by vincristine, colchicine, or delta 12-prostaglandin J2 on human osteosarcoma cells.

We examined the protective effects of cycloheximide against cytotoxicity induced by vincristine, colchicine, delta 12-prostaglandin J2, or other antitumor agents on the human osteosarcoma cell line, KSu. Vincristine at a concentration of 0.5 microgram/ml decreased the initial cell number to 34% during 4 days; however, when cycloheximide (0.5 to 10 micrograms/ml) was coexistent, the decrease of the cell number was suppressed and 68% of the initial cells remained viable at the maximum. Furthermore, 0.1 micrograms/ml of cycloheximide also reduced cytotoxicity of colchicine (0.1 to 5 microM) or delta 12-prostaglandin J2 (1 to 5 micrograms/ml) and reduced the cytotoxicity of 0.1 microgram/ml of doxorubicin or 1 micrograms/ml of mitomycin C, suggesting that protection by cycloheximide is shown against cytotoxicity of various types of antitumor agents even on human malignant cells. Next, protein synthesis was reduced to 52% of a control at 3 h by 0.1 micrograms/ml of cycloheximide, suggesting that protein synthesis inhibition precedes the protection. De novo protein synthesis analysis showed that vincristine (0.5 microgram/ml) does not induce any specific protein, whereas delta 12-prostaglandin J2 (3 or 4 micrograms/ml) induced Mr 70,000 and 90,000 proteins, and these were markedly inhibited by cycloheximide (0.1 microgram/ml). In a cell-cycle study, M-phase arrest by vincristine (0.5 microgram/ml) was inhibited in the presence of 0.1 microgram/ml of cycloheximide, suggesting that cell cycle arrest by cycloheximide may be important for protection. From these data, this protection by cycloheximide seems to be more general than expected before.

Cell Cycle↗

PubMed1977

HeLa cell DNA polymerases: the effect of cycloheximide in vivo and detection of a new form of DNA polymerase alpha.

Blockage of protein synthesis in HeLa cells by cycloheximide leads to selective effects on the levels of DNA polymerases alpha, beta, and gamma in the cell. The total activity of DNA polymerase alpha remains unchanged after 7 h exposure of cells to cycloheximide but drops to 50% of its original level after 24 h. The level of the beta-polymerase falls rapidly in the cell and is reduced to less than 30% of its initial value by 7 h after treatment of the cells with cycloheximide. The gamma-polymerase level is diminished by 30--40% during the 7 h cycloheximide treatment and reaches 50% of its original level after 24 h. Cells which have been exposed to cycloheximide for 7 h will regain normal levels of the beta- and gamma-polymerases within 90 min after removal of the drug. The cycloheximide-treated cells also show the presence of a new form of the alpha-polymerase, designated alpha1, which can be clearly detected as a separate entity in column chromatography. The level of alpha1 in the nucleus increases during the period that the cells are treated and cycloheximide so that after 24 h it represents almost 50% of the nuclear DNA polymerase activity. The presence of alpha1 in the cytoplasmic fraction can also be demonstrated in both cycloheximide-treated and normal, growing cells.

Cell Nucleus↗

PubMed1975

Nature of the increase in renal ornithine decarboxylase activity after cycloheximide administration in the rat.

The present study was designed to determine whether the increase in rat renal ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity after cycloheximide administration was a primary effect on the kidney or was a secondary effect of adrenal or pituitary hormones released in response to the drug. Renal ornithine decarboxylase activity was reduced approximately 70% 1 hr after intraperitoneal administration of doses of cycloheximide that also inhibited renal protein synthesis by 68-95% within 1 hr. Protein synthesis began to recover by the second hour, accompanied by a rise in decarboxylase activity that reached a peak about six times greater than pretreatment values at 8 hr, then gradually declined to preinjection levels by 16 hr. Peak ornithine decarboxylase activity was directly proportional to cycloheximide doses up to 250 mug; larger doses, which almost abolished protein synthesis for 8 hr, where inhibitory. Plasma corticosterone rose rapidly after cycloheximide, reached a peak at 2 hr, then fell to baseline by 8 hr. Corticosterone response was also dose-dependent up to 250 mug, but larger doses were inhibitorymadrenalectomy did not reduce decarboxylase activity response to cycloheximide, nor did cortisol administration enhance it. Hypophysectomy greatly reduced baseline renal decarboxylase activity within 9 hr and all but abolished the increase in enzyme activity normally seen after cycloheximide administration to the intact rat. The hypophysectomized animal exhibited apparent increased sensitivity to cycloheximide, since a smaller dose of the drug caused a reduction in renal protein synthesis similar to that seen with a larger dose in the intact rat. As protein synthesis was recovering in the hypophysectomized animals, renal decarboxylase activity responded adequately to the injection of a crude pituitary extract. These data suggest that renal ornithine decarboxylase turnover is rapid, that baseline activity is.maintained by new protein synthesis, and that the increase in renal enzyme activity after cycloheximide is in larger part dependent upon pituitary hormone action.

Adrenal Glands↗

PubMed1992

Cycloheximide protection against actinomycin D cytotoxicity.

Pretreatment plus concomitant treatment with 10 micrograms/ml cycloheximide protected Chinese hamster ovary cells and Swiss 3T3 cells against the cytotoxicity of actinomycin D. The cycloheximide treatment reduced the intracellular concentration of actinomycin D by reducing the level of actinomycin D bound to the acid precipitable fraction of the cell. Levels of unbound actinomycin D were unaffected by cycloheximide, indicating that the plasma membrane permeability to AD was not reduced. Actinomycin D inhibited total transcription but did not reduce cytoplasmic levels of rRNA nor of most tested mRNA; however, cytoplasmic levels of c-myc mRNA were reduced below detectability. Cycloheximide treatment further inhibited total transcription and had no effect on cytoplasmic levels of rRNA nor of most tested mRNA. Cytoplasmic levels of c-myc were elevated by cycloheximide and remained so even in the presence of actinomycin D. These data suggested that a reduction in cytoplasmic levels of short lived, essential mRNA, such as c-myc mRNA, was one lethal lesion of actinomycin D. Furthermore, cycloheximide's protection may result, in part, from its ability to stabilize and/or elevate cytoplasmic levels of these mRNA, thus counteracting their depletion by actinomycin D. Protection may also result from the cycloheximide-induced reduction of actinomycin D bound to the acid precipitable fraction of the cells.

Animals↗

PubMed1977

Cycloheximide produces adult-like retention deficits of prior learning in infant mice.

Utilizing a dosage of cycloheximide which was found to inhibit cerebral protein synthesis by almost 90% after injection, separate groups of 13-day-old mice received either cycloheximide or saline followed by 0 (control), 15, or 25 training trials in a discriminated shock-escape T-maze. Twenty-four hr later, each mouse was treated with cycloheximide or saline and tested for retention by an additional 25 trails in the T-maze. As reflected by correct choice-point turns, the results suggest that whereas salinetreated mice demonstrated reliable retention of prior learning, cycloheximide treated mice exhibited memory impairment; cycloheximide per se had no effect on performance during either original training or retest. A final experiment indicated that this memory impairment was not due to cycloheximide's general debilitating side effects at the time of retention testing. Taken together, these data suggest that protein synthesis inhibition during training impaired consolidation and/or retrieval processes involved in memory. The biochemical and behavioral effects following cycloheximide injection in 13-14-day-old mice in the present study parallel those reported with adult animals and lend indirect support to the hypothesis that the 24-hr memory capacity exhibited by these young mice reflects the early functioning of those processes involved in adult long-term memory.

Amnesia↗

PubMed1991

Mechanisms involved in the stimulation by cycloheximide of prostaglandin production in the guinea-pig uterus.

Cycloheximide produced a large increase in prostaglandin (PG) E2 output and smaller increases in PGF2 alpha and 6-keto-PGF1 alpha when superfused over the guinea-pig uterus for 20 min. This stimulation of the outputs of these 3 PGs by cycloheximide did not require extracellular calcium. TMB-8 (an intracellular calcium antagonist) had no effect on the stimulation of PGE2 output by cycloheximide, but it completely prevented the stimulation of PGF2 alpha and 6-keto-PGF1 alpha outputs. W-7 (a calmodulin antagonist) had no effect on the stimulation of PGE2 and PGF2 alpha outputs by cycloheximide, but it partially reduced and delayed the stimulation of 6-keto-PGF1 alpha output. Neomycin (a phospholipase C inhibitor) did not prevent the increases in PGE2 and 6-keto-PGF1 alpha outputs produced by cycloheximide. However, neomycin (5 and 10 mM, but not 1 mM) inhibited the small increases in PGF2 alpha caused by cycloheximide. On its own, neomycin produced a dose-dependent, transient increase in 6-keto-PGF1 alpha output without affecting the outputs of PGF2 alpha and PGE2. It is concluded that different mechanisms are involved in the processes by which cycloheximide stimulates the syntheses of PGE2, PGF2 alpha and 6-keto-PGF1 alpha in the guinea-pig uterus.

6-Ketoprostaglandin F1 alpha↗

PubMed1979

Inhibition by cycloheximide of degradation of cytochrome P-450 in primary cultures of adult rat liver parenchymal cells and in vivo.

Degradation of cytochrome P-450 was studied in adult rat liver parenchymal cells in primary monolayer culture. In cells incubated in standard culture medium, the amount of cytochrome P-450 decreased at an accelerated rate relative to either the rate of degradation of total protein in the cells or the turnover of cytochrome P-450 in vivo. This change was succeeded by a spontaneous increase in the activity of haem oxygenase, an enzyme system that converts haem into bilirubin in vitro, measured in extracts from the cultured cells. This finding suggests that the rate of cytochrome P-450 breakdown may be controlled by factor(s) other than the activity of haem oxygenase. The decline in cytochrome P-450 and the subsequent increase in haem oxygenase activity was prevented by incubation of hepatocytes in medium containing an inhibitor of protein synthesis such as cycloheximide, puromycin, actinomycin D, or azaserine. The effect of cycloheximide appeared to be due to decreased breakdown of microsomal (14)C-labelled haem. By contrast, cycloheximide was without effect on the degradation of total protein, measured either in homogenates or in microsomal fractions prepared from the cultured cells. These results suggest that the conditions of cell culture stimulate selective degradation of cytochrome P-450 by a process that is inhibited by cycloheximide and hence may require protein synthesis. The findings in culture were verified in parallel studies of cytochrome P-450 degradation in vivo. After administration of bromobenzene, the degradation of the haem moiety of cytochrome P-450 was accelerated in vivo in a manner resembling that observed in cultured hepatocytes. Administration of cycloheximide to either bromobenzene-treated rats or to untreated rats decreased the degradation of the haem moiety of cytochrome P-450. However, the drug failed to affect degradation of haem not associated with cytochrome P-450, suggesting that cycloheximide is not a general inhibitor of haem oxidation in the liver. These findings confirm that the catabolism of hepatic cytochrome P-450 haem is controlled by similar cycloheximide-sensitive processes in the basal steady state in vivo, as stimulated by bromobenzene in vivo, or in hepatocytes under the conditions of cell culture. We conclude that the rate-limiting step in this process appears to require protein synthesis and precedes cleavage of the haem ring.

Animals↗

PubMed1989

The enhancing effect of adrenocorticotropin on adrenal 3-hydroxy-3-methylglutaryl coenzyme A reductase messenger ribonucleic acid level is inhibited by aminoglutethimide but not by cycloheximide.

3-Hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase activity and reductase mRNA level were determined in adrenals from hamsters treated with ACTH, with or without cycloheximide or aminoglutethimide. Both reductase activity and reductase mRNA level were similarly enhanced by ACTH administration compared to levels in NaCl-treated animals. The administration of cycloheximide with ACTH resulted in a 73% decrease in reductase activity compared to control values, but did not prevent the enhancing effect of ACTH on the reductase mRNA level. Furthermore, the administration of cycloheximide alone diminished HMG-CoA reductase activity, but enhanced by 1.1- to 1.6-fold the reductase mRNA level. Coadministration of aminoglutethimide with ACTH also resulted in a decrease (65%) in reductase activity compared to that in NaCl-treated animals. However, coadministration of aminoglutethimide, in contrast to cycloheximide, with ACTH not only prevented the reductase mRNA level increase produced by ACTH, but also resulted in a 30% decrease in the reductase mRNA level compared to that in controls injected with 0.15 M NaCl. In addition, aminoglutethimide alone resulted in 50% and 54% decreases in reductase mRNA level and reductase activity, respectively. Thus, we have shown that both cycloheximide and aminoglutethimide can prevent the enhancing effect of ACTH on HMG-CoA reductase activity, but their modes of action differ. It is likely that the aminoglutethimide inhibition could be the result of a diminution of specific reductase gene transcription, whereas cycloheximide would result in inhibition of the synthesis of specific proteins, including HMG-CoA reductase. In this respect, since the adrenal free cholesterol content was increased in groups treated with ACTH-aminoglutethimide, we postulate that free cholesterol could be one of the important components involved in the regulation of HMG-CoA reductase gene transcription. As for the ACTH-cycloheximide-treated groups, the adrenal free cholesterol content was also increased, but the effect of ACTH on the reductase mRNA level was not prevented, presumably because this drug blocked the synthesis of a putative sterol regulatory protein that is required to repress HMG-CoA reductase gene transcription.

Adrenal Glands↗

PubMedDate not supplied

[Activation of transcription of the tyrosine aminotransferase gene in the rat McA-RN 7777 hepatoma cell line by cycloheximide].

The expression of the tyrosine aminotransferase (TAT) mRNA after cycloheximide treatment was analysed by Northern blotting method in Morris rat hepatoma cell lines. The level of TAT mRNA increased after 6-8 h of cycloheximide treatment only in the McA-RH 7777 cell line. McA-RH 7777 nuclear run-off assay showed that TAT transcription was induced by cycloheximide treatment. Both glucocorticoid and cycloheximide modulated TAT gene transcription in a synergistic way. There was no induction of TAT expression following cycloheximide or cycloheximide glucocorticoid simultaneous treatment in another cell line (McA-RH 8994), while c-myc and c-fos expression was superinduced by cycloheximide treatment. The possible mechanism of transcription regulation and its damage in hepatoma cells is discussed.

Animals↗

PubMed1990

Divergent effects of cycloheximide on the induction of class II and class III cytochrome P450 mRNAs in cultures of adult rat hepatocytes.

We have previously reported that when hepatocytes isolated from adult male rats are cultured in serum-free medium on matrigel, a reconstituted basement membrane gel, it is possible to elicit a stimulation of gene expression for both Class II cytochrome P450b/e and Class III cytochrome P450p by phenobarbital treatment (E.G. Schuetz et al., 1990 J. Biol. Chem. 265, 1188-1192). In the present study, an investigation of the requirement of protein synthesis for the rise in mRNAs for these cytochromes, pretreatment of the cells with cycloheximide prior to adding phenobarbital or "phenobarbital-like" inducers to the culture medium inhibited induction of P450b/e mRNA (46-90%), whereas the accumulation of P450p mRNA was enhanced (2- to 19-fold). Heme depletion did not appear to explain these observations because the inhibitory effects of cycloheximide on the induction of P450b/e mRNA were not overcome by supplementation of the medium with exogenous heme or with delta-aminolevulinic acid. Because Class IIIA P450s are regulated by gender as well as by phenobarbital, we examined the basal expression of P450p mRNA in cultures of hepatocytes derived from male rats and found that cycloheximide treatment was without effect. However, in cultures of hepatocytes isolated from female rats, where P450p mRNA is barely detectable, cycloheximide treatment greatly enhanced expression of P450p mRNA. As was observed in the cultured cells, the treatment of living female rats with cycloheximide also increased the amounts of P450p mRNA to levels comparable to those found in livers of untreated male rats. Analysis of Northern blots hybridized with oligonucleotides specific for P450PCN1(IIIA1) and P450PCN2(IIIA2), respectively, revealed that untreated male rat liver and cultures of hepatocytes prepared from these animals expressed readily detectable amounts of P450PCN1(IIIA1) mRNA. Such analyses confirmed that cycloheximide treatment selectively increased P450PCN1(IIIA1) mRNA in female rat liver, whereas the amount of mRNA for P450PCN2(IIIA2), a closely related male-specific family member, was unaffected. We conclude that the pathways for the induction of P450b/e and P450p by phenobarbital, and the pathways for the gender-specific basal expression of P450PCN1(IIIA1) and P450PCN2(IIIA2) are not the same and can be distinguished by their differential response to inhibition of ongoing protein synthesis.

Animals↗

PubMed1990

Dermal fibroblasts from Down's syndrome patients share a cycloheximide-induced deficiency in collagen adhesion responses with normal aging cells.

Human skin fibroblasts from three different Down's syndrome patients (trisomy 21) of very different ages have been tested for their adhesion responses on tissue culture substrata coated with type I collagen, fibronectin (FN), and their combination after or during treatment of cells with cycloheximide to evaluate limitations in specific responses. It was shown previously that in vitro-aged papillary and reticular dermal fibroblasts from normal individuals do not generate F-actin stress fibers when pretreated with cycloheximide on collagen substrata but do so on FN substrata, a deficiency linked to limiting amounts/function of collagen-specific receptors in aging cells. In these studies, all three Down's fibroblast populations demonstrated a similar deficiency in stress fiber formation, evaluated by rhodamine-phalloidin staining, upon cycloheximide treatment at all passage levels. They remained competent for stress fiber formation on FN substrata and for reorganization of microtubule and intermediate filament networks on all substrata, demonstrating the specificity for the collagen matrix and for the F-actin cytoskeleton in this deficiency. The cycloheximide-induced deficiency could be readily reversed in all three cell populations by further incubation of cells in drug-free medium and, in some cases, by prior growth of cells in ascorbate-supplemented medium to stimulate collagen and possibly collagen receptor production. However, several pieces of evidence indicate that reduced amounts of FN and collagen synthesized by fibroblasts do not contribute to the cycloheximide-induced deficiency, including the inability to reverse the effect by treatment of cells with TGF beta. Several conclusions are suggested from these studies: (a) Down's dermal fibroblasts become deficient in collagen-specific receptor(s) upon cycloheximide treatment, which leads to altered transmembrane signaling and inability to reorganize F-actin into stress fibers; (b) Down's dermal fibroblasts at all passage levels have matrix adhesive phenotypes similar to those of aging fibroblasts from normal individuals; and (c) these studies provide further support for cells from Down's patients as a genetic model of aging in normal populations.

Actins↗