S-adenosylmethionine decarboxylase structure-function relationships.
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Acanthamoeba↗
Biomedical subjects
L M Shantz
Publications and source records attributed to L M Shantz.
Regulation of S-adenosylmethionine decarboxylase activity by alterations in the intracellular polyamine content.
The effects of addition of exogenous spermidine and spermine and of two inhibitors of polyamine biosynthesis, alpha-difluoromethylornithine (DFMO), which decreases spermidine concentrations, and n-butyl-1,3-diaminopropane, which depletes spermine, on the expression of S-adenosylmethionine decarboxylase (AdoMetDC) activity were studied in mammalian cell lines (HT29, CHO and COS-7). AdoMetDC levels were inversely related to the polyamine content, and spermine was the more potent repressor of AdoMetDC activity, but only spermidine affected the amount of AdoMetDC mRNA. Transfection of COS-7 cells or CHO cells with plasmid constructs containing a chloramphenicol acetyltransferase (CAT) reporter gene driven by portions of the AdoMetDC promoter region indicated that CAT expression was altered by spermidine, but not by spermine, suggesting that there is a spermidine-responsive element in this promoter. Transient transfection of COS-7 cells with pSAMh1, a plasmid containing the AdoMetDC cDNA in a vector with the SV40 promoter and origin of replication, led to a large increase in AdoMetDC expression. Although treatment of COS-7 cells with n-butyl-1,3-diaminopropane greatly increased endogenous AdoMetDC activity, the spermine depletion brought about by this inhibitor did not stimulate AdoMetDC expression from pSAMh1. The pSAMh1 cDNA is missing 72 nucleotides from the 5' end of the AdoMetDC mRNA, and it is possible that translational regulation by spermine involves this region. The expression of AdoMetDC from pSAMh1 in COS-7 cells was greatly inhibited by DFMO treatment, although endogenous AdoMetDC activity was increased. The expression of other plasmids containing the SV40 origin of replication was also inhibited by DFMO in COS-7 cells, but not in CHO cells. DFMO treatment did not interfere with the expression of plasmids driven by the RSV promoter. These results suggest that low spermidine levels interfere with the replication of plasmids containing the SV40 origin of replication.
Adenosylmethionine Decarboxylase↗
Purification of human S-adenosylmethionine decarboxylase expressed in Escherichia coli and use of this protein to investigate the mechanism of inhibition by the irreversible inhibitors, 5'-deoxy-5'-[(3-hydrazinopropyl)methylamino]adenosine and 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine.
Human S-adenosylmethionine decarboxylase (AdoMetDC) was expressed in high yield in Escherichia coli using the pIN-III(lppP-5) expression vector and purified to apparent homogeneity using affinity chromatography on methylglyoxal bis(guanylhydrazone)-Sepharose. The inactivation of the purified enzyme by 5'-deoxy-5'-[(3-hydrazinopropyl)methylamino]adenosine (MHZPA) was accompanied by an increase in absorbance at 260 nm of the large subunit. This increase was equivalent to the addition of 1 molecule of MHZPA. After digestion with the protease Lys-C, a peptide that contained the bound MHZPA was isolated and found to have the amino acid composition consistent with that expected from the amino terminus of the large subunit. These results indicate that MHZPA inactivates AdoMetDC by forming a hydrazone derivative at the pyruvate prosthetic group. Inactivation of AdoMetDC by 5'-([(Z)-4-amino-2-butenyl]methylamino]-5'-deoxyadenosine (AbeAdo) led to the appearance of a new peptide peak in the Lys-C protease digest. This peptide had the sequence ASMFVSK. This agrees with the expected sequence from the amino terminus, which is pyruvoyl-SMFVSK, with the exception that the pyruvate has been converted to alanine. Direct gas-phase sequencing of the large subunit of the enzyme also indicated the presence of alanine at the amino terminus after inactivation with AbeAdo. These results indicate that this inhibitor leads to transamination of the pyruvate prosthetic group. Since the pyruvate is covalently linked to the protein, its replacement by alanine leads to an irreversible inactivation of AdoMetDC.
Adenosylmethionine Decarboxylase↗
Mechanism of inhibition of growth of 3T3-L1 fibroblasts and their differentiation to adipocytes by dehydroepiandrosterone and related steroids: role of glucose-6-phosphate dehydrogenase.
Dehydroepiandrosterone (DHEA) and certain structural analogues block the differentiation of 3T3-L1 mouse embryo fibroblasts to adipocytes. These steroids also are potent uncompetitive inhibitors of mammalian glucose-6-phosphate dehydrogenases (G6PDs). We provide direct evidence that treatment of the 3T3-L1 cells with DHEA and its analogues results in intracellular inhibition of G6PD, which is associated with the block of differentiation: (i) Levels of 6-phosphogluconate and other products of the pentose phosphate pathway are decreased; (ii) the magnitude of these decreases depends on the potency of steroids as inhibitors of G6PD and on concentration and duration of exposure, and it is accompanied by a proportionate block of differentiation; (iii) in cells exposed to 16 alpha-bromoepiandrosterone (a more potent inhibitor of G6PD than DHEA) at concentrations that block differentiation, introduction of exogenous 6-phosphogluconate in liposomes raises the levels of 6-phosphogluconate and other products of the pentose phosphate pathway and partially relieves the steroid block of cell growth and differentiation.
Adipose Tissue↗
Megestrol acetate-induced differentiation of 3T3-L1 adipocytes in vitro.
We recently observed significant weight gain in patients with advanced breast cancer treated with high-dose megestrol acetate. However, the mechanisms of this effect are completely unknown. As the analysis of the action of steroids in vivo is complex, we chose to examine the effects of megestrol acetate on the differentiation of a preadipocyte clone (L1) of the Swiss 3T3 mouse fibroblast cell line. Addition of insulin, fetal bovine serum, dexamethasone, and methyl-isobutylxanthine to confluent 3T3-L1 cells induced adipocyte differentiation, as verified by morphologic studies and analysis of the activity of the enzyme glycerol-3-phosphate dehydrogenase (G-3-PD), a specific and sensitive indicator of lipocyte function. Substitution of megestrol acetate for dexamethasone also resulted in greatly increased lipocyte differentiation. Tumor necrosis factor (TNF) alpha blocked the adipocyte differentiation induced by the combination of insulin, dexamethasone, and methyl-isobutylxanthine. Addition of megestrol acetate failed to reverse the inhibitory effect of TNF. Thus, megestrol acetate, in vitro, is a potent inducer of lipocyte differentiation. Further studies on the regulation of adipocyte differentiation and function in this model system may be important to clarify megestrol acetate's mechanism of action.
1-Methyl-3-isobutylxanthine↗
Modulation of growth, differentiation and carcinogenesis by dehydroepiandrosterone.
Dehydroepiandrosterone (3 beta-hydroxy-5-androsten-17-one; DHEA) and its conjugates are abundant circulating steroids that originate largely from the adrenal cortex. Their levels decline profoundly with age in human beings of both sexes, as the incidence of most cancers rises. Low levels of these steroids have been associated with the presence and risk of development of cancer. Administration of DHEA to rodents produces protection against spontaneous tumors and chemical carcinogenesis, suppresses weight gain without significantly affecting food intake, ameliorates the severity of diabetes in genetically diabetic mice, and restrains autoimmune processes. DHEA and related steroids also depress the mitogenic effects of carcinogens, tumor promoters and plant lectins, and block viral and carcinogen-induced cell transformations. DHEA and certain congeners are also potent and quite specific inhibitors of mammalian glucose-6-phosphate dehydrogenases. We have observed that the conversion of 3T3-L1 and 3T3-F442A preadipocyte clones to the adipocyte phenotype, in response to appropriate differentiation stimuli (fetal calf serum, insulin, dexamethasone, and 1-methyl-3-isobutylxanthine), is blocked by DHEA and other steroidal inhibitors of glucose-6-phosphate dehydrogenase. The structural requirements for blocking adipocyte differentiation and for inhibiting glucose-6-phosphate dehydrogenase are closely correlated. Evidence is reviewed suggesting that the inhibition of glucose-6-phosphate dehydrogenase is central to the anticarcinogenic and differentiation-blocking actions of DHEA and related steroids. The 3T3 preadipocyte clones provide a valuable system for the analysis of the mechanisms of the effects of DHEA on growth, differentiation and carcinogenesis.
Adipose Tissue↗
Inhibition of the conversion of 3T3 fibroblast clones to adipocytes by dehydroepiandrosterone and related anticarcinogenic steroids.
Dehydroepiandrosterone (3 beta-hydroxy-5-androsten-17-one; DHEA) and related steroids have widespread protective effects against spontaneous and chemically induced tumors, suppress weight gain without affecting food intake, and depress lipogenesis. We have observed that DHEA and 16 alpha-bromoepiandrosterone (16 alpha-bromo-3 beta-hydroxy-5 alpha-androstan-17-one) block the conversion to adipocytes of the 3T3-L1 and 3T3-F442A mouse embryo fibroblast clones. The arrest of lipogenic conversion was assessed by measurements of lipid biosynthesis and the specific activity of cytosolic glycerol-3-phosphate dehydrogenase. In the presence of 215 microM DHEA or 30 microM 16 alpha-bromoepiandrosterone, the increase in glycerol-3-phosphate activity was only 50% of that of fully differentiated control cells. The blocking effects were concentration dependent and were observed only if the differentiation stimuli and the blocking steroid were present simultaneously. Concentrations of these steroids that almost completely blocked conversion to adipocytes were not cytotoxic. Although the relation between structure and blocking activity of steroids is complicated by metabolism of DHEA in these cultures, a strong correlation exists between the structural requirements for blocking differentiation and for inhibition of glucose-6-phosphate dehydrogenase. The 3T3-L1 and 3T3-F442A preadipocyte clones are, therefore, appropriate and convenient model systems for the analysis of the mechanism of the anticarcinogenic effects of DHEA and related steroids.
Adipose Tissue↗