Downregulation of metallothionein-IIA expression occurs at immortalization.
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Metallothioneins (MTs) may modulate a variety of cellular processes by regulating the activity of zinc-binding proteins. These proteins have been implicated in cell growth regulation, and their expression is abnormal in some tumors. In particular, MT-IIA is expressed 27-fold less in human colorectal tumors and tumor cell lines compared with normal tissue (Zhang et al., 1997). Here we demonstrate that MT-IIA downregulation occurs when human cells become immortal, a key event in tumorigenesis. After immortalization MT-IIA expression remains inducible but the basal activity of the MT-IIA promoter is decreased. MT-IIA downregulation at immortalization is one of the most common immortalization-related changes identified to date, suggesting that MT-IIA has a role in this process. (+info)
Estrogen-dependent and independent activation of the P1 promoter of the p53 gene in transiently transfected breast cancer cells.
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Loss of p53 function by mutational inactivation is the most common marker of the cancerous phenotype. Previous studies from our laboratory have demonstrated 17 beta estradiol (E2) induction of p53 protein expression in breast cancer cells. Although direct effects of E2 on the expression of p53 gene are not known, the steroid is a potent regulator of c-Myc transcription. In the present studies, we have examined the ability of E2 and antiestrogens to regulate the P1 promoter of the p53 gene which contains a c-Myc responsive element. Estrogen receptor (ER)-positive T47D and MCF-7 cells were transiently transfected with the P1CAT reporter plasmid and levels of CAT activity in response to serum, E2 and antiestrogens were monitored. Factors in serum were noted to be the dominant inducers of chloramphenicol acetyltransferase (CAT) expression in MCF-7 cells. The levels of CAT were drastically reduced when cells were maintained in serum free medium (SFM). However, a subtle ER-mediated induction of CAT expression was detectable when MCF-7 cells, cultured in SFM, were treated with E2. In serum-stimulated T47D cells, the CAT expression was minimal. The full ER antagonist, ICI 182 780 (ICI) had no effect. Treatment with E2 or 4-hydroxy tamoxifen (OHT) resulted in P1CAT induction; OHT was more effective than E2. Consistent with c-Myc regulation of the P1 promoter, E2 stimulated endogenous c-Myc in both cell lines. Two forms of c-Myc were expressed independent of E2 stimuli. The expression of a third more rapidly migrating form was E2-dependent and ER-mediated since it was blocked by the full ER antagonist, ICI, but not by the ER agonist/antagonist OHT. These data demonstrate both ER-mediated and ER-independent regulation of c-Myc and the P1 promoter of the p53 gene, and show differential effects of the two classes of antiestrogens in their ability to induce the P1 promoter of the p53 gene in breast cancer cells. (+info)
JunB forms the majority of the AP-1 complex and is a target for redox regulation by receptor tyrosine kinase and G protein-coupled receptor agonists in smooth muscle cells.
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To understand the role of redox-sensitive mechanisms in vascular smooth muscle cell (VSMC) growth, we have studied the effect of N-acetylcysteine (NAC), a thiol antioxidant, and diphenyleneiodonium (DPI), a potent NADH/NADPH oxidase inhibitor, on serum-, platelet-derived growth factor BB-, and thrombin-induced ERK2, JNK1, and p38 mitogen-activated protein (MAP) kinase activation; c-Fos, c-Jun, and JunB expression; and DNA synthesis. Both NAC and DPI completely inhibited agonist-induced AP-1 activity and DNA synthesis in VSMC. On the contrary, these compounds had differential effects on agonist-induced ERK2, JNK1, and p38 MAP kinase activation and c-Fos, c-Jun, and JunB expression. NAC inhibited agonist-induced ERK2, JNK1, and p38 MAP kinase activation and c-Fos, c-Jun, and JunB expression except for platelet-derived growth factor BB-induced ERK2 activation. In contrast, DPI only inhibited agonist-induced p38 MAP kinase activation and c-Fos and JunB expression. Antibody supershift assays indicated the presence of c-Fos and JunB in the AP-1 complex formed in response to all three agonists. In addition, cotransfection of VSMC with expression plasmids for c-Fos and members of the Jun family along with the AP-1-dependent reporter gene revealed that AP-1 with c-Fos and JunB composition exhibited a higher transactivating activity than AP-1 with other compositions tested. All three agonists significantly stimulated reactive oxygen species production, and this effect was inhibited by both NAC and DPI. Together, these results strongly suggest a role for redox-sensitive mechanisms in agonist-induced ERK2, JNK1, and p38 MAP kinase activation; c-Fos, c-Jun, and JunB expression; AP-1 activity; and DNA synthesis in VSMC. These results also suggest a role for NADH/NADPH oxidase activity in some subset of early signaling events such as p38 MAP kinase activation and c-Fos and JunB induction, which appear to be important in agonist-induced AP-1 activity and DNA synthesis in VSMC. (+info)
Esterases in serum-containing growth media counteract chloramphenicol acetyltransferase activity in vitro.
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The spirochete Borrelia burgdorferi was unexpectedly found to be as susceptible to diacetyl chloramphenicol, the product of the enzyme chloramphenicol acetyltransferase, as it was to chloramphenicol itself. The susceptibilities of Escherichia coli and Bacillus subtilis, as well as that of B. burgdorferi, to diacetyl chloramphenicol were then assayed in different media. All three species were susceptible to diacetyl chloramphenicol when growth media were supplemented with rabbit serum or, to a lesser extent, human serum. Susceptibility of E. coli and B. subtilis to diacetyl chloramphenicol was not observed in the absence of serum, when horse serum was used, or when the rabbit or human serum was heated first. In the presence of 10% rabbit serum, a strain of E. coli bearing the chloramphenicol acetyltransferase (cat) gene had a fourfold-lower resistance to chloramphenicol than in the absence of serum. A plate bioassay for chloramphenicol activity showed the conversion by rabbit, mouse, and human sera but not bacterial cell extracts or heated serum of diacetyl chloramphenicol to an inhibitory compound. Deacetylation of acetyl chloramphenicol by serum components was demonstrated by using fluorescent substrates and thin-layer chromatography. These studies indicate that esterases of serum can convert diacetyl chloramphenicol back to an active antibiotic, and thus, in vitro findings may not accurately reflect the level of chloramphenicol resistance by cat-bearing bacteria in vivo. (+info)
The nucleoprotein of Marburg virus is target for multiple cellular kinases.
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The nucleoprotein (NP) of Marburg virus is phosphorylated at serine and threonine residues in a ratio of 85:15, regardless of whether the protein is isolated from virions or from eukaryotic expression systems. Phosphotyrosine is absent. Although many potential phosphorylation sites are located in the N-terminal half of NP, this part of the protein is not phosphorylated. Analyses of phosphorylation state and phosphoamino acid content of truncated NPs expressed in HeLa cells using the vaccinia virus T7 expression system led to the identification of seven phosphorylated regions (region I*, amino acids 404-432; II*, amino acids 446-472; III*, amino acids 484-511; IV*, amino acids 534-543; V*, amino acid 549; VI*, amino acids 599-604; and VII*, amino acid 619) with a minimum of seven phosphorylated amino acid residues located in the C-terminal half of NP. All phosphothreonine residues and consensus recognition sequences for protein kinase CKII are located in regions I*-V*. Regions VI* and VII* contain only phosphoserine with three of four serine residues in consensus recognition motifs for proline-directed protein kinases. Mutagenesis of proline-adjacent serine residues to alanine or aspartic acid did not influence the function of NP in a reconstituted transcription/replication system; thus it is concluded that serine phosphorylation in the most C-terminal part of NP is not a regulatory factor in viral RNA synthesis. (+info)
Identification of an enhancer element of class Pi glutathione S-transferase gene required for expression by a co-planar polychlorinated biphenyl.
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3,3',4,4',5-Pentachlorobiphenyl (PenCB), one of the most toxic co-planar polychlorinated biphenyl congeners, specifically induces class Pi glutathione S-transferase (GSTP1) as well as cytochrome P-450 1A1 in primary cultured rat liver parenchymal cells [Aoki, Matsumoto and Suzuki (1993) FEBS Lett. 333, 114-118]. However, the 5'-flanking sequence of the GSTP1 gene does not contain a xenobiotic responsive element, to which arylhydrocarbon receptor binds. Using a chloramphenicol acetyltransferase assay we demonstrate here that the enhancer termed GSTP1 enhancer I (GPEI) is necessary for the stimulation by PenCB of GSTP1 gene expression in primary cultured rat liver parenchymal cells. GPEI is already known to contain a dyad of PMA responsive element-like elements oriented palindromically. It is suggested that a novel signal transduction pathway activated by PenCB contributes to the stimulation of GSTP1 expression. (+info)
Transcriptional regulation of the mouse ferritin H gene. Involvement of p300/CBP adaptor proteins in FER-1 enhancer activity.
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We previously identified a major enhancer of the mouse ferritin H gene (FER-1) that is central to repression of the ferritin H gene by the adenovirus E1A oncogene (Tsuji, Y., Akebi, N., Lam, T. K., Nakabeppu, Y., Torti, S. V., and Torti, F. M. (1995) Mol. Cell. Biol. 15, 5152-5164). To dissect the molecular mechanism of transcriptional regulation of ferritin H, E1A mutants were tested for their ability to repress FER-1 enhancer activity using cotransfection with ferritin H-chloramphenicol acetyltransferase (CAT) reporter constructs. Here we report that p300/CBP transcriptional adaptor proteins are involved in the regulation of ferritin H transcription through the FER-1 enhancer element. Thus, E1A mutants that failed to bind p300/CBP lost the ability to repress FER-1, whereas mutants of E1A that abrogated its interaction with Rb, p107, or p130 were fully functional in transcriptional repression. Transfection with E1A did not affect endogenous p300/CBP levels, suggesting that repression of FER-1 by E1A is not due to repression of p300/CBP synthesis, but to E1A and p300/CBP interaction. In addition, we have demonstrated that transfection of a p300 expression plasmid significantly activated ferritin H-CAT containing the FER-1 enhancer, but had a marginal effect on ferritin H-CAT with FER-1 deleted. Furthermore, both wild-type p300 and a p300 mutant that failed to bind E1A but retained an adaptor function restored FER-1 enhancer activity repressed by E1A. Sodium butyrate, an inhibitor of histone deacetylase, mimicked p300/CBP function in activation of ferritin H-CAT and elevation of endogenous ferritin H mRNA, suggesting that the histone acetyltransferase activity of p300/CBP or its associated proteins may contribute to the activation of ferritin H transcription. Recruitment of these broadly active transcriptional adaptor proteins for ferritin H synthesis may represent an important mechanism by which changes in iron metabolism are coordinated with other cellular responses mediated by p300/CBP. (+info)
Anti-rheumatic compound aurothioglucose inhibits tumor necrosis factor-alpha-induced HIV-1 replication in latently infected OM10.1 and Ach2 cells.
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NF-kappaB is a potent cellular activator of HIV-1 gene expression. Down-regulation of NF-kappaB activation is known to inhibit HIV replication from the latently infected cells. Gold compounds have been effectively used for many decades in the treatment of rheumatoid arthritis. We previously reported that gold compounds, especially aurothioglucose (AuTG) containing monovalent gold ion, inhibited the DNA-binding of NF-kappaB in vitro. In this report we have examined the efficacy of the gold compound AuTG as an inhibitor of HIV replication in latently infected OM10.1 and Ach2 cells. Tumor necrosis factor (TNF)-alpha-induced HIV-1 replication in OM10.1 or Ach2 cells was significantly inhibited by non-cytotoxic doses of AuTG (>10 microM in OM10.1 cells and >25 F.M in Ach2 cells), while 25 microM of the counter-anion thioglucose (TG) or gold compound containing divalent gold ion, HAuCl3, had no effect. The effect of AuTG on NF-kappaB-dependent gene expression was confirmed by a transient CAT assay. Specific staining as well as electron microscopic examinations revealed the accumulation of metal gold in the cells, supporting our previous hypothesis that gold ions could block NF-kappaB-DNA binding by a redox mechanism. These observations indicate that the monovalent gold compound AuTG is a potentially useful drug for the treatment of patients infected with HIV. (+info)