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ABSTRACT: Induced transcription of a battery of stress response genes in mammals, including several phase I and phase II drug-metabolizing enzymes, is regulated by the electrophile responsive element (EpRE). Because previous directed mutagenesis of nucleotide motifs within the large, composite EpRE were shown to affect transcription factor binding and associated induced expression of dependent genes, we hypothesized that naturally-occurring variation or polymorphism in the EpRE sequence, if found, could affect the induced expression of important protective genes like glutathione S-transferases, and that this could be an important determinant of cancer risk in humans and other mammals. To determine whether this occurred in nature, 32 strains and species of inbred mice were screened to examine the EpRE sequence present in the mGSTa1 promoter. Two species, Mus caroli and Mus spretus, showed TGAC-->TGGC mutations in the tandem TGAC motif. Inducibility (15-fold) of the variant Mus spretus EpRE sequence in a reporter gene construct in HepG2 cells was significantly increased versus the wild-type EpRE sequence (8-fold). A comparison of mGSTa1-induced expression in the livers of Mus spretus, Mus caroli, and BALB/cJ mice showed the highest level of mGSTa1 mRNA in livers from the Mus spretus and Mus carolimice. This naturally-occurring polymorphism within the EpRE domain is the first mutation with an associated phenotype to be reported within a promoter regulatory element of a drug metabolizing gene.
Cancer Letters 04/2001; 164(2):113-8. · 4.24 Impact Factor
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ABSTRACT: Cells of most tissues, with the exception of hematopoietic cells, require adhesion to an appropriate surface to grow. Cyclin A is needed for cell cycle progression at the G1-S transition, and appearance of cyclin A mRNA and protein in late G1 has been shown to be dependent on adhesion-initiated signals in normal rat kidney fibroblasts. Previously, we have reported that the adhesion-dependent activation of cyclin A transcription in late G1 is mediated by CBP/cycA (CCAAT-binding protein for cyclin A gene), a novel CCAAT-binding protein. Specific binding of CBP/cycA, a Mr 30,000/40,000/115,000 heterotrimeric protein complex, to the CCAAT element of the cyclin A promoter was detectable in growing but not in G0-arrested or nonadherent normal rat kidney cells. Here, we demonstrate that the Mr 30,000/40,000 subunits of CBP/cycA are identical with NF-YA and NF-YB, the two subunits of NF-Y. In addition, we show that, aside from CBP/cycA, NF-Y itself also binds to the CCAAT element of the cyclin A promoter. But, whereas the binding of CBP/cycA is adhesion and cell cycle dependent and correlates with the expression of cyclin A in late G1 phase, NF-Y itself seems to bind in a cell cycle-independent manner.
Cancer Research 12/1997; 57(22):5117-21. · 7.86 Impact Factor
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ABSTRACT: Transcriptional activation of the mouse glutathione S-transferase Ya gene by chemoprotective molecules is mediated through the interaction of trans-acting factors with an antioxidant responsive element (ARE) in the promoter region of this gene. In a step toward identifying those factors which bind productively to the GST Ya ARE, all of the discernible, specific ARE-binding proteins (ARE-BP) in nuclear extracts from HepG2 cells were systematically characterized. By gel-mobility-shift analysis, seven specific ARE-BPs, termed ARE-BP-1 through 7 in order of increasing mobility, were observed that did not vary in concentration or migration between induced and uninduced cell extracts. The molecular weights of the individual ARE-BP subunits were determined by a two-dimensional electrophoresis protocol. Ferguson gel analysis of native protein size indicated that several of the ARE-BP-DNA complexes are composed of multiple protein subunits. Wild-type AREs and GST Ya ARE fragments and mutant sequences were evaluated for their ability to mediate induction in a reporter gene system in HepG2 cells. This same panel of sites was tested in an in vitro binding assay for the ability to compete for the ARE-BPs. A binding profile for each ARE-BP was compiled. Correlation between the ARE-BP binding profiles and induction results indicated that: (i) the ARE-BP-1 and ARE-BP-2 complexes formed only with AREs that supported induction, and (ii) the ARE-BP-4 complex formed with all inducible AREs, but it also bound to ARE mutants that failed to support induction. Based on the studies, an early composite regulatory element model for ARE-mediated expression is presented. ARE-BP-1 is proposed to be the mediator of the ARE's unique induction response to chemoprotective agents.
Archives of Biochemistry and Biophysics 09/1997; 344(2):387-96. · 2.93 Impact Factor
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ABSTRACT: Exposure of human and rodent cells to a wide variety of chemoprotective compounds confers resistance against a broad set of carcinogens. For a subset of the chemoprotective compounds, protection is generated by an increase in the abundance of protective enzymes like glutathione S-transferases (GST). Antioxidant responsive elements (AREs) mediate the transcriptional induction of a battery of genes which comprise much of this chemoprotective response system. Past studies identified a necessary ARE "core" sequence of RTGACnnnGC, but this sequence alone is insufficient to mediate induction. In this study, the additional sequences necessary to define a sufficient, functional ARE are identified through systematic mutational analysis of the murine GST Ya ARE. Introduction of the newly identified necessary nucleotides into the regions flanking a nonresponsive, ARE-like, GST-Mu promoter sequence produced an inducible element. A screen of the GenBank database with the newly identified ARE consensus identified 16 genes which contained the functional ARE consensus sequence in their promoters. Included within this group was an ARE sequence from the murine ferritin-L promoter that mediated induction when tested. In an electrophoretic mobility-shift assay, the ferritin-L ARE was bound by ARE-binding protein 1, a protein previously identified as the likely mediator of the chemoprotective response. A three-level ARE classification system is presented to account for the distinct induction strengths observed in our mutagenesis studies. A model of the ARE as a composite regulatory site, where multiple transcription factors interact, is presented to account for the complex characteristics of ARE-mediated chemoprotective gene expression.
Proceedings of the National Academy of Sciences 06/1997; 94(10):5361-6. · 9.68 Impact Factor
