Regulation of apolipoprotein A-I gene expression in Hep G2 cells depleted of copper by cupruretic tetramine.
Committee ChairLei, David K. Y.
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PublisherThe University of Arizona.
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AbstractTwo cupruretic chelators, 2,3,2-tetramine and diamsar were used to deplete copper from Hep G2 cells. Studies using incremental concentrations of chelators, an average maximal depletion of 45% of cellular Cu amounting to 2.4 or 2.3 pmol Cu/μg DNA were attained after 24 h of incubation with 10 μM of tetramine or diamsar in basal medium containing 0.63 μM of Cu. In time-course studies, maximal depletion of Cu was rapidly reached after only 5 h of treatment with 50 μM of these chelators for cells cultured in basal medium. In long-term Cu depletion studies, cellular Cu was reduced more than 40% as compared to controls when cells were cultured in basal medium containing 20 μM tetramine for 4 passages. At the end of the second passage, the synthesis of total protein and albumin was not altered for tetramine-treated cells. After cells were treated with tetramine for 2 passages, pulse-chase studies were performed to determine apolipoprotein synthesis and secretion. Cells were pulsed for 10 min with (³H) leucine and chased for up to 2 h. Trichloroacetic acid precipitation or immunoprecipitation and SDS-PAGE were used to isolate the nascent total protein or specific apolipoproteins, respectively. A two-fold increase in apo A-I synthesis was observed at the end of 10 min pulse in tetramine-treated cells. Although the amount of nascent apo A-I degraded was not affected, that secreted into the medium was increased 56% by tetramine treatment between 30 and 120 min of the chase. A reporter construct -256 A-I.CAT was transiently transfected into cells treated with tetramine for 2 to 3 passages to determine the influence of Cu depletion on apo A-I gene transcriptional regulation. In these Cu depleted cells, a 40% increase in CAT activity indicated that the apo A-I gene promoter activity was enhanced. In addition, the ability of apo A-I regulatory protein 1 to repress the CAT activity was not affected by tetramine treatment. Furthermore, gel shift assays indicated that the increased apo A-I gene expression was probably mediated by enhanced binding of hepatocyte nuclear factor 4 and other unknown nuclear factors to site A of the apo A-I gene promoter.
Degree ProgramNutritional Sciences