Regulation of apolipoprotein A-I gene expression in Hep G2 cells depleted of copper by cupruretic tetramine.

Persistent Link:
http://hdl.handle.net/10150/186914
Title:
Regulation of apolipoprotein A-I gene expression in Hep G2 cells depleted of copper by cupruretic tetramine.
Author:
Zhang, Jin.
Issue Date:
1994
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Two 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.
Type:
text; Dissertation-Reproduction (electronic)
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Nutritional Sciences; Graduate College
Degree Grantor:
University of Arizona
Committee Chair:
Lei, David K. Y.

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleRegulation of apolipoprotein A-I gene expression in Hep G2 cells depleted of copper by cupruretic tetramine.en_US
dc.creatorZhang, Jin.en_US
dc.contributor.authorZhang, Jin.en_US
dc.date.issued1994en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.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.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineNutritional Sciencesen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.chairLei, David K. Y.en_US
dc.contributor.committeememberWeber, Charles W.en_US
dc.contributor.committeememberReid, Bobbyen_US
dc.contributor.committeememberMcCaughey, W.F.en_US
dc.identifier.proquest9517530en_US
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