The effect of cyclosporine A on cytokeratin intermediate filament phosphorylation.

Persistent Link:
http://hdl.handle.net/10150/185577
Title:
The effect of cyclosporine A on cytokeratin intermediate filament phosphorylation.
Author:
Vernetti, Lawrence Allen
Issue Date:
1991
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:
MCF7 cell cultures exposed to cyclosporine A (CsA) from 1-5 μM and from 0-48 hr have decreased viability and clonogenic ability. When these cells are examined by indirect immunofluorescence for cytoskeletal filaments, there is altered cell shape, but no alteration in microfilaments or microtubules. However, there is a collapse in cytokeratin intermediate filament arrays resulting in a peri-nuclear reorganization at 24-48 hr incubation (5 μM). When MCF7 cells are pre-treated in 5 μM CsA for 24 hr, and then placed in non-drug media for 12-24 hr, there is recovery of cytokeratin arrays and cell shape. Analysis of cytokeratin proteins by 2-D electrophoresis depict decreased phosphorylated proteins at 2-5 μM CsA (24 hr). CsA (1-5 μM CsA, 1-48 hr) inhibits phosphate content on cytokeratin protein to 35% levels on cytokeratin #8, and to 50% levels on cytokeratins #18, and #19. CsA decreases phosphate content on cytokeratin protein during incorporation, at steady state, and during turnover. Determinations of phosphate loss indicate that CsA increases turnover at greater levels than it inhibits incorporation, suggesting the activation of a phosphatase enzyme. CsA-induced decreased phosphorylation levels can be rescued by addition of 100 nM 12-o-tetradecanoylphorbol-13-acetate (TPA), but not upon addition of 50 μM forskolin. This indicates a CsA-sensitive protein kinase C site on cytokeratin protein. An in vitro urea assay was utilized to examine assembly and disassembly or cytokeratin protofilaments. CsA (5 μM, 48-72 hr) does not alter assembly of cytokeratin protofilaments from monomeric proteins. Analysis of disassembly of protofilaments into monomeric units resulted in CsA-induced (5 μM, 48-72 hr) inhibition of disassembly. Regulation of phosphorylation may therefore be important in the assembly and disassembly of cytokeratin filaments, and a resistance to disassembly may indicate a CsA interaction that is toxic to the cell.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Dissertations, Academic; Toxicology; Pharmacology
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Pharmacology and Toxicology; Graduate College
Degree Grantor:
University of Arizona
Advisor:
Gandolfi, A. Jay

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleThe effect of cyclosporine A on cytokeratin intermediate filament phosphorylation.en_US
dc.creatorVernetti, Lawrence Allenen_US
dc.contributor.authorVernetti, Lawrence Allenen_US
dc.date.issued1991en_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.abstractMCF7 cell cultures exposed to cyclosporine A (CsA) from 1-5 μM and from 0-48 hr have decreased viability and clonogenic ability. When these cells are examined by indirect immunofluorescence for cytoskeletal filaments, there is altered cell shape, but no alteration in microfilaments or microtubules. However, there is a collapse in cytokeratin intermediate filament arrays resulting in a peri-nuclear reorganization at 24-48 hr incubation (5 μM). When MCF7 cells are pre-treated in 5 μM CsA for 24 hr, and then placed in non-drug media for 12-24 hr, there is recovery of cytokeratin arrays and cell shape. Analysis of cytokeratin proteins by 2-D electrophoresis depict decreased phosphorylated proteins at 2-5 μM CsA (24 hr). CsA (1-5 μM CsA, 1-48 hr) inhibits phosphate content on cytokeratin protein to 35% levels on cytokeratin #8, and to 50% levels on cytokeratins #18, and #19. CsA decreases phosphate content on cytokeratin protein during incorporation, at steady state, and during turnover. Determinations of phosphate loss indicate that CsA increases turnover at greater levels than it inhibits incorporation, suggesting the activation of a phosphatase enzyme. CsA-induced decreased phosphorylation levels can be rescued by addition of 100 nM 12-o-tetradecanoylphorbol-13-acetate (TPA), but not upon addition of 50 μM forskolin. This indicates a CsA-sensitive protein kinase C site on cytokeratin protein. An in vitro urea assay was utilized to examine assembly and disassembly or cytokeratin protofilaments. CsA (5 μM, 48-72 hr) does not alter assembly of cytokeratin protofilaments from monomeric proteins. Analysis of disassembly of protofilaments into monomeric units resulted in CsA-induced (5 μM, 48-72 hr) inhibition of disassembly. Regulation of phosphorylation may therefore be important in the assembly and disassembly of cytokeratin filaments, and a resistance to disassembly may indicate a CsA interaction that is toxic to the cell.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectDissertations, Academicen_US
dc.subjectToxicologyen_US
dc.subjectPharmacologyen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplinePharmacology and Toxicologyen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorGandolfi, A. Jayen_US
dc.contributor.committeememberNagle, Raymond B.en_US
dc.contributor.committeememberCress, Anne E.en_US
dc.contributor.committeememberBrendel, Klausen_US
dc.contributor.committeememberAposhian, H. Vaskenen_US
dc.identifier.proquest9200028en_US
dc.identifier.oclc711709663en_US
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