P-glycoprotein: Expression and function in normal circulating leukocytes
AuthorKlimecki, Walter Thomas.
Committee ChairDalton, William
MetadataShow full item record
PublisherThe University of Arizona.
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.
AbstractP-glycoprotein (P-gly) is a well characterized membrane protein, expressed in cancer cells, functioning as an efflux pump. This function confers drug-resistance. P-gly is also expressed in normal tissues such as the liver and kidney. In normal tissues P-gly has restricted expression. In the kidney P-gly is expressed in tubular brush border. This suggests a physiologic role for P-gly. A goal of this work was to determine whether P-gly, if present in leukocytes, followed the cell-type restriction seen in other P-gly positive normal tissues. Assays measuring P-gly included immunofluorescence, immunocytochemistry, and immunoblot analysis. Northern blot analysis and RT-PCR were used to measure mdr1 mRNA. P-gly function was assayed by measuring the verapamil-sensitive retention of rhodamine 123 (rh123). Immunofluorescent staining of leukocytes for lineage and P-gly revealed high levels of P-gly in CD56+ cells. CD8+ cells followed in staining, with CD4+ and CD19+ cells at intermediate levels, and CD14+ and CD15+ cells staining at background. RNA analysis by RT-PCR confirmed the immunofluorescence data, except for CD15+ cells, which demonstrated mdr1 mRNA similar to CD4+ cells. Function assays confirmed the immunofluorescence results, with efficient clearing of dye from CD56+ cells, followed by CD8+, CD4+, and CD19+ cells. CD14+ and CD15+ cells did not demonstrate P-gly function. Immunoblot analysis of membranes and immunocytochemical analysis of CD15+ cells demonstrated P-gly. The high level of functional P-gly observed in CD56+ cells prompted experiments to determine whether P-gly was involved in the CD56+ mediated cytolytic response. Using 4 inhibitors of P-gly mediated efflux, cyclosporine A, PSC 833, R-verapamil, and S-verapamil, NK cells were assayed for cytolytic function. Each compound demonstrated dose-response relationships in inhibiting NK-mediated cytolysis. Each compound also demonstrated a dose-response in inhibition of P-gly mediated efflux, although there was not an exact correlation between efflux inhibition and cytolysis inhibition. Nevertheless, the data in this study demonstrate a relatively high level of P-gly expression in CD56+ and CD8+ cells. In addition, the data support a role for P-gly in the cytolytic function of NK cells, although the point of P-gly involvement in the process of cytolysis remains to be defined.
Degree ProgramPharmacology & Toxicology