Phase I: Multidrug resistance (MDR) secondary to overexpression of (P-gp) is an important cause of therapeutic failure in the treatment of cancer. Pharmacologic reversal of MDR by a variety of different drugs has been the subject of over seven hundred publications. However, the ability of noncytotoxic drugs to induce MDR has not been investigated.
Specific Aim A- To determine if clinically important, noncytotoxic drugs can induce MDR. Methods-Several human tumor cell lines will be incubated in media alone and with doxorubicin (positive control) doxycycline, cefoperazone, piperacillin, or prednisolone. Cell lines will be evaluated for mdr1 gene expression, P-gp expression, and the MDR phenotype. Phase II: Pharmacologic modulation of MDR has been accomplished by a wide variety of structurally and functionally diverse drugs (calcium channel blockers, hormones, phenothiazines, calmodulin inhibitors, antibiotics, and immunosuppressives). Cyclosporin A and similar drugs are among the most clinically useful MDR-modulating drugs. The mechanism by which they reverse MDR has not been determined. Calcineurin (CaN), a serine-threonine protein phosphatase, has recently been identified as a molecular target for cyclosporins and related drugs. The role, if any, that CaN plays in the regulation of P-gp has not been identified.
Specific Aim B- To determine (1) what effect CaN has on P-gp phosphorylation and (2) if CaN phosphatase activity correlates with the ability to reverse MDR for seven different compounds (cyclosporins and related drugs). Methods-(1) Phosphorylation of P-gp will be assessed in the presence and absence of exogenous CaN, both with and without CaN inhibitors. (2_) Calcineurin phosphatase activity will be assessed in the presence of each of the selected modulating drugs. MDR reversal will also be evaluated for each of the modulating drugs, using at least two established MDR cell lines.
Specific Aim C- To determine if CaN is overexpressed in MDR cells. Methods-Calcineurin expression will be assessed quantitatively both at the mRNA and protein level. The cell lines induced in Phase I and their respective negative controls will be used for these studies.
