Abstract
The interaction between P-glycoprotein (140-180 kDa) from the multidrug-resistant Chinese hamster ovary cell line CHRC5 and cyclosporin A was characterized using three different photoactivable cyclosporin A analogs. Two monoclonal antibodies, which are able to discriminate between two major domains of cyclosporin A (the cyclophilin and calcineurin binding domains), were used to detect the photolabeled proteins. A protein of 155 kDa corresponding to P-glycoprotein was much more strongly photolabeled in membranes of CHRC5 cells than in membranes of their drug-sensitive parent cell line AuxB1. The antitumor drug vinblastine and the reversal agents verapamil and cyclosporin A inhibited the photolabeling, and the nonimmunosuppressive derivative PSC-833 caused a stronger inhibition than cyclosporin A. P-glycoprotein photolabeled with cyclosporin A analogs was only detected with the monoclonal antibody that recognizes cyclosporin A and its metabolites, indicating that the calcineurin binding domain recognized specifically by the other antibody is not exposed. These results suggest that the portion of cyclosporin A that binds to calcineurin plays a role in the interaction of cyclosporin A with P-glycoprotein.
Highlights
The interaction between P-glycoprotein (140 –180 kDa) from the multidrug-resistant Chinese hamster ovary cell line CHRC5 and cyclosporin A was characterized using three different photoactivable cyclosporin A analogs
P-glycoprotein photolabeled with cyclosporin A analogs was only detected with the monoclonal antibody that recognizes cyclosporin A and its metabolites, indicating that the calcineurin binding domain recognized by the other antibody is not exposed. These results suggest that the portion of cyclosporin A that binds to calcineurin plays a role in the interaction of cyclosporin A with P-glycoprotein
Two anti-cyclosporin A (CsA) monoclonal antibodies (mAbs) directed against CsA, which are able to discriminate between two portions of CsA, were used (Fig. 2)
Summary
P-gp, P-glycoprotein; CsA, cyclosporin A; mAb, monoclonal antibody; IAAP, [125I]iodoarylazido prazosin; TBS-T, Tris buffered saline-Tween 20; CsD, cyclosporin D; SDZ, Sandoz; CyP, cyclophilin; CAL, calcineurin. Cyclosporin A (CsA) was previously reported to be a substrate for P-gp in renal cell lines [11]. For the reversal activity of CsA in multidrug resistance the exact molecular mechanism remains unknown, and the molecular events implicated in the interaction of CsA and its analogs with P-gp remain to be established. One recognizes the calcineurin binding domain (anti-CsACAL) and the other recognizes the cyclophilin binding domain of CsA (anti-CsACyP). The use of these mAbs allowed us to bring new information on the molecular mechanism implicated in the interaction of CsA with P-gp
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