PKC-independent modulation of multidrug resistance in cells with mutant (V185) but not wild-type (G185) P-glycoprotein by bryostatin 1.

Bryostatin 1 is a new antitumor agent which modulates the enzyme activity of protein kinase C (PKC, phospholipid-Ca2+-dependent ATP:protein transferase, EC 2.7.1.37). Several reports have suggested that the pumping activity of the multidrug resistance gene 1 (MDR1)-encoded multidrug transporter P-glycoprotein (PGP) is enhanced by a PKC-mediated phosphorylation. It was shown here that bryostatin 1 was a potent modulator of multidrug resistance in two cell lines over-expressing a mutant MDR1-encoded PGP, namely KB-C1 cells and HeLa cells transfected with an MDR1-V185 construct (HeLa-MDR1-V185) in which glycine at position 185 (G185) was substituted for valine (V185). Bryostatin 1 is not able to reverse the resistance of cells over-expressing the wild-type form (G185) of PGP, namely CCRF-ADR5000 cells and HeLa cells transfected with a MDR1-G185 construct (HeLa-MDR1-G185). Treatment of HeLa-MDR1-V185 cells with bryostatin 1 was accompanied by an increase in the intracellular accumulation of rhodamine 123, whereas no such effect could be observed in HeLa-MDR1-G185 cells. HeLa-MDR1-V185 cells expressed the PKC isoforms alpha, delta and zeta. Down-modulation of PKC alpha and delta by 12-O-tetradecanoylphorbol-13-acetate (TPA) did not affect the drug accumulation by bryostatin 1. Bryostatin 1 depleted PKC alpha completely and PKC delta partially. In HeLa-MDR1-V185 cells, short-term exposure to bryostatin 1, which led to a PKC activation, was as efficient in modulating the pumping activity of PGP as long-term exposure leading to PKC depletion. Bryostatin 1 competed with azidopine for binding to PGP in cells expressing the MDR1-V185 and MDR1-G185 forms of PGP. It is concluded that bryostatin 1: i) interacts with both the mutated MDR1-V185 and the wild-type MDR1-G185; ii) reverses multidrug resistance and inhibits drug efflux only in PGP-V185 mutants; and iii) that this effect is not due to an interference of PKC with PGP. For gene therapy, it is important to reverse the specific resistance of a mutant in the presence of a wild-type transporter and vice versa. Our results show that it is possible to reverse a specific mutant PGP.