Decreased sensitivity to N-Benzyladriamycin-14-valerate (AD 198) in non-dividing LLC-PK1 renal epithelial cells corresponds with decreased PKC-ϵ expression.

3232 In vivo analyses of AD 198 in rats indicate no significant organ toxicity at therapeutic doses, with the dose limiting toxicity of AD 198 being myelosuppression. AD 198 exerts its cytotoxic effect in proliferating cells by binding to the C1b regulatory domain of conventional and novel isoforms of PKC and rapidly triggering apoptosis in a manner that is unaffected by changes in Bcl-2, p53, or NF-κB activity. PKC is active in non-dividing, differentiated cells as well as proliferating cells and, consequently, could provide a cytotoxic target for AD 198. However, we have previously observed ex vivo in rodent hearts that AD 198 has a cardioprotective effect through activation of PKC-ϵ in cardiomyocytes. In this current study, we assessed the cytotoxicity of AD 198 in LLC-PK1 pig renal epithelial cells as they progressed from a proliferative to a non-dividing functionally differentiated state. LLC-PK1 cells grow in monolayer until confluency, whereupon they exhibit typical epithelial functionality: providing a cellular barrier and polarity of fluid transport. These aspects are observed in culture through tight junction formation and transepithelial transport of medium to form fluid-filled epithelial domes. Both functions, in turn, are reported to be modulated by PKCs -α and -δ, with increased expression resulting in tight junction permeability. Confluent and non-dividing LLC-PK1 cells demonstrate a 6-fold decrease in sensitivity to AD 198 compared with proliferating cells. LLC-PK1 cells that have formed tight junctions and produce fluid domes exhibit a 13-fold decreased sensitivity to AD 198. Decreased sensitivity to AD 198 does not correlate with altered drug uptake, since dome-producing cells contain a slightly increased amount of AD 198. PKC-α and PKC-δ protein levels remain unchanged as cells progress from proliferating to dome-producing status, suggesting that changes in AD 198 cytotoxicity do not correspond to modulation of these isoforms. In fact, increased PKC-α and PKC-δ expression at all growth phases from tet-responsive vectors transfected into LLC-PK1 cells failed to alter AD 198 cytotoxicity. By contrast, we observe a 10-fold decrease in the expression of PKC-ϵ protein as cells become confluent, stop proliferating, and form tight junctions. These results suggest that AD 198 targets PKC-ϵ in proliferating LLC-PK1 cells to trigger apoptosis and that loss of drug sensitivity in functionally differentiated cells is due to loss of target protein. (Supported by NCI grant CA100093)