| Apoptosis induced by various chemotherapeutic agents is associated with an increased level of intracellular ceramide. Ceramide is a lipid second messenger in the regulation of cell growth. The objective of this study was to identify the signal transduction cascade involving ceramide, culminating in lymphoma cell apoptosis induced by P-glycoprotein (P-gp) inhibitors. The P-gp inhibitors PSC 833 (PSC, 0.1--2muM, a non-immunosuppressive cyclosporin D derivative) and Verapamil (V, 10--100muM) were used in the present study. The range of concentrations of PSC used in this study includes levels present in the blood of patients treated experimentally with this drug. In the studies described here, inhibitors of the P-gp efflux pump inhibited P-gp-mediated rhodamine 123 (Rh123) efflux and induced apoptosis in Molt-4 T lymphoma cells in a dose- dependent fashion. However, P-gp inhibitors did not induce apoptosis in normal peripheral blood T cells regardless of whether these cells were quiescent, activated or proliferating. Apoptosis was assessed by radioactive quantitation of DNA fragmentation, flow cytometry, and fluorescence microscopy. P-gp inhibitors, resembling ceramide, induced an increase in the DNA-binding activities of nuclear factor-kappa B (NF-kB) and activator protein 1 (AP-1) (as measured by the electrophoretic mobility shift assay (EMSA)). To further determine the role of ceramide in our system, we assessed the effects of inhibition of ceramide biosynthesis on apoptosis induced in Molt-4 cells by P-gp inhibitors. Cells were pretreated 1--2 h with agents, Fumonisin (Fum) and L-Cycloserine (CS), which inhibit different steps of ceramide formation and distinct portions of the ceramide-mediated apoptotic pathway. These treated cells exhibited lower levels of apoptosis upon exposure to PSC or V. As mentioned before, increased levels of intracellular ceramide have been measured when different inducers of apoptosis were used. This holds true for the two P-gp inhibitors: PSC and V, as determined by the Diacylglycerol (DAG) kinase assay. Since ceramide was associated with similar changes in cells, these observations support the suggestion that ceramide is involved in the induction of apoptosis by P-gp inhibitors and that NF-kB and AP-1 may also be involved in the regulation of apoptosis induced by P-gp inhibitors in Molt-4 T lymphoma cells. |
