Transforming growth factor-β mediated apoptosis in the Ramos B-lymphoma cell line is accompanied by caspase activation and Bcl-XL downregulation

Upon transforming growth factor-beta (TGF-beta) treatment, Ramos cells, a B-cell lymphoma cell line, undergo apoptosis, as measured by annexin V labeling, DNA fragmentation, and propidium iodide staining. Apoptosis could be observed by 24 h after TGF-beta exposure and occurred before the development of a significant blockage of cell cycle progression. TGF-beta-mediated apoptosis was also accompanied by a strong induction of caspase-3 subfamily activity. Incubation of cells with the caspase inhibitor Z-VAD.FMK at 20 mu M, but not at 10 mu M, prevented TGF-beta-induced apoptosis from occurring. By comparison, caspase-3 subfamily activity was 87% inhibited at 10 mu M Z-VAD.FMK and completely inhibited at 20 mu M. Because of TGF-beta's well-established role of regulating gene transcription, the mRNA levels for proteins associated with apoptosis (Fas- and Fas-associated proteins, Bcl-2 family members, IAP proteins, and I kappa B) were also studied. After 24 h of TGF-beta treatment, the most significant mRNA changes occurred with Bcl-X-L (twofold decrease) and Bik (twofold increase). TGF-beta treatment also resulted after 48 h in a fivefold decrease in Bcl-X-L protein levels, based on immunoblotting analysis. Therefore, TGF-beta-mediated apoptosis involves the activation of caspases. In addition, TGF-beta transcriptionally regulates Bcl-2 family members, Bcl-X-L and Bik, to further influence the apoptosis process, (C) 1998 Academic Press.