TRANSFORMING-GROWTH-FACTOR-BETA MEDIATED APOPTOSIS IN THE RAMOS B-LYMPHOMA CELL-LINE IS ACCOMPANIED BY CASPASE ACTIVATION AND BCL-X-L DOWN-REGULATION

Upon transforming growth factor-beta (TGF-beta) treatment, Ramos cells , a B-cell lymphoma cell line, undergo apoptosis, as measured by annex in V labeling, DNA fragmentation, and propidium iodide staining. Apopt osis could be observed by 24 h after TGF-beta exposure and occurred be fore the development of a significant blockage of cell cycle progressi on. TGF-beta-mediated apoptosis was also accompanied by a strong induc tion of caspase-3 subfamily activity. Incubation of cells with the cas pase 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 subfa mily activity was 87% inhibited at 10 mu M Z-VAD.FMK and completely in hibited at 20 mu M. Because of TGF-beta's well-established role of reg ulating gene transcription, the mRNA levels for proteins associated wi th 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 (t wofold decrease) and Bik (twofold increase). TGF-beta treatment also r esulted after 48 h in a fivefold decrease in Bcl-X-L protein levels, b ased on immunoblotting analysis. Therefore, TGF-beta-mediated apoptosi s involves the activation of caspases. In addition, TGF-beta transcrip tionally regulates Bcl-2 family members, Bcl-X-L and Bik, to further i nfluence the apoptosis process, (C) 1998 Academic Press.