Transforming growth factor-beta-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 microM, but not at 10 microM, prevented TGF-beta-induced apoptosis from occurring. By comparison, caspase-3 subfamily activity was 87% inhibited at 10 microM Z-VAD.FMK and completely inhibited at 20 microM. 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-XL (two-fold decrease) and Bik (twofold increase). TGF-beta treatment also resulted after 48 h in a fivefold decrease in Bcl-XL 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-XL and Bik, to further influence the apoptosis process.
Rhône-Poulenc Rorer Central Research, Gene Medicine Department, Collegeville, Pennsylvania 19426, USA.
A
A
Saltzman
R
R
Munro
G
G
Searfoss
C
C
Franks
M
M
Jaye
Y
Y
Ivashchenko
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D001402:B-Lymphocytes / Q000166:cytology* / Q000201:enzymology; D002051:Burkitt Lymphoma; D053148:Caspase 3; D020169:Caspases; D002453:Cell Cycle; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004247:DNA / Q000032:analysis; D004789:Enzyme Activation; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D020672:I-kappa B Proteins; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D011506:Proteins / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D051636:X-Linked Inhibitor of Apoptosis Protein; D051020:bcl-X Protein; D019014:fas Receptor / Q000235:genetics
D000590:Amino Acid Chloromethyl Ketones; D017304:Annexin A5; D051017:Apoptosis Regulatory Proteins; C494811:BCL2L1 protein, human; C096820:BIK protein, human; D015853:Cysteine Proteinase Inhibitors; D020672:I-kappa B Proteins; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D051020:bcl-X Protein; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D019014:fas Receptor; D004247:DNA; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases