BMP-4 and retinoic acid synergistically induce activation of caspase-9 and cause apoptosis of P19 embryonal carcinoma cells cultured as a monolayer.
In monolayer cultures of P19 EC cells treated with both all-trans retinoic acid (RA) and bone morphogenetic protein (BMP)-4 (RA/BMP-4 treatment), many non-adherent apoptotic cells and activated caspase-3-positive cells were observed, but they were not observed in cells treated with RA or BMP-4 alone. Consistent with the appearance of activated caspase-3-positive cells, BMP-4 and RA together induced processing of caspase-9, Ac-DEVD-MCA cleavage activity and DNA fragmentation. These three activities were observed infrequently or not at all when cells were treated with RA or BMP-4 alone. In the RA/BMP-4 treatment-induced apoptosis, caspase-9 was upstream of caspase-3 in the enzyme cascade, and the caspase-9 to -3 step was key in the apoptotic pathway. Bcl-xL inhibited processing of caspase-9, Ac-DEVD-MCA cleavage activity and DNA fragmentation induced by RA/BMP-4 treatment. However, unlike staurosporine-induced apoptosis, cytochrome c, which activates caspase-9, was not detected in the cytosol of RA/BMP-4-treated cells. RA and BMP-4 may activate caspase-9 through an apoptotic pathway other than the Apaf-1/cytochrome c pathway. The prominent decrease of X-chromosome-linked inhibitory apoptosis protein (XIAP) in the cytosol may explain the activation of caspase-9 induced by RA and BMP-4 treatment.
Fujita
E
E
Division of Development and Differentiation, National Institute of Neuroscience, NCNP, Kodaira, Tokyo 187-8502, Japan.
Soyama
A
A
Kawabata
M
M
Momoi
T
T
D000818:Animals; D017209:Apoptosis; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins / Q000378:metabolism* / Q000494:pharmacology; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000235:genetics / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism / Q000494:pharmacology; D004357:Drug Synergism; D004789:Enzyme Activation; D051379:Mice; D009842:Oligopeptides / Q000378:metabolism / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism / Q000494:pharmacology; D014212:Tretinoin / Q000378:metabolism* / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D051020:bcl-X Protein
C494812:Bcl2l1 protein, mouse; C525810:Bmp4 protein, mouse; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins; D015853:Cysteine Proteinase Inhibitors; D009842:Oligopeptides; D019253:Proto-Oncogene Proteins c-bcl-2; C099158:acetyl-aspartyl-glutamyl-valyl-aspartal; D051020:bcl-X Protein; D014212:Tretinoin; C505409:Casp3 protein, mouse; C506434:Casp9 protein, mouse; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases