BMP-4 and retinoic acid synergistically induce activation of caspase-9 andcause 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 o bserved, 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 ac tivity and DNA fragmentation. These three activities were observed infreque ntly or not at all when cells were treated with RA or BMP-4 alone, In the R A/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 act ivity and DNA fragmentation induced by RA/BMP-4 treatment. However, unlike staurosporine-induced apoptosis, cytochrome c, which activates caspase-9, w as 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/cytoc hrome c pathway, The prominent decrease of X chromosome-linked inhibitory a poptosis protein (XIAP) in the cytosol may explain the activation of caspas e-9 induced by RA and BMP-4 treatment.