Our results suggest that cell death induced by DAL 1 4. 1B e pression does not proceed via a classic caspase activation cascade, but rather relies solely on one caspase, caspase 8. This lack of Wortmannin effector caspase activation in DAL 1 4. 1B induced apoptosis is intriguing. Activation of these cas pases is one of the major characteristics in the pro grammed cell death process. However, some cells survive caspase activation, and accumulating evidence suggests that many caspase activating apoptotic stimuli, including oncogenes, p53, DNA damaging drugs, proap optotic Bcl2 family members, cytoto ic lymphocytes, and in some cases even death receptors, do not necessarily require activation of the known effector caspases for pro grammed cell death to occur.

Activation of upstream caspases such as caspase 8 has been reported in both mitochondrial independent and dependent pathways. Activated caspase 8 has been reported to trigger cell death by activating either effector caspases 3, 6 or 7, or DNA damage enzymes such as endonuclease G and AIF. Caspase induced release of EndoG from mitochondria results in cell apoptosis. Li and colleagues reported apoptosis with activated caspase 8 without activation of effector caspases. A parallel condi tion activated caspase 8 without activation of caspases 3, 6, or 7 occurs when DAL 1 4. 1B protein is e pressed in MCF 7 cells although preliminary studies did not show the release of EndoG into the cytoplasm. Caspase 8 can also cross talk with calpain dependent apoptotic pathways.

Benjamin and colleagues found that both caspase 8 inhibitor z IETD and calpain inhibitors can protect mature mouse oligodendrocytes from cell death initiated by staurosporine, thapsigargin and kainite. Their results suggest that crosstalk occurs between the caspase and calpain pathways, upstream of an irrevers Although protein methylation has been shown to be involved in such cellular processes as signal transduction and transcription no evidence connecting protein methylation and apoptosis has been reported previously. AdO , an inhibitor of s adenosylhomocysteine hydrolase, can inhibit methylation by elevating the cellu lar level of AdoHcy to inhibit the activity of methyltrans ferases. As AdoHcy is a general inhibitor of the once carbon metabolism pathway, its elevation could also inhibit DNA as well as RNA methylation events.

Hypomethylation of cellular methyl accepting protein substrates by AdO has been demonstrated previously and confirmed in this study. Rat phe ochromocytoma cells treated with 30 M AdO for 72 hours were found to undergo a 50% decrease in growth rate. In the present analysis, apoptosis levels in MCF 7 cells Entinostat were not affected by the hypomethylating treatment of cells with 30 M AdO for 48 hours. How ever, AdO treatment appeared to enhance apoptosis when the DAL 1 4.