all three TGF-beta single agents at the concentrations used did not induce apoptosis above Pemirolast ic50 background levels. Whereas in HL 60/Bcl2 cells, apoptosis above history was only caused when ABT 737 was put into the doxorubicin/AN 9 combination, the combination of doxorubicin/AN 9 was synergistic in HL 60/Puro cells with the addition of ABT 737 causing a further escalation in apoptosis. Two other separate apoptosis assays were also performed to show that the traditional hallmarks of apoptosiswere discovered inresponse to the double therapy. After 6 h treatment, caspase 3 activation was apparent in HL 60/ Puro cells treated with the doxorubicin/AN 9 combination but not in HL 60/Bcl2 cells. Also, the addition of ABT 737 in the treatment more increased caspase 3 activity in HL 60/Puro cells and overcame Bcl 2 resistance in HL 60/Bcl2 cells. Similar results were also received in themorphology analysis inwhich cells were scored as being apoptotic on the basis of the existence of chromatin condensation noticed by Hoechst staining. Specific chromatin region was obvious in HL 60/Puro cells treated with doxorubicin/AN 9 for 6 h,whereas the nuclei Metastatic carcinoma of HL 60/Bcl2 cells appeared normal. Only in the current presence of ABT 737 did chromatin aggregation become evident in HL 60/Bcl2 cells. These three separate apoptosis assays all indicated that ABT 737 was able to overcome the apoptosis stop in cells in which Bcl 2 was overexpressed, thus restoring sensitivity to doxorubicin/AN 9 treatments. To verify that cell kill concerned caspase dependent apoptosis, the wide spectrum caspase inhibitor ZVAD fmk was used to prevent apoptosis. Cells were pre treated order Geneticin with 30 mM ZVAD fmk for 1 h before being treated with the treatment. Pre treatment with ZVAD fmk paid down the apoptotic levels to near background levels, indicating that cell kill in a reaction to the double treatment was mediated by caspase dependent apoptosis. To verify that the cytotoxicity of the triple treatment was not limited to only HL 60 cells, another leukemic cell line, U937 was used. The mix of doxorubicin and AN 9 was shown to be complete, and the addition of 10 nM ABT737 was able to boost cell kill more in the treatment. The use of higher ABT 737 attention in the double therapy in the U937 cells compared to HL 60/Puro cells is attributed to the fact that U937 cells show higher endogenous levels of Mcl 1 and as such are more resistant to ABT 737. These results show that ABT 737 is able to overcome Bcl2 mediated resistance to doxorubicin/AN 9 treatments, therefore making previously immune cells exquisitively sensitive and painful to cell kill via adduct damage response pathways.