To analyze the nature of nelfinavir mediated cell death, a propidium iodide permeability and anne in binding assay was performed. FACScan analysis showed that a concentration of 8 ug ml nelfina vir induced a significant increase in the number of apoptotic and necrotic or dead leukemia cells, but had no detectable effects STA-9090 on the morphology or apoptosis of the rather heterogeneous BMC cell population. Nelfinavir downregulates cyclin B and cdk1 e pression and interferes with cell cycle progression It has previously been shown by both our group and others that nelfinavir induces the endoplasmic reti culum stress response in solid human cancer cells, resulting in upregulation of BiP, phosphorylation of eIF2, upregulation of ATF3, and autophagy.

In contrast to our results for ovarian cancer cells, Western blot analysis did not shown upregulation of BiP or ATF3 in nelfinavir treated leukemia cells, and cells e hibited no signs of autophagy as shown by a lack of LC3B upregu lation. However, nelfinavir induced a slight increase in eIF2 phosphorylation, suggesting an influence on cell cycle progression, which was further indicated by reduced e pression of cyclin B and cdk1. Cell cycle analysis by FACScan revealed a reduced G2 M peak, suggesting interference with cell cycle progression. However, the most promi nent effect of nelfinavir appeared to be the induction of apoptosis, as indicated by a significant increase in the number of cells in the sub G1 phase.

Nelfinavir induces caspase activation and mcl 1 upregulation despite partial caspase 8 mediated mcl 1 cleavage To gain better insight into the mechanism by which nel finavir induced apoptosis and the e tent of caspase involvement, we performed Western blot analysis for several apoptosis related proteins. In accordance with the FACS analyses presented in Figs. 1 and 2B, induc tion of apoptosis by nelfinavir was confirmed by clea vage of PARP, a specific substrate of effector caspases 3 and 7, whose activation is shown by the appearance of their specific cleavage products. Caspases 3 and 7 are cleaved and activated by initiator caspase 9. Caspase 9 cleavage GSK-3 was observed in nelfinavir treated leukemia cells by Western blot analysis, but the bands were rather faint. In contrast, significant acti vation of initiator caspase 8 was observed, suggesting potential involvement of an additional, mitochondria independent apoptotic pathway. Activation of caspase 12, an initiator caspase downstream of ER stress, was not detected by Western blot analysis. To further investigate the mechanism leading to nelfi navir induced apoptosis, the e pression of several apop tosis regulatory proteins was analyzed. Nelfinavir did not increase the e pression of p53 in IM9 cells.