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Apoptosis was monitored morphologically and by flow cytometry.In the case of PC and DU, saquinavir sensitized the surviving cells to ionizing radiation.We conclude that saquinavir inhibits proteasome activity in mammalian cells as well as acting on the HIVI protease.Because saquinavir induced apoptosis in human cancer cells, HIVI protease inhibitors might become a new class of cytotoxic drugs, alone or in combination with radiation or chemotherapy.INTRODUCTION HIVI encodes for a protease required for the cleavage of the viral gagpol polyprotein, and its inhibition leads to the release of noninfectious virus particles. The costs of publication of this article were defrayed in part by the payment of page charges.The cleavage sites of Calcitriol action for HIVI protease were once thought to be unique and distinct from those of mammalian proteases.However, recently, the s proteasome has been shown to cleave the same sites. This led us to investigate whether saquinavir is an inhibitor of the s proteasome, as was previously reported for ritonavir. Transfected cells were maintained in DMEM, and clones were obtained.INDUCTION OF APOPTOSIS BY SAQUINAVIR therefore, used to determine whether simultaneous addition of different concentrations of saquinavir affected the activation process.This inhibition coincided with an accumulation of IB, which indicated involvement of the classical activation pathway of NFB by proteasomedependent degradation of IB. Similar results were observed for human PC prostate cancer cells using M concentrations of saquinavir. A negative control was prepared for one sample by adding unlabeled oligonucleotide in fold excess.Briefly, cells were washed with PBS, then with buffer I, and pelleted by centrifugation.Glass beads and homogenization buffer were added and cells were vortexed for min.Beads and cell debris were removed by centrifugation at, gfor min, and the supernatant was further clarified by spinning at, gfor min.Twenty g of protein from each sample was diluted with buffer I to a final volume of l.To assess s function, buffer I was replaced by buffer containing SDS. Proteolytic activity was monitored continuously using a fluorescence plate reader at nm by release of the fluorescent group AMC.Ten g of protein were separated on SDS gel and blotted to polyvinylidene difluoride membranes at C.NFB signaling is normally dependent on proteasomal degradation of the binding inhibitor IB and is cystine prevented by treatment of cells with proteasome inhibitors. Cleavage activity was monitored for min and expressed as relative fluorescence units, DU cells were incubated with different concentrations of saquinavir.Release of the fluorogenic compound AMC was monitored continuously in a fluorescence plate reader. The specificity of the cleavage reaction was demonstrated by the addition of the proteasome inhibitor MG. Saquinavir inhibited chymotryptic s proteasome activity in all three of the cell lines in a concentrationdependent manner.Saquinavir inhibited the function of both chymotryptic s proteasome activity in a concentrationdependent fashion.The ability of saquinavir treatment to achieve this end point was, therefore, tested.By h, all of the cell lines showed typical morphological criteria of apoptosis.Most PC cells tolerated up to M for over h, but M induced considerable apoptosis by this time point.By h, PC cells showed an increase of the apoptotic.

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