tosylphenylalanyl-chloromethyl-ketone and Leukemia--Promyelocytic--Acute

Using an episomal eucaryotic expression vector, we derived three stable transfected human leukemic U-937 variant lines showing differential expression of the Bcl-xL protein. Preventive effect of Bcl-xL on cell death induced by various concentrations of camptothecin (DNA topoisomerase I inhibitor; CPT) was observed in the three lines with most pronounced effect in cells containing the highest level of Bcl-xL expression. These results show that increased cell death protection by Bcl-xL is correlated with its level of expression. The extent of DNA strand break formation and DNA synthesis inhibition following CPT treatments was similar in control and transfected U-937 cells, suggesting that Bcl-xL acts downstream of CPT-DNA topoisomerase I-mediated DNA strand breaks. Modulation of cell death by Bcl-xL was also observed in cells treated with etoposide, vinblastine, paclitaxel, and cisplatinum (II) diammine dichloride. To define whether Bcl-xL functions downstream or upstream of apoptogenic proteolytic cascade activation, we compared kinetics of DNA fragmentation in treated cells with kinetics of caspase 1-like, caspase 3-like, and N-tosyl-L-phenylalanylchloromethyl ketone (TPCK)-sensitive activities. In CPT-treated U-937 cells, caspase 3-like and TPCK-sensitive activities promoting DNA fragmentation in a cell-free system were detected much more rapidly in extracts obtained from CPT-treated U-937 cells compared to those obtained from CPT-treated U-937-Bcl-xL variant cells. These results suggest that Bcl-xL delays their activation that correlates with the occurrence of DNA fragmentation. Addition of recombinant Bcl-xL in extracts containing DEVDase and TPCK-sensitive activities did not inhibit these activities, suggesting that Bcl-xL acts primarily upstream of their activation in the apoptotic process. Taken together, these results suggest that Bcl-xL is a primary checkpoint that can block or delay transmission of cell death signals emerging from DNA damage and prevents activation of an apoptogenic proteolytic cascade.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-X Protein; Camptothecin; Cell-Free System; Cysteine Proteinase Inhibitors; DNA Fragmentation; Endopeptidases; Enzyme Activation; Humans; Leukemia, Promyelocytic, Acute; Oligopeptides; Peptide Hydrolases; Proto-Oncogene Proteins c-bcl-2; Serine Proteinase Inhibitors; Tosylphenylalanyl Chloromethyl Ketone; Transfection; Tumor Cells, Cultured

Cell death plays an essential role in cell homeostasis and the pathological process in cancer. Apoptosis has been identified by the internucleosomal DNA cleavage which appears to be associated with endonuclease activation. Proteolysis is considered to be an early event in apoptosis. We studied the effects of proteolysis on early apoptotic events, such as chromatin condensation, nuclear breakdown, DNA breakage and sensitivity to denaturation induced by anticancer drugs (camptothecin: CAM, 5-azacytidine: AZA) on HL-60 cells. CAM induced apoptosis on S phase and AZA on G1 phase. The internucleosomal DNA cleavage shown by both the presence of DNA fragments during gel electrophoresis and a large number of in situ DNA strands breaks (revealed in high intensity fluorescence FITC of cells in the TdT reaction) was prevented by the protease inhibitor, TPCK (N-tosyl-L-phenylalanine chlorometyl-ketone), as well as by an inhibitor of the apoptosis-associated endonuclease, ZnSO4. The protective effects were observed under conditions in which apoptosis was induced by agents with a different mechanism of action, such as the DNA damaging drug. CAM (topo-isomerase inhibitor), and an RNA antimetabolite, AZA. The protease inhibitor inhibits early events of apoptosis such as chromatin condensation, nuclear breakdown, DNA breakage and sensitivity to denaturation, which have different structures and a different mechanism of interaction with drugs. The results suggest that control of protease inhibitor may be a useful strategy to treat cancers.

Topics: Antimetabolites, Antineoplastic; Antineoplastic Agents, Phytogenic; Apoptosis; Azacitidine; Camptothecin; Cell Cycle; HL-60 Cells; Humans; Leukemia, Promyelocytic, Acute; Tosylphenylalanyl Chloromethyl Ketone