Abstract
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, andcis-platinum(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, andN-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.
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