Involvement of Bcl-2 family proteins in paclitaxel-induced cell death. (A) Bcl-2 protects cells against the apoptosis-inducing effects of paclitaxel. Left panel shows immunoblot analysis of the status of Bcl-2 expression in Jurkat cells that were either stably transfected with Bcl-2 or the corresponding vector control. Right panel shows that Bcl-2–overexpressing cells and the corresponding vector control cells were subjected to increasing concentrations of paclitaxel for 48 hours as indicated. Flow cytometric analysis of hypodiploid cell nuclei revealed a strong protective effect of Bcl-2 against up to 10 μM paclitaxel. Results show the mean values (± SD). (B) Single knock-out of Bax or Bim confers partial resistance, while the double knock-out of Bax and Bak completely inhibits paclitaxel-induced apoptosis. MEFs derived from Bim, Bid, Bak, and Bax single knock-out as well as from Bak/Bax double knock-out mice were incubated with increasing concentrations of paclitaxel for 48 hours. Results from subsequent flow cytometric analysis of hypodiploid cell nuclei are depicted as a bar diagram of mean values (± SD; left panel). The right panel shows a time course analysis of apoptosis in Bax and Bak single and double knock-out MEFs that were treated for the indicated times with 1 μM paclitaxel. (C) Mitochondrial depolarization in response to paclitaxel is blocked in Bak/Bax double knock-out cells, and decreased in Bax or Bim single knock-out cells. MEFs were treated for 24 hours with the indicated concentrations of paclitaxel, stained with DiOC6/PI, and analyzed by flow cytometry. Results are presented as mean values (± SD). (D) Clonogenicity assays of wild-type and knock-out MEFs were performed as described in Figure 6A. After 7 days in normal medium, proliferation of paclitaxel-treated cells was measured by crystal violet staining and calculated as the mean (± SD) relative to the untreated controls.
