Schmitt E, Cimoli G, Steyaert A, Bertrand R
Hospital Research Center of University of Montreal (CHUM), Montreal Cancer Institute, Quebec, Canada.
Exp Cell Res. 1998 Apr 10;240(1):107-21. doi: 10.1006/excr.1998.4003.
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.
我们使用一种附加型真核表达载体，获得了三个稳定转染的人白血病U - 937变异株系，这些株系显示出Bcl - xL蛋白的差异表达。在这三个株系中均观察到Bcl - xL对不同浓度喜树碱（DNA拓扑异构酶I抑制剂；CPT）诱导的细胞死亡具有预防作用，在Bcl - xL表达水平最高的细胞中效果最为显著。这些结果表明，Bcl - xL增强的细胞死亡保护作用与其表达水平相关。CPT处理后，对照和转染的U - 937细胞中DNA链断裂形成和DNA合成抑制的程度相似，这表明Bcl - xL在CPT - DNA拓扑异构酶I介导的DNA链断裂下游起作用。在用依托泊苷、长春碱、紫杉醇和顺铂（II）二氯二氨处理的细胞中也观察到了Bcl - xL对细胞死亡的调节作用。为了确定Bcl - xL在凋亡蛋白水解级联激活的下游还是上游发挥作用，我们比较了处理细胞中DNA片段化的动力学与半胱天冬酶1样、半胱天冬酶3样和N - 甲苯磺酰 - L - 苯丙氨酰氯甲基酮（TPCK）敏感活性的动力学。在CPT处理的U - 937细胞中，与从CPT处理的U - 937 - Bcl - xL变异细胞中获得的提取物相比，在从CPT处理的U - 937细胞中获得的提取物中，在无细胞系统中促进DNA片段化的半胱天冬酶3样和TPCK敏感活性被检测到的速度要快得多。这些结果表明，Bcl - xL延迟了它们的激活，这与DNA片段化的发生相关。在含有DEVD酶和TPCK敏感活性的提取物中添加重组Bcl - xL并没有抑制这些活性，这表明Bcl - xL在凋亡过程中主要在它们激活的上游起作用。综上所述，这些结果表明Bcl - xL是一个主要的检查点，它可以阻断或延迟由DNA损伤产生的细胞死亡信号的传递，并防止凋亡蛋白水解级联的激活。
