Sané A T, Schmitt E, Steyaert A, Meyer D, Bertrand R
Louis-Charles Simard Research Center, Montreal Cancer Institute, CHUM, QC, Canada.
Biochem Cell Biol. 1997;75(4):359-68.
Abstract: In human B lymphoma Namalwa variant cells expressing the serpin-like CrmA protein, the kinetics of oligonucleosome-sized DNA fragmentation was retarded compared with that of control Namalwa cells following camptothecin treatment. However, no difference in the kinetics of high molecular weight DNA fragmentation was observed between the two lines after camptothecin treatment. Similar delay and inhibition of the oligonucleosome-sized DNA fragmentation was observed in human B lymphoma Namalwa and monocytic-like leukemia U-937 cells coincubated in the presence of various concentrations of N-tosyl-L-phenylalanyl chloromethylketone and camptothecin. The effect of N-tosyl-L-phenylalanyl chloromethylketone was similar to that of CrmA and did not prevent the appearance of high molecular weight DNA fragments. Similar suppression of camptothecin-induced internucleosomal DNA fragmentation was also observed in a cell-free system when cytosolic extracts obtained from camptothecin-treated Namalwa and U-937 cells were coincubated with untreated nuclei in the presence of N-tosyl-L-phenylalanyl chloromethylketone. Furthermore, N-tosyl-L-phenylalanyl chloromethylketone had no significant effects on caspase-3-like activities in camptothecin-treated Namalwa and U-937 cells. Hydrolysis of Ac-Asp-Glu-Val-Asp-amino-4-methylcoumarin, a fluorogenic substrate with caspase-3-like activities, was detected in extracts prepared from camptothecin-treated Namalwa and U-937 cells with no apparent difference in the time courses of caspase-3-like activation in the absence or presence of N-tosyl-L-phenylalanyl chloromethylketone. Similarly, N-tosyl-L-phenylalanyl chloromethylketone was a weak inhibitor of caspase-3-like activities in vitro. Taken together, these observations suggest that the pathway sensitive to N-tosyl-L-phenylalanyl chloromethylketone is involved in camptothecin-induced oligonucleosome-sized DNA fragmentation. Furthermore, inhibition of this pathway had no effect on caspase-3-like activation and on the occurrence of high molecular weight DNA fragmentation.
在表达丝氨酸蛋白酶抑制剂样CrmA蛋白的人B淋巴瘤Namalwa变异细胞中，喜树碱处理后，寡核小体大小的DNA片段化动力学与对照Namalwa细胞相比有所延迟。然而，喜树碱处理后，两条细胞系在高分子量DNA片段化动力学方面未观察到差异。在存在不同浓度的N-对甲苯磺酰-L-苯丙氨酰氯甲基酮和喜树碱的情况下共同孵育的人B淋巴瘤Namalwa细胞和单核细胞样白血病U-937细胞中，也观察到了类似的寡核小体大小的DNA片段化延迟及抑制现象。N-对甲苯磺酰-L-苯丙氨酰氯甲基酮的作用与CrmA相似，且不阻止高分子量DNA片段的出现。当在N-对甲苯磺酰-L-苯丙氨酰氯甲基酮存在的情况下，将喜树碱处理的Namalwa细胞和U-937细胞的胞质提取物与未处理的细胞核共同孵育时，在无细胞体系中也观察到了喜树碱诱导的核小体间DNA片段化的类似抑制现象。此外，N-对甲苯磺酰-L-苯丙氨酰氯甲基酮对喜树碱处理的Namalwa细胞和U-937细胞中的半胱天冬酶-3样活性无显著影响。在喜树碱处理的Namalwa细胞和U-937细胞制备的提取物中检测到了具有半胱天冬酶-3样活性的荧光底物Ac-Asp-Glu-Val-Asp-氨基-4-甲基香豆素的水解，在不存在或存在N-对甲苯磺酰-L-苯丙氨酰氯甲基酮的情况下，半胱天冬酶-3样激活的时间进程没有明显差异。同样，N-对甲苯磺酰-L-苯丙氨酰氯甲基酮在体外是半胱天冬酶-3样活性的弱抑制剂。综上所述，这些观察结果表明，对N-对甲苯磺酰-L-苯丙氨酰氯甲基酮敏感的途径参与了喜树碱诱导的寡核小体大小的DNA片段化。此外，该途径的抑制对半胱天冬酶-3样激活和高分子量DNA片段化的发生没有影响。
