Meier H L, Millard C B
U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010, USA.
Biochim Biophys Acta. 1998 Sep 16;1404(3):367-76. doi: 10.1016/s0167-4889(98)00078-0.
Changes in genomic DNA caused by exposure to the cytotoxic alkylating agent, 2,2'-dichlorodiethyl sulfide (sulfur mustard; HD), alone or in combination with selective inhibitors of poly(ADP-ribose) polymerase (PARP), were analyzed as a function of HD concentration and post-exposure time. Preparations of human peripheral blood lymphocytes were exposed to HD (1x10(-8) M-1x10(-3) M), and incubated at 37 degrees C for 0-24 h. Total genomic DNA was extracted from these cells and compared with DNA from control cells of the same donor using agarose gel electrophoresis. The effects of HD on genomic DNA depended on the HD concentration and the length of the post-exposure time interval. DNA fragmentation was detected as early as 2 h after exposure to 3x10(-4) M HD, or at 24 h after exposure to 6x10(-6) M HD. The qualitative DNA pattern, as well as the extent of DNA fragmentation, changed with post-exposure time. Exposure to HD caused a time-dependent shift in the DNA cleavage pattern from an oligonucleosome-sized 'DNA ladder' characteristic of apoptotic cell death, to a 'broad band' pattern characteristic of necrotic cell death. DNA fragmentation was not observed if cells were killed with heat or with Lewisite. Treatment of cells with selective PARP inhibitors consistently altered the DNA fragmentation caused by HD exposure. The inhibitors arrested DNA fragmentation at the DNA ladder stage. This effect only was observed if the PARP inhibitors were applied within 8 h of HD exposure. We conclude that early inhibition of PARP activity can induce a switch in the mechanism of cell death caused by HD. Such a switch may be useful therapeutically to convert a lytic, pro-inflammatory cell death that includes the disintegration of dying cells (necrosis), into a slower, programmed cell death that includes absorption of dying cells (apoptosis).
分析了单独暴露于细胞毒性烷基化剂2,2'-二氯二乙硫醚（硫芥；HD）或与聚（ADP-核糖）聚合酶（PARP）选择性抑制剂联合使用时，基因组DNA的变化与HD浓度和暴露后时间的关系。将人外周血淋巴细胞制剂暴露于HD（1×10⁻⁸M - 1×10⁻³M），并在37℃孵育0 - 24小时。从这些细胞中提取总基因组DNA，并使用琼脂糖凝胶电泳与同一供体的对照细胞的DNA进行比较。HD对基因组DNA的影响取决于HD浓度和暴露后时间间隔的长度。在暴露于3×10⁻⁴M HD后2小时或暴露于6×10⁻⁶M HD后24小时，最早检测到DNA片段化。DNA的定性模式以及DNA片段化程度随暴露后时间而变化。暴露于HD导致DNA切割模式随时间发生变化，从凋亡性细胞死亡特征性的寡核小体大小的“DNA梯带”模式，转变为坏死性细胞死亡特征性的“宽带”模式。如果用热或路易氏剂杀死细胞，则未观察到DNA片段化。用PARP选择性抑制剂处理细胞始终会改变HD暴露引起的DNA片段化。这些抑制剂使DNA片段化停滞在DNA梯带阶段。仅当在HD暴露后8小时内应用PARP抑制剂时才观察到这种效果。我们得出结论，早期抑制PARP活性可诱导HD引起的细胞死亡机制发生转变。这种转变在治疗上可能有助于将包括死亡细胞解体（坏死）的溶解性、促炎性细胞死亡，转变为包括死亡细胞吸收（凋亡）的较慢的程序性细胞死亡。
