Catchpoole D R, Stewart B W
Children's Leukaemia and Cancer Research Centre, University of New South Wales, Prince of Wales Children's Hospital, Sydney, Australia.
Cancer Res. 1993 Sep 15;53(18):4287-96.
Features of the apoptotic response evident in glucocorticoid-treated thymocytes are not uniformly observed in cell lines exposed to anticancer drugs. The significance of such variation has been assessed by monitoring molecular and cellular processes induced by etoposide (VP-16) in the human lymphoblastoid T-cell lines CCRF-CEM (CEM) and MOLT-4 contrasted, where appropriate, with those induced by necrotizing injury. Cytotoxic concentrations of the drug were determined to be 5-100 microM on the basis of tetrazolium reduction assay. The two lines were equally sensitive to VP-16; no difference in concentration of drug which inhibited cell growth by 50% with respect to control (i.e., drug free) cultures was apparent irrespective of exposure times from 3-72 h. DNA strand breaks were evident in both populations within 3 h of exposure to VP-16. Morphological change, assessed microscopically, involving nuclear condensation and cell shrinkage was qualitatively and quantitatively similar in VP-16-treated CEM and MOLT-4 cells. Flow cytometric analysis indicated that the G2/M fraction of the randomly dividing MOLT-4 population was approximately one-third that of CEM cells, but each line exhibited a decrease in this fraction 3-6 h after treatment. Despite these similarities, marked differences in the response to VP-16 were evident in the two populations. Internucleosomal fragmentation, detected electrophoretically 3 h after treatment in DNA isolated from CEM cells, was not detected under any condition in MOLT-4 DNA. Apoptotic bodies, also evident within 3 h of VP-16 treatment of CEM cells, were not readily apparent in MOLT-4 cells under the same conditions. Treatment causing necrosis resulted in trypan blue uptake within 1 h in a similar high proportion of cells from both lines. Exposure to VP-16 resulted in such a loss of membrane integrity by 6 h in CEM cells, while change in this parameter occurred only after 24 h in the case of MOLT-4 cells. The findings indicate a wide scope of difference in apoptotic response and suggest delayed loss of membrane permeability, rather than DNA fragmentation, as the clearest indicator of programmed cell death in these populations.
在糖皮质激素处理的胸腺细胞中明显的凋亡反应特征，在暴露于抗癌药物的细胞系中并非都能观察到。通过监测依托泊苷（VP - 16）在人淋巴母细胞T细胞系CCRF - CEM（CEM）和MOLT - 4中诱导的分子和细胞过程，并在适当情况下与坏死性损伤诱导的过程进行对比，评估了这种差异的意义。基于四唑盐还原试验，确定该药物的细胞毒性浓度为5 - 100微摩尔。这两个细胞系对VP - 16同样敏感；无论暴露时间为3 - 72小时，相对于对照（即无药物）培养物，抑制细胞生长50%的药物浓度没有明显差异。在暴露于VP - 16后3小时内，两个细胞群体中均出现DNA链断裂。通过显微镜评估的形态学变化，包括核浓缩和细胞收缩，在经VP - 16处理的CEM和MOLT - 4细胞中在定性和定量上相似。流式细胞术分析表明，随机分裂的MOLT - 4群体的G2/M期比例约为CEM细胞的三分之一，但每个细胞系在处理后3 - 6小时该比例均下降。尽管有这些相似之处，但两个群体对VP - 16的反应仍存在明显差异。在从CEM细胞分离的DNA中，处理3小时后通过电泳检测到的核小体间断裂，在MOLT - 4 DNA的任何条件下均未检测到。在VP - 16处理CEM细胞3小时内也明显可见的凋亡小体，在相同条件下MOLT - 4细胞中并不容易观察到。导致坏死的处理在1小时内使两个细胞系中相似高比例的细胞摄取台盼蓝。暴露于VP - 16使CEM细胞在6小时内出现这种膜完整性丧失，而在MOLT - 4细胞中，该参数的变化仅在24小时后发生。这些发现表明凋亡反应存在广泛差异，并表明膜通透性的延迟丧失而非DNA片段化，是这些群体中程序性细胞死亡最明显的指标。
