Clarke Alan A, Gibson Frances M, Scott Julie, Myatt Nyree, Rutherford Tim R
St George's Hospital Medical School, Department of Cellular and Molecular Medicine, Division of Hematology, Cranmer Terrace, London, UK.
Haematologica. 2004 Jan;89(1):11-20.
Fanconi anemia (FA) cells are characteristically hypersensitive to bifunctional alkylating agents, notably mitomycin C (MMC), causing increased programmed cell death (PCD). FA cells also have abnormalities in mitochondrial function. We hypothesized that the abnormalities in PCD are mitochondrially mediated. We examined mitochondrial function in FA cells, comparing the intrinsic death pathway induced by MMC with the extrinsic pathway via Fas antibody, which can bypass the mitochondria.
Normal and FA lymphoblastoid cell lines were treated with MMC or agonistic anti-Fas antibody. PCD was assessed using flow cytometry, Western blot analysis, and DNA gel electrophoresis.
FA cells showed hypersensitivity to MMC, but slight resistance to Fas-mediated PCD. MMC induced chromatin condensation, but not apoptotic body formation. Fas induced classical apoptosis. MMC failed to induce mitochondrial depolarization, while some depolarization occurred with anti-Fas. These results suggested that MMC failed to induce caspase activity in FA cells. No cleavage of caspase 3 was observable and PCD was not inhibited by the caspase inhibitor zVAD-fmk. Fas-induced caspase 3 cleavage, and cell death was inhibited by zVAD-fmk. There were common downstream abnormalities in the execution phase of PCD, as both agonists failed to cleave PARP, or to induce nucleosomal fragmentation.
Our results suggest that mitochondrial function in FA cells is abnormal, resulting in necrotic or caspase independent PCD, but that further abnormalities may exist downstream of the mitochondria. This may have implications in explaining in vivo aspects of FA.
范可尼贫血（FA）细胞的特征是对双功能烷化剂高度敏感，尤其是丝裂霉素C（MMC），这会导致程序性细胞死亡（PCD）增加。FA细胞的线粒体功能也存在异常。我们推测PCD异常是由线粒体介导的。我们检测了FA细胞中的线粒体功能，比较了MMC诱导的内源性死亡途径与通过Fas抗体的外源性途径，后者可绕过线粒体。
用MMC或激动性抗Fas抗体处理正常和FA淋巴母细胞系。使用流式细胞术、蛋白质印迹分析和DNA凝胶电泳评估PCD。
FA细胞对MMC表现出高度敏感性，但对Fas介导的PCD有轻微抗性。MMC诱导染色质浓缩，但不诱导凋亡小体形成。Fas诱导典型的凋亡。MMC未能诱导线粒体去极化，而抗Fas处理则出现了一些去极化。这些结果表明MMC未能在FA细胞中诱导半胱天冬酶活性。未观察到半胱天冬酶3的切割，并且PCD不受半胱天冬酶抑制剂zVAD-fmk的抑制。Fas诱导半胱天冬酶3切割，并且细胞死亡受zVAD-fmk抑制。在PCD的执行阶段存在共同的下游异常，因为两种激动剂均未能切割聚（ADP-核糖）聚合酶（PARP）或诱导核小体片段化。
我们的结果表明FA细胞中的线粒体功能异常，导致坏死性或半胱天冬酶非依赖性PCD，但线粒体下游可能还存在其他异常。这可能对解释FA的体内情况有意义。
