Apoptosis, a major form of cell death, is characterized by chromatin condensation, a reduction in cell volume and endonuclease cleavage of DNA into oligonucleosomal length fragments. The detection of these fragments by gel electrophoresis, as a DNA ladder, is currently used as the major biochemical index of apoptosis. Here we report that key morphological changes of apoptosis can be dissociated experimentally from the DNA fragmentation produced by endonuclease activity. Internucleosomal cleavage of DNA is thus likely to be a later event in the apoptotic process.

Primary B cells from B cell chronic lymphocytic leukaemia (B-CLL) were resistant to the novel selective cytotoxic agent, TNF-related apoptosis-inducing ligand (TRAIL). Low levels of the death-inducing TRAIL receptors, TRAIL-R1 and TRAIL-R2 but not the putative 'decoy' receptors, TRAIL-R3 and TRAIL-R4, were expressed on the surface of B-CLL cells. Resistance to TRAIL was upstream of caspase-8 activation, as little or no caspase-8 was processed in TRAIL-treated B-CLL cells. Low levels of a TRAIL death-inducing signalling complex (DISC) were formed in these cells, accompanied by the recruitment of endogenous FADD, caspase-8 and c-FLIP L but not c-FLIP S . Both caspase-8 and c-FLIP L were cleaved to form two stable intermediates of *43 kDa, which remained associated with the DISC. Caspase-8 was not further processed to its active heterotetramer. Thus the resistance of B-CLL cells to TRAIL may be due partly to low surface expression of the death receptors resulting in low levels of DISC formation and also to the high ratio of c-FLIP L to caspase-8 within the DISC, which would prevent further activation of caspase-8. Our results highlight the possibility of sensitising B-CLL cells to TRAIL by modulation of c-FLIP levels or by upregulation of surface expression of death receptors.

Apoptosis in megakaryocytes results in the formation of platelets. The role of apoptotic pathways in platelet turnover and in the apoptotic-like changes seen after platelet activation is poorly understood. ABT-263 (Navitoclax), a specific inhibitor of antiapoptotic BCL2 proteins, which is currently being evaluated in clinical trials for the treatment of leukemia and other malignancies, induces a doselimiting thrombocytopenia. In this study, the relationship between BCL2/BCL-X L inhibition, apoptosis, and platelet activa- IntroductionAll nucleated cells in multicellular organisms are genetically programmed to undergo apoptosis to remove unnecessary or damaged cells from the whole organism. This program has been recognized as the central mechanism of platelet production from megakaryocytes. 1 However, the role of apoptosis in anuclear, mature platelets is less well characterized, with apoptotic-like changes seen in both aging platelets and in the formation of procoagulant microparticles after agonist stimulation.Two main pathways lead to the execution of apoptosis: the extrinsic and the intrinsic (or mitochondrial) pathways. Both converge into the activation of caspases, which are proteases that cleave Ͼ 500 cellular targets and induce typical morphologic changes associated with apoptosis in nucleated cells. A critical step in the intrinsic pathway is the loss of mitochondrial membrane potential (MMP) and the release of cytochrome c into cytosol, where it triggers the activation of caspase-9. Therefore, the release of cytochrome c from mitochondria needs to be tightly regulated: a function that is fulfilled by the BCL2 protein family, which consists of proapoptotic and antiapoptotic members that promote or block the release of cytochrome c, respectively. 2,3 The proapoptotic family members BAX and BAK play an essential role in directly mediating the release of cytochrome c by forming a pore in the outer mitochondrial membrane. Antiapoptotic BCL2 proteins, including BCL2, BCL-X L , BCL-w, MCL1, and BCL2A1, prevent the activation of BAX and BAK. Besides their function in regulating mitochondrial cytochrome c release, BCL2 proteins have also been implicated in the regulation of intracellular calcium homeostasis at the endoplasmic reticulum (ER), possibly by interacting with inositol triphosphate receptors. 4,5 Because of their key role, the antiapoptotic BCL2 proteins are attractive targets for anticancer therapy, with several small molecule inhibitors currently in preclinical testing or early clinical trials. 6,7 Among these, the most promising and specific inhibitors are ABT-263 (Navitoclax) and 9 ABT-737 shows promising antitumor activity in animal models of leukemia and lymphoma. A related compound, ABT-263, is metabolically more stable and currently in phase 1 and 2 clinical trials for leukemia and other malignancies. 10 Both compounds have often been regarded as interchangeable because they bind with high affinity to BCL2, BCL-X L , and BCL-w but do not inhibit MCL1 or BCL2A1. 11 Early results from the clinica...