The activity of ICE‐like proteases or caspases is essential for apoptosis. Multiple caspases participate in apoptosis in mammalian cells but how many caspases are involved and what is their relative contribution to cell death is poorly understood. To identify caspases activated in apoptotic cells, we developed an approach to simultaneously detect multiple active caspases. Using tumor cells as a model, we have found that CPP32 (caspase 3) and Mch2 (caspase 6) are the major active caspases in apoptotic cells, and are activated in response to distinct apoptosis‐inducing stimuli and in all cell lines analyzed. Both CPP32 and Mch2 are present in apoptotic cells as multiple active species. In a given cell line these species remained the same irrespective of the apoptotic stimulus used. However, the species of CPP32 and Mch2 detected varied between cell lines, indicating differences in caspase processing. The strategy described here is widely applicable to identify active caspases involved in apoptosis.
During stress-induced apoptosis, the initiator caspase-9 is activated by the Apaf-1 apoptosome and must remain bound to retain significant catalytic activity. Nevertheless, in apoptotic cells the vast majority of processed caspase-9 is paradoxically observed outside the complex. We show herein that apoptosome-mediated cleavage of procaspase-9 occurs exclusively through a CARD-displacement mechanism, so that unlike the effector procaspase-3, procaspase-9 cannot be processed by the apoptosome as a typical substrate. Indeed, procaspase-9 possessed higher affinity for the apoptosome and could displace the processed caspase-9 from the complex, thereby facilitating a continuous cycle of procaspase-9 recruitment/activation, processing, and release from the complex. Owing to its rapid autocatalytic cleavage, however, procaspase-9 per se contributed little to the activation of procaspase-3. Thus, the Apaf-1 apoptosome functions as a proteolytic-based 'molecular timer', wherein the intracellular concentration of procaspase-9 sets the overall duration of the timer, procaspase-9 autoprocessing activates the timer, and the rate at which the processed caspase-9 dissociates from the complex (and thus loses its capacity to activate procaspase-3) dictates how fast the timer 'ticks' over.
We have recently developed a method for the separation and quantification of viable apoptotic cells without the need for permeabilisation or fixation of the cells. The method is based on the observation that apoptotic rat thymocytes fluoresce more brightly than normal cells after a brief incubation with the DNA binding dye, Hoechst 33342. In order to understand these differences, we have investigated the uptake of Hoechst 33342 by normal and apoptotic thymocytes. By staining with fluorescein diacetate, we have shown that the efflux of fluorescein from apoptotic cells is more rapid than that from normal thymocytes. This result demonstrated an increase in the permeability of the plasma membrane of the apoptotic thymocytes and it is this change which probably results in the more rapid uptake of Hoechst 33342. The data also revealed two populations of apoptotic thyrnocytes. o 1993 Wiley-Liss, Inc. Key terms: Apoptosis, flow cytometry, thymocytesEukaryotic cells are thought to die either by necrosis or apoptosis. Loss of integrity of the cell membrane is an early event in necrosis while in apoptosis this is preceded morphologically by condensation of nuclear chromatin, compaction of cytoplasmic organelles, and changes at the cell surface (1,421. Biochemically, the most striking change in apoptosis is the digestion of nuclear DNA into oligonucleosomal length fragments (41). Apoptosis can be induced in immature thymocytes in vitro by a variety of agents including glucocorticoids, such as dexamethasone (41,431 and compounds reactive with DNA topoisomerase 11, for example, etoposide (37).In some types of cell, apoptotic and normal cells can be distinguished by a brief incubation with the bisbenzimidazole dye, Hoechst 33342; the apoptotic cells fluoresce more brightly on excitation of the dye-DNA complex by uv radiation (9,23). In the same assay, cells which have lost the integrity of their plasma membrane can be separately quantified by their failure to exclude another DNA binding dye, propidium iodide (PI). We have adapted this method to quantify viable apoptotic rat thymocytes (30); the method has also assisted us in the recognition, isolation, and characterisation of a transitional preapoptotic population (4).Hoechst 33342 is one of a family of bis-benzimadazoles which stain DNA specifically, binding preferentially to repetitive AT sequences in the minor groove of the helix (2,191. The fluorescence of the dye in this environment is an order of magnitude greater than its fluorescence in aqueous solution. At high concentrations of dye, there is a second mode of binding which leads to fluorescence quenching (2) and a red shift of the fluorescence spectrum (28,29,39). Hoechst 33342 is taken up by viable cells although the rate of uptake into the nucleus strongly depends on the type of cell studied and this property has been used to distinguish different types of lymphoid cell (16,391. The dye is actively pumped out of cells by an energy dependent pump which is probably the p-glycoprotein pump responsible for multiple drug...
Understanding how oncogenic transformation sensitizes cells to apoptosis may provide a strategy to kill tumor cells selectively. We previously developed a cell-free system that recapitulates oncogene dependent apoptosis as ref lected by activation of caspases, the core of the apoptotic machinery. Here, we show that this activation requires a previously identified apoptosis-promoting complex consisting of caspase-9, APAF-1, and cytochrome c. As predicted by the in vitro system, preventing caspase-9 activation blocked druginduced apoptosis in cells sensitized by E1A, an adenoviral oncogene. Oncogenes, such as E1A, appear to facilitate caspase-9 activation by several mechanisms, including the control of cytochrome c release from the mitochondria.
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