No abstract

Several recent studies have implicated proteases as important triggers of apoptosis. Thus far, substrates that are cleaved during apoptosis have been elusive. In this report we demonstrate that cleavage of alpha-fodrin (non-erythroid spectrin) accompanies apoptosis, induced by activation via the CD3/T cell receptor complex in a murine T cell hybridoma, ligation of the Fas (CD95) molecule on a human T cell lymphoma line and other Fas-expressing cells, or treatment of cells with staurosporine, dexamethasone, or synthetic ceramide. Furthermore, inhibition of activation-induced apoptosis by pretreatment of T hybridoma cells with antisense oligonucleotides directed against c-myc also inhibited fodrin proteolysis, confirming that this cleavage process is tightly coupled to apoptosis. Fodrin cleavage during apoptosis may have implications for the membrane blebbing seen during this process.

No abstract

Apoptosis plays an important role in the survival of an organism, and substantial work has been done to understand the signaling pathways that regulate this process. Characteristic changes in chromatin organization accompany apoptosis and are routinely used as markers for cell death. We have examined the organization of chromatin in apoptotic PC12 and HeLa cells by indirect immunofluorescence and electron spectroscopic imaging. Our results indicate that de novo chromatin condensation normally seen during mitosis does not occur when cells undergo apoptosis. Instead, the condensed chromatin typically observed results from aggregation of the heterochromatin. We present evidence that, early in apoptosis, there is a rapid degradation of the nuclease-hypersensitive euchromatin that contains hyperacetylated histones. This occurs coincident with the loss of nuclear integrity due to degradation of lamins and reorganization of intranuclear protein matrix. These events lead to collapse of the nucleus and aggregation of heterochromatin to produce the appearance of condensed apoptotic chromatin. This heterochromatin aggregate is then digested by nucleases to produce the oligonucleosomal DNA ladder that is a hallmark of late apoptosis. Unlike mitosis, we have not seen any evidence for the requirement of phosphorylated histones H1 and H3 to maintain the chromatin in the condensed state.Apoptosis, or programmed cell death, is a tightly regulated mechanism that multicellular organisms employ to replace damaged cells and to remove cells no longer needed during normal growth and development (1-4). The generation of large condensed chromatin bodies and degradation of DNA into oligonucleosomal fragments are features that are seen in many cells undergoing programmed cell death. Although commonly used as markers of apoptosis, the biochemical mechanism responsible for these features is not known. De novo chromosome condensation occurs during mitosis in cycling cells, and this condensation has been extensively studied. Many of the genes involved in regulating mitosis have been identified (5-7). The cdc2 gene, which encodes a histone H1 kinase, was identified as the major regulator of mitosis and has also been implicated in apoptosis. This raises the question of whether the same mechanisms regulating the formation of the mitotic chromosome also apply to apoptotic chromatin condensation. The requirement for histone H1 kinase activity in both processes led to the suggestion that cells could be triggering apoptosis by entering into an unscheduled mitosis and that a major player in this process could be the p34 cdc2 kinase (1, 4). Activation of the p34 cdc2 kinase was first demonstrated to accompany apoptosis in YAC-1 lymphoma cells (8). Subsequently, it was observed that temperatureinduced inactivation of p34 cdc2 in FT210 cells prevented the induction of apoptosis by treatment with fragmentin and perforin. Since then, the up-regulation of p34 cdc2 during apoptosis has been reported in other cell lines (8 -12), including PC12 (13-15) and He...