An endogenous calcium-dependent, caspase-independent intranuclear degradation pathway in thymocyte nuclei: antagonism by physiological concentrations of K(+) ions.

Calcium ions have been implicated in apoptosis for many years, however the precise role of this ion in the cell death process remains incomplete. We have extensively examined the role of Ca(2+) on nuclear degradation in vitro using highly purified nuclei isolated from non-apoptotic rat thymocytes. We show that these nuclei are devoid of CAD (caspase-activated DNase), and DNA degradation occurs independent of caspase activity. Serine proteases rather than caspase-3 appear necessary for this Ca(2+) -dependent DNA degradation in nuclei. We analyzed nuclei treated with various concentrations of Ca(2+) in the presence of both a physiological (140 mM) and apoptotic (40 mM) concentration of KCl. Our results show that a 5-fold increase in Ca(2+) is required to induce DNA degradation at the physiological KCl concentration compared to the lower, apoptotic concentration of the cation. Ca(2+) -induced internucleosomal DNA degradation was also accompanied by the release of histones, however the apoptotic-specific phosphorylation of histone H2B does not occur in these isolated nuclei. Interestingly, physiological concentrations of K(+) inhibit both Ca(2+) -dependent DNA degradation and histone release suggesting that a reduction of intracellular K(+) is necessary for this apoptosis-associated nuclear degradation in cells. Together, these data define an inherent caspase-independent catabolic pathway in thymocyte nuclei that is sensitive to physiological concentrations of intracellular cations.

Isolation and characterization of mammalian cells that are undergoing apoptosis by a bovine serum albumin density gradient.

When cells are treated with cytotoxic agents, they enter apoptosis asynchronously to yield cells at various stages of cellular deterioration. This mixture makes it difficult to study the biochemical pathways leading to cell death. We have fractionated apoptotic mammalian cells in a simple discontinuous bovine serum albumin (BSA) density gradient centrifugation into five layers, each containing cells at different stages of apoptosis, (1) nonapoptotic, (2) undergoing apoptosis, and (3) mature apoptotic cells, as judged by light and electron microscopy of chromatin condensation and by the extent of DNA fragmentation. Modifications of apoptosis markers including c-Jun N-terminal kinase/stress-activated protein kinase and procaspase 3 cleavage were apparent in those cells that are undergoing apoptosis. Apoptosis-specific histone H2B phosphorylation was highly elevated and DNA fragmentation activity in the cytoplasm was observed in those cells that are undergoing apoptosis, but not much was observed in the cells of other fractions. Results show that apoptotic cells can be fractionated easily by the BSA gradient method, and this method will be invaluable for studying the biochemical processes that drive apoptosis.

Apoptotic phosphorylation of histone H2B is mediated by mammalian sterile twenty kinase.

DNA in eukaryotic cells is associated with histone proteins; hence, hallmark properties of apoptosis, such as chromatin condensation, may be regulated by posttranslational histone modifications. Here we report that phosphorylation of histone H2B at serine 14 (S14) correlates with cells undergoing programmed cell death in vertebrates. We identify a 34 kDa apoptosis-induced H2B kinase as caspase-cleaved Mst1 (mammalian sterile twenty) kinase. Mst1 can phosphorylate H2B at S14 in vitro and in vivo, and the onset of H2B S14 phosphorylation is dependent upon cleavage of Mst1 by caspase-3. These data reveal a histone modification that is uniquely associated with apoptotic chromatin in species ranging from frogs to humans and provide insights into a previously unrecognized physiological substrate for Mst1 kinase. Our data provide evidence for a potential apoptotic "histone code."