Chromatin Condensation Is Not Associated with Apoptosis

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...

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

“…In this study, we found little evidence for chromatin condensation in nuclei treated with CaCl 2 at all KCl concentrations examined (0, 40 and 140 mM), supporting the notion that chromatin condensation may not be necessarily linked to or is independent of DNA degradation [45]. Previous studies have shown that histones are released in various apoptotic cells [46,47].…”

Hyperphosphorylation of histone H2A.X and dephosphorylation of histone H1 subtypes in the course of apoptosis

“…Activation of proteases and degradation of both the nuclear lamina and components of the intranuclear protein matrix enable aggregation of chromatin into apoptotic spherical bodies. 39 Furthermore, it is known that inhibition of lamin or nuclear scaffold degradation prevents nuclei from acquiring complete apoptotic features. 40,41 Whereas hyperphosphorylation of H2A.X in apoptotic cells depends indirectly on activation of caspases and nuclear scaffold (lamin) proteases, as shown in zVAD-(OMe)-fmk or zAPF-cmktreated cells, H1 dephosphorylation can be inhibited only by the caspase inhibitors zVAD-(OMe)-fmk and Ac-YVADcmk and not by the used nuclear scaffold protease inhibitor.…”

The anthracycline antibiotics: antitumor drugs that alter chromatin structure

“…(58,59) Chromatin aggregation (23) may facilitate or promote the DNA fragmentation (68,69) by specific apoptotic endonucleases. Indeed, it has been recently shown that chromatin aggregation precedes oligonucleosomal fractionation (70) and that DNA is supercoiled in apoptotic chromatin. (71) Despite all this, the reason(s) why or how anthracyclines exert their preferential killing of tumor versus non-carcinogenic cells are still not well understood.…”