Apoptosis in C3H/10T1/2 mouse embryonic cells: evidence for internucleosomal DNA modification in the absence of double-strand cleavage.

Abstract

Apoptosis in embryonic C3H/10T/2 (done 8) cells is marked by specific changes in morphology and DNA fragmentation that differ from those found in apoptotic thymocytes. These results demonstrate that ultrastructural changes within the nucleus associated with endonucleolytic degradation are linked with structural degradation at higher levels of chromatin organization. Strand modifications within the internucdeosomal linker region are shown to involve alkaline-sensitive sites that appear to be sensitive to Si endonudease. Our results suggest that apoptosis is not dependent upon internucleosomal cleavage and may reveal the penultimate step and the nature of the metabolic cascade that leads to cell death.Apoptosis is a term originally proposed by Kerr, Wyllie, and Currie (1) to describe a process of controlled cell deletion in tissues following either physiological or pathological stimuli. The concept of apoptosis implies that a common metabolic pathway leading to cell death may be initiated by a wide variety of signals, including hormones, serum growth factor deprivation, chemotherapeutic agents, and ionizing radiation (2)(3)(4)(5)(6). Understanding this process of apoptosis is critical to understanding the mechanisms of cell death common to embryonic development, cellular immunology, and protection against the expression of heritable genotypic changes in cells linked with mutagenesis and carcinogenesis (see ref. 7).Much of the basic research on apoptosis has focused upon the phenomenon as it occurs in thymocytes and lymphocytederived cell lines. Perhaps the most widely accepted markers of apoptotic cell death are morphological changes observed by electron microscopy (EM) and the nonrandom nature of the fragmentation of nuclear DNA. We have focused our research on the study of regulation of cell proliferation and death as it is expressed in the pluripotent, untransformed mouse embryonic cell, C3H/1OTY2 clone 8 (10T1/2). Previ-ously published evidence has shown that this cell exhibits the ability to modulate proliferation, differentiation, and death. Its phenotype is subject to transformation by various physical and chemical agents and it has been used to study the process of multistage carcinogenesis in vitro (see ref. 8).There has been considerable debate over the precise role of DNA fragmentation in apoptosis and the mechanism by which the integrity of the genome is irreversibly damaged.This report demonstrates that apoptosis does not require double-strand DNA cleavage in the internucleosomal linker region and that apoptosis in the 1OTY2 cell is marked by specific changes in morphology and DNA fragmentation unlike that in thymocytes. Our results suggest that ultrastructural changes previously associated with apoptosis are actually linked with DNA structural degradation at levels ofDNA organization above that of the nucleosome. Furthermore, although it is generally assumed that DNA degradation in apoptotic cells progresses through a series of steps or metabolic cascade, the evidence presented here demonstr...

Mapping the initial DNA breaks in apoptotic Jurkat cells using ligation-mediated PCR

“…The existence of ss breaks in apoptotic DNA fragments has been clearly demonstrated (Figure 1). 7,51,52 The rate of nuclear proteolysis might also be a factor determining the extent of DNA fragmentation, since rapid protein degradation and collapse of nuclear structure might stop DNA cleavage at any given stage. This would explain the fact that DNA degradation never proceeds to completion and that the DNA ladder is not always observed even in cells containing nucleases capable of internucleosomal DNA cleavage.…”

Evidence that DNA fragmentation in apoptosis is initiated and propagated by single-strand breaks

“…The DNA within domains is more protected by virtue of its interaction with core histones and histone H1. Evidence that the internucleosomal linker region becomes somehow modified, increasing its nuclease sensitivity, prior to cleavage was provided by Tomei et al (1993) who showed that the larger fragments of DNA from apoptotic C3H/10T cells (which do not normally degrade their DNA to small fragments) appeared to contain a modification in the linker region that was sensitive to alkali and to S1 nuclease. In another study, Pietsch et al (1993) analyzed the small oligonucleosomal fragments generated at the completion of DNA degradation in apoptotic thymocytes and showed that numerous singlestrand breaks (ssb) were present in the linker regions.…”

Caspases: the executioners of apoptosis

“…These DNA ladders are derived from large fragments of DNA of 30-50 and 200-300 kbp, which may in terms of higher-order chromatin structure represent loops and rosettes of DNA [10][11][12]. Internucleosomal cleavage of DNA now appears to be a relatively late event in the apoptotic process, which in some models may be dissociated from early critical steps [13,14]. Nevertheless, its measurement is simple and it is often used as a major criterion to determine whether a cell is apoptotic.…”