DNase I mediates internucleosomal DNA degradation in human cells undergoing drug-induced apoptosis

Abstract

Internucleosomal DNA fragmentation following the activation of endonucleases is the common end point of apoptosis. DNase I, a Ca2+ / Mg2+‐dependent endonuclease ubiquitously expressed in mammalian tissues, is believed to play a role in this process. To analyze the in vivo function of this enzyme in human cells, we have generated a cell line with targeted disruption of the DNase I gene, as well as several stable cell lines which overexpress the DNase I gene. Inactivation of the human DNase I gene was obtained in the Jurkat T cell clone JA3, characterized by high susceptibility to apoptotic cell death induced by pharmacological stimuli. JA3 cells, after disruption of the DNase I gene, became resistant to apoptotic stimuli. DNase I was overexpressed in the human cell lines JA3, K562 (erythroleukemia), M 14 (melanoma) and CEM (T cell lymphoma). Remarkably, stable overexpression of DNase I gene resulted in accelerated apoptosis in JA3 cells and induced apoptosis in K562, CEM and M14 cell lines, which are otherwise resistant to internucleosomal DNA degradation following pharmacological stimuli. Our study provides the first in vivo evidence that DNase I mediates internucleosomal DNA degradation in human cells undergoing drug‐induced apoptosis.

Action of Recombinant Human Apoptotic Endonuclease G on Naked DNA and Chromatin Substrates

“…chromatin also exhibits a high internucleosomal DNA background, which is not nearly as sharp as the reported DNase I-dependent apoptotic ladder (26). Taken together, these observations prompted us to test whether nicks generated by DNase I would be targets for endoG action because of their singlestranded character, and whether exonuclease gapping of nicks generated by endoG or DNase I would also stimulate DNA processing under physiological ionic strengths.…”

“…Indeed, we demonstrated that exonuclease and DNase I each can cooperate with endoG to facilitate DNA processing. It is significant that DNase I recently has been reported to be required for DNA laddering under certain apoptotic conditions that are DFF independent (26). Taken together with our results, we suggest that endoG may participate with DNase I (and possibly exonucleases) in vivo for apoptotic DNA processing.…”

“…Third, there is a high internucleosomal DNA background upon endoG digestion of isolated nuclei, not completely characteristic of the DNA laddering pattern seen during apoptosis in DFF knockout cells (9). Fourth, DNase I knockout cells fail to ladder chromatin under apoptotic conditions that block DFF activation (26). Fifth, the ladder generated by DNase I digestion of FIG.…”

“…AIF (8), topo II (14), and caspase-treated DFF/CAD-ICAD (15)(16)(17) have each been implicated in the higher order DNA cleavage reaction. Nucleosomal DNA laddering, on the other hand, has been associated with several endonucleases, including caspase-activated DFF/CAD-ICAD (18 -25), endoG (9,10), and DNase I (26). Although some of the catalytic properties of endoG have been reported previously, nucleic acid, not chromatin substrates, had been employed.…”

Coordinate involvement of cysteine protease and nuclease in the executive phase of plant apoptosis

“…Several apoptogenic effectors, such as caspases (cysteine proteases) [9][10][11][12], CAD/DFF40 (caspase-activated DNase/40 kDa DNA fragmentation factor) [9][10][11][12], endoG (mitochondrial endonuclease G) [13], DNase I [14], DNase II [15], AIF (apoptosis-inducing factor) [16] and Acinus (apoptosis chromatin condensation inducer in the nucleus) [17], have been implicated in mediating these nuclear disintegration processes using a cell-free system. In plants, chromatin condensation and DNA laddering are induced during PCD in response to biotic/abiotic stresses and developmental factors [4,6,[18][19][20][21][22].…”