Glucocorticoid-induced lymphocytolysis is not mediated by an induced endonuclease

The mechanism of glucocorticoid-induced internucleosomal DNA cleavage and cytolysis of lymphatic cells is not known. Recent data (Compton, M.M., and Cidlowski, J.A. (1987) J. Biol. Chem. 262, 8288-8292) suggested that in vivo treatment of rat thymocytes with glucocorticoids induces a nucleolytic "lysis gene" product(s) responsible for lymphocytolysis. In this paper, the possibility that lymphocytolysis may result from glucocorticoid-induced nuclease(s) was examined. Using the rat thymocytes as a model system, we have shown by electrophoretic, enzymatic, and amino acid sequence analysis that the putative glucocorticoid-induced nucleases identified recently by Compton and Cidlowski are in fact H1, H1(0), and core histones, and their gross appearance is not the result of new histone protein synthesis, but a result of the release of histone-containing nucleosomes during chromatin breakdown. Evidence presented here shows that the putative induced nuclease activity is an artifact of the assay system employed. Because our data do not support induction of a glucocorticoid-induced nuclease(s), we examined the possibility that DNA cleavage might be induced by activation of a constitutive endogenous endonuclease. We have shown that it is possible to produce characteristic internucleosomal DNA cleavage of rat thymocytes, merely by incubating intact nuclei from untreated adrenalectomized rat thymocytes with Ca2+ and Mg2+ for a short period of time. However, in glucocorticoid-sensitive human CEM-C7 lymphocytes activation of internucleosomal DNA cleavage was independent of calcium uptake. We conclude that glucocorticoid induction of internucleosomal DNA fragmentation does not necessarily require expression of a new nuclease(s), but is the result of the activation of a constitutive endogenous endonuclease(s). Also, our data suggest that the mechanism which controls activation of internucleosomal DNA cleavage in rat thymocytes differs from that which operates in CEM-C7 lymphocytes.     

Action of apoptotic endonuclease DNase gamma on naked DNA and chromatin substrates

The internucleosomal cleavage of genomic DNA is a biochemical hallmark of apoptosis. DNase gamma, a Mg2+/Ca2+-dependent endonuclease, has been suggested to be one of the apoptotic endonucleases, but its biochemical characteristic has not been fully elucidated. Here, using recombinant DNase gamma, we showed that DNase gamma is a Mg2+/Ca2+-dependent single-stranded DNA nickase and has a high activity at low ionic strength. Under higher ionic strength, such as physiological buffer conditions, the endonuclease activity of DNase gamma is restricted, but its activity is enhanced in the presence of linker histone H1, which explains DNA cleavage at linker regions of apoptotic nuclei.     

Cleavage preferences of the apoptotic endonuclease DFF40 (caspase-activated DNase or nuclease) on naked DNA and chromatin substrates

Here we report the co-factor requirements for DNA fragmentation factor (DFF) endonuclease and characterize its cleavage sites on naked DNA and chromatin substrates. The endonuclease exhibits a pH optimum of 7.5, requires Mg(2+), not Ca(2+), and is inhibited by Zn(2+). The enzyme generates blunt ends or ends with 1-base 5'-overhangs possessing 5'-phosphate and 3'-hydroxyl groups and is specific for double- and not single-stranded DNA or RNA. DFF endonuclease has a moderately greater sequence preference than micrococcal nuclease or DNase I, and the sites attacked possess a dyad axis of symmetry with respect to purine and pyrimidine content. Using HeLa cell nuclei or chromatin reconstituted on a 5 S rRNA gene tandem array, we prove that the enzyme attacks chromatin in the internucleosomal linker, generating oligonucleosomal DNA ladders sharper than those created by micrococcal nuclease. Histone H1, high mobility group-1, and topoisomerase II activate DFF endonuclease activity on naked DNA substrates but much less so on chromatin substrates. We conclude that DFF is a useful reagent for chromatin research.     

Action of recombinant human apoptotic endonuclease G on naked DNA and chromatin substrates: cooperation with exonuclease and DNase I

Endonuclease G (endoG) is released from mitochondria during apoptosis and is in part responsible for internucleosomal DNA cleavage. Here we report the action of the purified human recombinant form of this endonuclease on naked DNA and chromatin substrates. The addition of the protein to isolated nuclei from non-apoptotic cells first induces higher order chromatin cleavage into DNA fragments > or = 50 kb in length, followed by inter- and intranucleosomal DNA cleavages with products possessing significant internal single-stranded nicks spaced at nucleosomal ( approximately 190 bases) and subnucleosomal ( approximately 10 bases) periodicities. We demonstrate that both exonucleases and DNase I stimulate the ability of endoG to generate double-stranded DNA cleavage products at physiological ionic strengths, suggesting that these activities work in concert with endoG in apoptotic cells to ensure efficient DNA breakdown.     

The DFF40/CAD endonuclease and its role in apoptosis

The sequential generation of large-scale DNA fragments followed by internucleosomal chromatin fragmentation is a biochemical hallmark of apoptosis. One of the nucleases primarily responsible for genomic DNA fragmentation during apoptosis is called DNA Fragmentation Factor 40 (DFF40) or Caspase-activated DNase (CAD). DFF40/CAD is a magnesium-dependent endonuclease specific for double stranded DNA that generates double strand breaks with 3'-hydroxyl ends. DFF40/CAD is activated by caspase-3 that cuts the nuclease's inhibitor DFF45/ICAD. The nuclease preferentially attacks chromatin in the internucleosomal linker DNA. However, the nuclease hypersensitive sites can be detected and DFF40/CAD is potentially involved in large-scale DNA fragmentation as well. DFF40/CAD-mediated DNA fragmentation triggers chromatin condensation that is another hallmark of apoptosis.     

Propidium iodide staining correlates with the extent of DNA degradation in isolated nuclei.

Gradual degradation of internucleosomal DNA is a hallmark of apoptosis and can be simulated by incubating isolated thymocyte nuclei in the presence of 5 mM Mg2+ and 5 mM Ca2+ at 37 degrees C. Staining of nuclei with the DNA binding fluorescent dye propidium iodide (PI) showed that intensity of fluorescence correlated with the extent of DNA degradation. PI fluorescence was increased in the presence of DNase I. Thus it seems that the cleavage of chromatin DNA by DNase 1 or by the endogenous enzyme increases the accessibility of DNA for the dye. No increase of fluorescence was observed in the presence of the known inhibitors of the endogenous endonuclease: Zn2+ and EGTA. However, the presence of Zn2+ led to decreased staining of the nuclei by PI and caused a shift in the scatter profile of the nuclei, suggesting that a conformational change of chromatin is induced by this ion. This correlation between intensity of PI staining and DNA degradation should be useful to compare endogenous nuclease levels in lymphocyte populations.     

Ca<Superscript>2+</Superscript>, Mg<Superscript>2+</Superscript>-dependent DNase Involvement in Apoptotic Effects in Spermatozoa of Sea Urchin <Emphasis Type="Italic">Strongylocentrotus intermedius</Emphasis> Induced by Two-Headed Sphingolipid Rhizochalin

Previously, we have purified three distinct DNases from spermatozoa of sea urchin Strongylocentrotus intermedius and we suppose the role of Ca²⁺, Mg²⁺-dependent DNase (Ca, Mg-DNase) in apoptosis of spermatozoa. Two-headed sphingolipid rhizochalin (Rhz) induced characteristic apoptotic nuclear chromatin changes, internucleosomal DNA cleavage, and activation of caspase-9, caspase-8, and caspase-3 in spermatozoa as was shown by fluorescence Hoechst 33342/PI/FDA analysis, DNA fragmentation assay, and fluorescence caspase inhibitors FAM-LEHD-fmk, FAM-IETD-fmk, and FAM-DEVD-fmk, respectively. Inhibitor of caspase-3 z-DEVD-fmk subdued Rhz-induced internucleosomal ladder formation, which confirmed the major role of caspase-3 in apoptotic DNA cleavage probably through Ca, Mg-DNase activation. Participation of sea urchin Ca, Mg-DNase in apoptosis of spermatozoa was demonstrated by ions Zn²⁺ blocking of Rhz-induced DNA fragmentation due to direct inhibition of the Ca, Mg-DNase and internucleosomal cleavage of HeLa S and Vero E6 cell nuclei chromatin by highly purified Ca, Mg-DNase.     

DNA fragmentation during thymic apoptosis is catalyzed by DNase γ

The physiological and pathological importance of cell death by apoptosis has recently been recognized. One of the hallmarks of apoptosis is the enzymatic cleavage of genomic DNA into nucleosomal oligomers. The identification of an endonuclease responsible for apoptosis might help to explain how this cell suicide is regulated and why DNA is cleaved. Here, we found that γ type of DNase was retained in apoptotic rat thymocyte nuclei. Homogeneously purified DNase γ (Mr = 33 kDa) from the apoptotic nuclei was revealed to be a Ca²⁺/Mg²⁺-dependent endonuclease and inhibited by Zn²⁺. This enzyme cleaved chromosomal DNA with 3′-hydroxyl (OH) and 5′-phosphoryl (P) ends. The cleavage ends and its divalent cation dependencies match those observed in apoptotic thymocytes induced by X-irradiation or glucocorticoid treatment, indicating that this endonuclease is a central component of the thymic apoptosis machinery.     

Novobiocin induces the in vivo cleavage of active gene sequences in intact cells

When the topoisomerase II inhibitor, novobiocin, is administered to embryonic chicken red blood cells, it induces the in vivo release of an endogenous nuclease which cleaves specifically within internucleosomal spacer DNA and within nuclease-hypersensitive sites in the active chromatin of intact cells. This in vivo released nuclease activity is induced by novobiocin only in metabolically active immature red blood cells. Little induction occurs in mature erythrocytes and no induction occurs in cells previously treated with 2,4-dinitrophenol. Although novobiocin is required to induce release and/or activation of the nuclease, the activity of the nuclease, once activated, is independent of novobiocin. Analysis of the cleaved DNA in drug-treated immature cells demonstrates that the novobiocin-induced nuclease has an unusual blunt-ended double-stranded mode of cleavage. Because of its special properties and apparent chromatin related function in vivo, the novobiocin-induced nuclease activity offers a novel and useful in vivo and in vitro probe of chromatin structure.     

A novel inhibitor that protects apoptotic DNA fragmentation catalyzed by DNase gamma

The internucleosomal cleavage of genomic DNA is the biochemical hallmark of apoptosis. DNase gamma, a Ca(2+)/Mg(2+)-dependent endonuclease, has been suggested to be one of the apoptotic endonucleases. We identified here 4-(4,6-dichloro-[1,3,5]-triazin-2-ylamino)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoic acid (DR396) as a novel and potent DNase gamma inhibitor using stable HeLa S3 transfectants of DNase gamma (HeLa-gamma cells). DR396 inhibited apoptotic DNA fragmentation in HeLa-gamma cells induced by staurosporine (STS) and in rat splenocytes exposed to gamma-ray irradiation in a dose-dependent manner. This compound potently and selectively inhibited DNase gamma activity with an IC(50) value of 3.2 microM. DR396 did not delay the apoptotic processes as judged by the morphological changes and the cleavage of a death substrate, poly(ADP-ribose) polymerase (PARP). Furthermore, the compound did not prevent apoptotic DNA fragmentation in Jurkat cells induced by anti-Fas antibody (Ab), which is catalyzed by caspase-activated DNase (CAD). These findings clearly indicate that DR396 exerts chemical knockdown effect of DNase gamma on cells, suggesting that the compound could be an attractive tool for understanding of the physiological significance of DNase gamma.     

Gammaretroviral integration into nucleosomal target DNA in vivo

Some of the earliest studies of retroviral integration targeting reported that sites of gammaretroviral DNA integration were positively correlated with DNase I-hypersensitive sites in chromatin. This led to the suggestion that open chromatin was favorable for integration. More recent deep sequencing experiments confirmed that gammaretroviral integration sites and DNase I cleavage sites are associated in genome-wide surveys. Paradoxically, in vitro studies of integration show that nucleosomal DNA is actually favored over naked DNA, raising the question of whether integration target DNA in chromosomes is wrapped in nucleosomes or nucleosome free. In this study we examined gammaretroviral integration by infecting primary human CD4(+) T lymphocytes with a murine leukemia virus (MLV)-based retroviral vector or xenotropic murine leukemia virus-related virus (XMRV), and isolated 32,585 unique integration sites using ligation-mediated PCR and 454 pyrosequencing. CD4(+) T lymphocytes were chosen for study because of the particularly dense genome-wide mapping of chromatin features available for comparison. Analysis relative to predicted nucleosome positions showed that gammaretroviruses direct integration into outward-facing major grooves on nucleosome-wrapped DNA, similar to the integration pattern of HIV. Also, a suite of histone modifications correlated with gene activity are positively associated with integration by both MLV and XMRV. Thus, we conclude that favored integration near DNase I-hypersensitive sites does not imply that integration takes place exclusively in nucleosome-free regions.     

Functional characterization of DNase X, a novel endonuclease expressed in muscle cells

The activation of endonucleases resulting in the degradation of genomic DNA is one of the most characteristic changes in apoptosis. Here, we report the characterization of a novel endonuclease, termed DNase X due to its X-chromosomal localization. The active nuclease is a 35 kDa protein with 39% identity to DNase I. When incubated with isolated nuclei, recombinant DNase X was capable of triggering DNA degradation at internucleosomal sites. Similarly to DNase I, the nuclease activity of DNase X was dependent on Ca(2+) and Mg(2+) and inhibited by Zn(2+) ions or chelators of bivalent cations. Overexpression of DNase X caused internucleosomal DNA degradation and induction of cell death associated with increased caspase activation. Despite the presence of two potential caspase cleavage sites, DNase X was processed neither in vitro nor in vivo by different caspases. Interestingly, after initiation of apoptosis DNase X was translocated from the cytoplasm to the nuclear compartment and aggregated as a detergent-insoluble complex. Abundant expression of DNase X mRNA was detected in heart and skeletal muscle cells, suggesting that DNase X may be involved in apoptotic or other biological events in muscle tissues.     

Identification and characterization of glucocorticoid-regulated nuclease(s) in lymphoid cells undergoing apoptosis

Apoptosis is a physiological process by which selected cells are deleted from a population in response to specific regulatory signals. A hallmark of apoptosis is the internucleosomal degradation of DNA prior to cell death. We are studying glucocorticoid-induced lymphocytolysis as a model system for apoptosis within the immune system. In rat thymocytes, the internucleosomal DNA cleavage which occurs following glucocorticoid treatment is both time- and dose-dependent, and is blocked by the glucocorticoid antagonist RU 486, indicating that this effect is mediated by the glucocorticoid receptor. Similar experiments using glucocorticoid-responsive (wt) and glucocorticoid-resistant (nt⁻) S49.1 lymphoma cell lines confirm that internucleosomal DNA degradation and cell death are glucocorticoid receptor-mediated events and thus reflect the direct effects of glucocorticoids on lymphocytes. In an effort to identify the nuclease(s) responsible for the DNA degradation, we have developed two assays to detect nucleases whose activity is altered by glucocorticoid treatment. The first assay involves electrophoresing extracts of nuclear protein from control and glucocorticoid-treated lymphoid cells into SDS-polyacrylamide gels containing [³²P]DNA within the gel matrix. This assay is used to estimate the molecular mass of the nuclease, based on the observed in situ nuclease activity. The second assay uses HeLa nuclei as a substrate to detect internucleosomal cleavage activity present in nuclear extracts of control and glucocorticoid-treated lymphoid cells. Using these assays we have identified a novel Ca²⁺, Mg²⁺-dependent nuclease with an apparent molecular weight of 18 kDa in both S49 wt cells and rat thymocytes treated with glucocorticoids. Furthermore, nuclear extracts of glucocorticoid-treated, but not control, rat thymocytes and S49 wt cells were capable of cleaving HeLa chromatin at internucleosomal sites. In an effort to determine the identity of the nuclease capable of internucleosomal cleavage of DNA, nuclear extracts from dex-treated rat thymocytes were fractionated by gel filtration chromatography under non-denaturing conditions, and the fractions were analyzed using the [³²P]DNA SDS-PAGE and HeLa nuclei assays. When analyzed under native conditions, the 18 kDa nuclease described previously appears to exist as a 25 kDa protein which may be part of a high molecular weight complex. Interestingly, only the 25 kDa form of the protein was associated with internucleosomal DNA cleavage activity where as the high molecular weight form of the enzyme was devoid of this activity.     

Utilization of an in vitro assay to evaluate chromatin degradation by candidate apoptotic nucleases

Apoptosis is commonly associated with the catabolism of the genome in the dying cell. The chromatin degradation occurs in essentially two forms: (1) internucleosomal DNA cleavage to generate oligonucleosomal-length fragments (180-200 bp and multiples thereof), and (2) cleavage of higher order chromatin structures to generate approximately 30-50 Kb fragments. To investigate this component of apoptosis and identify the nuclease(s) responsible, we have developed and utilized an in vitro assay that recapitulates the genomic destruction seen during apoptosis in vivo and allows the simultaneous analysis of both forms of DNA degradation from the same sample. Using this assay we evaluated the digestion patterns of several candidate apoptotic nucleases: DNase I, DNase II, and cyclophilin (NUC18) as well as the bacterial enzyme micrococcal nuclease (not thought to be involved in apoptosis). Chromatin degraded by DNase I formed a smear of DNA on conventional static-field agarose gels and approximately amp;30 - 50 Kb DNA fragments on pulsed field gels. In contrast, DNase II, at a physiologically relevant pH, had no effect on the integrity of HeLa chromatin in either analysis. Similar to DNase I, cyclophilin C produced only approximately 30-50 Kb DNA fragments but did not generate internucleosomal fragments. In contrast, micrococcal nuclease generated both oligonucleosomal and approximately 30-50 Kb DNA fragments. Nuclear extracts from glucocorticoid-treated apoptotic thymocytes generated oligonucleosomal DNA fragments and the larger approximately 30-50 Kb DNA fragments, fully recapitulating both types of apoptotic DNA degradation. Previously, differential sensitivity of nucleases to inhibition by Zn2+ was used to argue that two distinct enzymes mediate approximately 30-50 Kb DNA cleavage and internucleosomal DNA degradation. While, the nuclease activity present in thymocyte nuclear extracts was differentially sensitive to inhibition by Zn2+ during short term incubations it was not during prolonged digestions, suggesting that differences in DNA detection are likely to account for previous results. Together our studies show that none of the nucleases commonly associated with apoptosis could fully recapitulate the DNA degradation seen in vivo.     

Acid DNases and their interest among apoptotic endonucleases

Apoptosis is characterized by cell shrinkage, nuclear condensation and internucleosomal DNA cleavage. Besides the central role of caspases and other proteases, cell death triggers DNA degradation so that DNases have an active role in apoptotic cell death. The best-characterized apoptotic DNase is CAD, a neutral Mg-dependent endonuclease. Its activity is regulated by its inhibitor, ICAD, which is cleaved by caspases. Other neutral DNases have been shown to cleave nuclear DNA in apoptotic conditions: endonuclease G, GADD. In cells, the cytosolic pH is maintained to 7.2, mostly due to the activity of the Na(+)/H(+) exchanger. In many apoptotic conditions, a decrease of the intracellular pH has been shown. This decrease may activate different acid DNases, mostly when pH decreases below 6.5. Three acidic DNases II are so far known: DNase II alpha, DNase II beta and L-DNase II, a DNase II, derived from the serpin LEI (Leukocyte Elastase Inhibitor). Their activation during cell death is discussed in this review.     

Calcium-activated DNA fragmentation kills immature thymocytes

Glucocorticoid hormones kill immature thymocytes by activating a self-destructive process that involves extensive DNA fragmentation. It has been demonstrated that thymocyte suicide is dependent on an early, sustained increase in cytosolic Ca2+ concentration, and new protein synthesis, but the biochemical lesion that leads to cell death has not been established. To determine whether endonuclease activation or activation of another Ca2+-dependent process could mediate cell killing, we treated thymocytes with the glucocorticoid methylprednisolone in the presence of inhibitors of various Ca2+-dependent degradative enzymes. The role of poly(ADP-ribose) polymerase, an enzyme known to be activated by DNA damage, was also assessed. Glucocorticoid-induced chromatin cleavage and cell killing were blocked by the endonuclease inhibitor aurintricarboxylic acid, whereas inhibitors of other Ca2+-dependent degradative processes or of poly(ADP-ribose) polymerase did not abrogate cell death. In addition, stimulation of thymocyte DNA fragmentation by the Ca2+ ionophore A23187 resulted in cell killing that could be blocked by the endonuclease inhibitor. Together, our results suggest that thymocyte suicide is caused by extensive Ca2+-stimulated DNA fragmentation.     

DNase γ Is the Effector Endonuclease for Internucleosomal DNA Fragmentation in Necrosis

Apoptosis and necrosis, two major forms of cell death, can be distinguished morphologically and biochemically. Internucleosomal DNA fragmentation (INDF) is a biochemical hallmark of apoptosis, and caspase-activated DNase (CAD), also known as DNA fragmentation factor 40 kDa (DFF40), is one of the major effector endonucleases. DNase γ, a Mg²⁺/Ca²⁺-dependent endonuclease, is also known to generate INDF but its role among other apoptosis-associated endonucleases in cell death is unclear. Here we show that (i) INDF occurs even during necrosis in cell lines, primary cells, and in tissues of mice in vivo, and (ii) DNase γ, but not CAD, is the effector endonuclease for INDF in cells undergoing necrosis. These results document a previously unappreciated role for INDF in necrosis and define its molecular basis.     

Characterization of the endogenous deoxyribonuclease involved in nuclear DNA degradation during apoptosis (programmed cell death).

Cell death by apoptosis occurs in a wide range of physiological events including repertoire selection of lymphocytes and during immune responses in vivo. A hallmark of apoptosis is the internucleosomal DNA degradation for which a Ca2+,Mg(2+)-dependent endonuclease has been postulated. This nuclease activity was extracted from both rat thymocyte and lymph node cell nuclei. When incubated with nuclei harbouring only limited amounts of endogenous nuclease activity, the ladder pattern of DNA fragments characteristic of apoptosis was induced. This extractable nucleolytic activity was immunoprecipitated with antibodies specific for rat deoxyribonuclease I (DNase I) and was inhibited by actin in complex with gelsolin segment 1, strongly pointing to the presence of a DNase I-type enzyme in the nuclear extracts. COS cells transiently transfected with the cDNA of rat parotid DNase I expressed the enzyme, and their nuclei were able to degrade their DNA into oligosome-sized fragments. PCR analysis of mRNA isolated from thymus, lymph node cells and kidney yielded a product identical in size to that from rat parotid DNase I. Immunohistochemical staining with antibodies to rat DNase I confirmed the presence of DNase I antigen in thymocytes and lymph node cells. The tissue distribution of DNase I is thus extended to tissues with no digestive function and to cells which are known to be susceptible to apoptosis. We propose that during apoptosis, an endonuclease indistinguishable from DNase I gains access to the nucleus due to the breakdown of the ER and the nuclear membrane.     

N-(2-mercaptoethyl)-1,3-propanediamine (WR-1065) protects thymocytes from programmed cell death.

Gamma-irradiation, glucocorticoid hormones, and calcium ionophores stimulate a suicide process in thymocytes, known as apoptosis or programmed cell death, that involves internucleosomal DNA fragmentation by a Ca(2+)- and Mg(2+)-dependent nuclear endonuclease. In this study we report that N-(2-mercaptoethyl)-1,3-propanediamine (WR-1065) blocked DNA fragmentation and cell death in thymocytes exposed to gamma-radiation, dexamethasone, or calcium ionophore A23187. WR-1065 protected the thymocytes from radiation-induced apoptosis when incubated with cells after irradiation but not before and/or during irradiation. WR-1065 inhibited Ca(2+)- and Mg(2+)-dependent DNA fragmentation in isolated thymocyte nuclei. Our results suggest that WR-1065 protects thymocytes from apoptosis by inhibiting Ca(2+)- and Mg(2+)-dependent nuclear endonuclease action.     

Lack of internucleosomal DNA fragmentation is related to Cl(-) efflux impairment in hematopoietic cell apoptosis.

The heterodimeric DNA fragmentation factor (DFF) is responsible for DNA degradation into nucleosomal units during apoptosis. This process needs the caspase-dependent release of ICAD/DFF-45, the inhibitory subunit of DFF. Here we report that triggering apoptosis via a hyperosmotic shock in hematopoietic cells causes the appearance of mitochondrial and cytosolic alterations, activation of caspases, chromatin condensation, nuclear disruption, and DNA fragmentation. However, oligonucleosomal but not high molecular weight (50-150 kb) DNA cleavage is abolished if Cl(-) efflux is prevented by using NaCl to raise extracellular osmolarity or by Cl(-) channel blockers, even when apoptosis is initiated by other agents (staurosporine, anti-Fas antibody). In these conditions, all the apoptosis hallmarks investigated remain detectable, including the cleavage of ICAD/DFF-45. In vitro assays with lysates of cells in which Cl(-) efflux is blocked confirm the lack of internucleosomal DNA degradation. These findings establish that neither caspase activation nor ICAD/DFF-45 processing per se is sufficient to induce oligonucleosomal DNA fragmentation and that high molecular weight DNA degradation and chromatin condensation appear independently of it. Finally, they suggest that Cl(-) efflux is a necessary cofactor that intervenes specifically in the activation of the DFF endonuclease.