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.     

Similar actions of glucocorticoids and calcium on the regulation of apoptosis in S49 cells

Glucocorticoid-induced lymphocyte cell death is a programmed process which is thought to involve the calcium-dependent degradation of DNA into multiples of 180 basepairs, characteristic of internucleosomal degradation. We have used the glucocorticoid-sensitive mouse lymphoma cell line S49.1 [wild-type (wt)] and the glucocorticoid-resistant cell line S49.22r (nt-) to evaluate the role of both glucocorticoid receptors and calcium in the regulation of internucleosomal DNA degradation and expression of calcium-dependent deoxyribonuclease activity. DNA was isolated from untreated (control) and dexamethasone (dex)-treated viable cells and analyzed for internucleosomal DNA degradation by agarose gel electrophoresis, followed by ethidium bromide staining. Glucocorticoid treatment resulted in substantial internucleosomal DNA degradation in wt cells, but not in nt- cells. This effect was inhibited by coincubation of cells with dex and the glucocorticoid receptor antagonist RU486. In contrast to the glucocorticoid response, administration of either of two calcium ionophores, ionomycin or A23187, produced internucleosomal degradation of DNA in both wt and nt- cells, although the latter were less sensitive to ionophore treatment. Interestingly, A23187 treatment also resulted in a loss of cell viability in HeLa S3 cells, a cell line that does not exhibit glucocorticoid-induced apoptosis. No internucleosomal DNA degradation was detected in HeLa S3 cells killed by A23187. To determine whether similar nucleases are associated with this internucleosomal DNA degradation resulting from both glucocorticoid and calcium ionophore treatment, 0.3 M NaCl nuclear protein extracts were prepared from control and treated cells and analyzed for protein composition or nuclease activity. To assay for nuclease activity, nuclear extracts were electrophoresed in sodium dodecyl sulfate-polyacrylamide gels impregnated with [32P]DNA. Nuclease activity was detected by removal of sodium dodecyl sulfate from the gel, activation with calcium, and subsequent visualization of the loss of [32P]DNA by autoradiography. Dex treatment of wt cells resulted in the appearance of several proteins within the mol wt range of 12-18 kDa, only one of which (16-18 kDa) exhibited calcium-dependent nuclease activity. The appearance of these proteins in nuclear extracts was inhibited by coincubation of glucocorticoid-treated cells with RU 486. Glucocorticoid treatment did not result in the appearance of nuclease activity in nuclear extracts from nt- cells. Interestingly, A23187 or ionomycin treatment resulted in an increase in activity of the 16- to 18-kDa nuclease in both wt and nt- cells. These findings indicate that both glucocorticoid receptors and calcium may share common features in the regulation of apoptosis in lymphoid cells.     

Characterization of apoptosis-like endonuclease activity in avian thymocytes.

In the current study the internucleosomal DNA cleavage activity associated with apoptosis was investigated in avian thymocytes. Thymocyte nuclear proteins from glucocorticoid-treated chickens were incubated with chicken red blood cell (cRBC) nuclei, and DNA degradation was analyzed by agarose gel electrophoresis and fluorescence-activated flow cytometry. The thymocyte nuclear extract contained an endonuclease activity that degraded cRBC chromatin at internucleosomal sites as detected by agarose gel electrophoresis. Flow cytometry analysis of cRBC nuclei that were treated with thymocyte nuclear proteins demonstrated a loss of cellular DNA as a function of the amount of added nuclease activity. Furthermore, it was demonstrated that the thymocyte nuclear extract contained a nuclease activity that was capable of degrading radiolabelled naked 32P-DNA into acid soluble DNA fragments. All three assay methods demonstrate that the thymocyte nuclease activity can be inhibited by EDTA, zinc ions and the nuclease inhibitor aurintricarboxylic acid. Based on the analysis of cofactor requirement of this nuclease activity and its susceptibility to inhibitors, the endonuclease activity present in avian apoptotic thymocytes appears to be identical to the mammalian counterpart.     

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.     

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.     

Identification of a glucocorticoid-induced nuclease in thymocytes. A potential "lysis gene" product

Glucocorticoids initiate a cytolytic process in lymphoid cells that is characteristic of programmed cell death. In vivo treatment of adrenalectomized rats with glucocorticoids results in the rapid degradation of the thymocyte genome at internucleosomal sites. This DNA degradation occurs prior to cell death, and considerable evidence indicates that this nucleolytic event is central to the initiation of lymphocytolysis. To further characterize this process, we have searched for the gene products in thymocytes which may be responsible for steroid-induced DNA degradation. Adrenalectomized rats were treated in vivo with dexamethasone or a vehicle control; nuclear thymocyte proteins were extracted with 0.6 M NaCl and analyzed for protein content or nuclease activity on sodium dodecyl sulfatepolyacrylamide gels containing calf thymus DNA. Glucocorticoid treatment resulted in the induction of two major protein families, a 30-32-kDa protein doublet and a series of 3-4 proteins of 12-19 kDa, both of which express prominent DNase activity. Induction of the lower molecular weight nucleases increased with time after steroid treatment and paralleled the time course of glucocorticoid-mediated DNA degradation. Nuclease induction was blocked by the glucocorticoid antagonist RU 486, indicating a steroid receptor-mediated process. When nuclei from glucocorticoid-resistant cells were incubated with nuclear extracts from glucocorticoid-treated rats, the DNA was cleaved at internucleosomal sites, whereas extracts from vehicle-treated animals were virtually inactive. Based on these findings we propose that glucocorticoids, acting via a receptor-mediated pathway, induce a nucleolytic "lysis gene" product(s) responsible for lymphocytolysis.     

Identification, purification, and characterization of a calcium-dependent endonuclease (NUC18) from apoptotic rat thymocytes. NUC18 is not histone H2B

Glucocorticoids stimulate apoptosis in rat thymocytes that is characterized by internucleosomal DNA degradation. We have previously identified an 18-kDa calcium-dependent nuclease whose activity is associated with this DNA degradation. The existence of this nuclease has been challenged by Alnemri and Litwack (1989) J. Biol. Chem. 264, 4104-4111, who suggest that the nuclease we observed was histone H2B. We report here a modified nuclease assay which uses [32P] DNA as a substrate that has enabled the purification and characterization of the 18-kDa nuclease (NUC18). Using Bio-Rex 70 chromatography in conjunction with this assay, we show that NUC18 can be separated from histone H2B. Enzymatically active NUC18, purified to apparent homogeneity, failed to react with two different anti-histone H2B antibodies. NUC18 was inactive in the absence of calcium and known inhibitors of apoptosis, i.e. zinc and aurintricarboxylic acid inhibit its activity. Although NUC18 activity was detected in nuclear extracts of thymocytes of both control and glucocorticoid-treated thymocytes, these activities were distinct. Gel filtration analysis revealed that NUC18 was present as a high molecular weight complex (greater than 100 kDa) in both groups of cells, whereas it also existed as a low molecular weight form in glucocorticoid-treated cells. Thus, NUC18 remains a candidate for the endonuclease responsible for the DNA degradation component of the apoptotic process.     

Different sublines of Jurkat cells respond with varying susceptibility of internucleosomal DNA degradation to different mediators of apoptosis

The ability of two different Jurkat sublines, termed standard and JM, to form DNA ladders was investigated after various apoptotic stimuli. Exposure to a broad spectrum of drugs interfering with signal transduction or cellular metabolism revealed distinct differences between both Jurkat sublines with regard to the pattern of DNA degradation. In standard Jurkat cells, internucleosomal DNA cleavage occurred only after treatment with the protein kinase inhibitor staurosporine. In contrast, the JM subline responded with internucleosomal DNA fragmentation to exposure to gemcitabine, cycloheximide or staurosporine. All drugs induced the formation of DNA fragments of about 50 kb in both sublines, as revealed by pulse field electrophoresis, except H2O2, which caused unspecific DNA degradation. The staurosporine-induced DNA ladder formation was accompanied by an increase in caspase-3 activity in both lines which, however, was considerably lower in Jurkat JM cells after gemcitabine or cycloheximide exposure. When the analysis of internucleosomal DNA degradation was carried out after mycoplasma infection, both Jurkat lines responded with DNA ladder formation after exposure to all drugs used (here only shown for the standard subline). Employing the zymogram technique, nuclease activities of 47 kDa and 54 kDa were detected in culture supernatants, cell homogenates and nuclear extracts only when mycoplasma-infected, whereas the samples obtained from mycoplasma-free sublines were nuclease-negative using this technique, indicating that these endonucleases were of mycoplasmal origin. After drug exposure, the mycoplasmal nucleases must have gained access to the cytoplasm and nuclei of their host cells by an unknown mechanism.     

A role of the Ca2+/Mg2+-dependent endonuclease in apoptosis and its inhibition by Poly(ADP-ribose) polymerase

Apoptosis is characterized by various cell morphological and biochemical features, one of which is the internucleosomal degradation of genomic DNA. The role of the human chromatin-bound Ca(2+)- and Mg(2+)-dependent endonuclease (CME) DNAS1L3 and its inhibition by poly(ADP-ribosyl)ation in the DNA degradation that accompanies apoptosis was investigated. The nuclear localization of this endonuclease is the unique feature that distinguishes it from other suggested apoptotic nucleases. Purified recombinant DNAS1L3 was shown to cleave nuclear DNA into both high molecular weight and oligonucleosomal fragments in vitro. Furthermore, exposure of mouse skin fibroblasts expressing DNAS1L3 to inducers of apoptosis resulted in oligonucleosomal DNA fragmentation, an effect not observed in cells not expressing this CME, as well as in a decrease in cell viability greater than that apparent in the control cells. Recombinant DNAS1L3 was modified by recombinant human poly(ADP-ribose) polymerase (PARP) in vitro, resulting in a loss of nuclease activity. The DNAS1L3 protein also underwent poly(ADP-ribosyl)ation in transfected mouse skin fibroblasts in response to inducers of apoptosis. The cleavage and inactivation of PARP by a caspase-3-like enzyme late in apoptosis were associated with a decrease in the extent of DNAS1L3 poly(ADP-ribosyl)ation, which likely releases DNAS1L3 from inhibition and allows it to catalyze the degradation of genomic DNA.     

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.     

Glucocorticoid action on the immune system

Glucocorticoids have profound effects on immune function that are mediated, in part, by steroid-induced cell death. Our studies have been aimed at identifying the mechanism of this lymphocytolytic process using the rat thymocyte as a model system. Administration of glucocorticoids in vivo resulted in internucleosomal cleavage of the lymphocyte genome that was detectable within 2 h of treatment and increased with time after hormone administration. Six h after steroid treatment greater than 50% of the genome was degraded, yet cell viability remained greater than 90% indicating that this event preceded cell death. Furthermore, this process appeared to be mediated by the glucocorticoid receptor since the antagonist RU 486 blocked glucocorticoid-mediated DNA degradation. To further characterize this lymphocytolysis we have analyzed glucocorticoid-treated thymocytes for nucleases. Two families of nuclear proteins have been identified, a 30-32 kDa doublet and a series of 3-4 proteins that are 12-19 kDa, both of which are induced by glucocorticoid treatment (137 +/- 6% and 342 +/- 24%, respectively) and have prominent nuclease activity. These nucleases can also be induced in vitro indicating that glucocorticoids act directly on thymocytes to mediate this response. Moreover, this nuclease induction, like glucocorticoid-mediated DNA degradation, could be blocked by RU 486. Based on these findings we propose a working model of glucocorticoid-mediated lymphocytolysis in which these steroids, acting via a receptor mediated process, induce the expression of a lysis gene product (nuclease) which degrades the genome and results in cell death.     

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.     

Apoptotic DNA degradation: evidence for novel enzymes

Apoptosis is characterized by multiple morphological and biochemical changes. One biochemical change that has been primarily associated with apoptosis is the cleavage of chromatin in the internucleosomal regions. We have taken two independent approaches to investigating the enzyme(s) responsible for such cleavage. First, using SDS-PAGE gels with (32)P-labelled DNA incorporated into the matrix, we identified a nuclease activity (termed NUC18) from apoptotic thymocytes. This enzyme has been purified to homogeneity and the activity of the pure protein is dependent on Ca(2+) and Mg(2+) while inhibited by Zn(2+) and aurintricarboxylic acid. This protein is found in the nucleus of apoptotic and nonapoptotic cells but is maintained in nondying cells in a large-molecular-weight inactive complex. NUC18 has a denatured molecular weight of 18 Kd but elutes from gel filtration columns with a native molecular weight of approximately 25 Kd. Although an exhaustive search has not been performed, NUC18 has been identified in several cell lines and tissues. Our second approach is designed specifically to detect internucleosomal cleavage of DNA, an obvious requirement for an apoptotic nuclease. By examining the degradation of HeLa chromatin, we have identified a low-molecular-weight of approximately 23 Kd native molecular weight) internucleosomal cleavage enzyme active in nuclear extracts from glucocorticoid-treated thymocytes. This activity is also dependent upon Ca(2+)and Mg(2+) and is inhibited by Zn(2+) as well as aurintricarboxylic acid. It is present in a variety of cell lines and tissues and is maintained in control cells in a latent state prior to apoptosis. In addition to similarities in physical properties, the two enzymes appear to be immunologically related to one another by virtue of their ability to interact with the same antibody. Overall, using independent approaches, we have identified two nucleases with similar biochemical properties whose activity correlates with apoptosis. The current work suggests that these are novel and perhaps closely related enzymes.     

Cell death in Leishmania induced by stress and differentiation: programmed cell death or necrosis?

Unicellular organisms, such as the protozoan parasite Leishmania, can be stimulated to show some morphological and biochemical features characteristic of mammalian apoptosis. This study demonstrates that under a variety of stress conditions such as serum deprivation, heat shock and nitric oxide, cell death can be induced leading to genomic DNA fragmentation into oligonucleosomes. DNA fragmentation was observed, without induction, in the infectious stages of the parasite, and correlated with the presence of internucleosomal nuclease activity, visualisation of 45 to 59 kDa nucleases and detection of TUNEL-positive nuclei. DNA fragmentation was not dependent on active effector downstream caspases nor on the lysosomal cathepsin L-like enzymes CPA and CPB. These data are consistent with the presence of a caspase-independent cell death mechanism in Leishmania, induced by stress and differentiation that differs significantly from metazoa.     

Identification of potassium-dependent and -independent components of the apoptotic machinery in mouse ovarian germ cells and granulosa cells

Recent studies with thymocytes have suggested a critical role for intracellular potassium in the regulation of apoptosis. In this study, we examined the pathways of K(+) regulation during ovarian cell death. In initial studies, fluorographic analysis demonstrated a significant loss of K(+) during apoptosis stimulated by doxorubicin in oocytes and trophic hormone deprivation in granulosa cells. In oocytes, suppression of potassium efflux by potassium-enriched medium prevented condensation, budding, and fragmentation, although it did not block DNA degradation, suggesting the existence of potassium-independent nucleases in oocytes. Culture of granulosa cells in potassium-enriched medium inhibited internucleosomal DNA cleavage, although high-molecular weight DNA cleavage was apparent, suggesting that the nuclease or nucleases responsible for generating 50-kilobase (kb) fragments in these cells is potassium independent. To address this directly, isolated granulosa cell nuclei were stimulated to autodigest their DNA, and internucleosomal, but not large-fragment, cleavage was completely blocked by 150 mM potassium. We next examined whether the proapoptotic caspases are targets for potassium regulation. In cell-free assays, processing of pro-interleukin-1beta and proteolysis of cellular actin by recombinant caspase-1 and caspase-3, respectively, were suppressed by the presence of 150 mM potassium. Other monovalent ions (NaCl, LiCl) exerted a similar effect in these cell-free assays. Thus, in oocytes and granulosa cells, potassium efflux appears to occur early in the cell death program and may regulate a number of apoptotic events including caspase activity and internucleosomal DNA cleavage. However, there also exist novel potassium-independent pathways in both ovarian germ cells and somatic cells that signal certain apoptotic events, such as large-fragment DNA cleavage.     

Ca2+/Mg(2+)-dependent nuclease: tissue distribution, relationship to inter-nucleosomal DNA fragmentation and inhibition by Zn2+.

Ca2+/Mg(2+)-dependent endonuclease has been implicated in the extensive internucleosomal DNA fragmentation that accompanies apoptosis (gene-directed cell death). We present further evidence that this enzyme is involved in apoptosis. Ca2+/Mg2+ nuclease activity was increased about 6-fold during colchicine-induced apoptosis in human chronic lymphocytic leukaemia cells. The increase in activity coincided with onset of DNA fragmentation. Spleen, liver, kidney and thymus expressed high levels of this enzyme while lung, brain, heart and testis contained little activity. Cells from tissues with high Ca2+/Mg2+ nuclease activity underwent rapid DNA fragmentation in response to a Ca2+ flux. Physiological concentrations of Zn2+ known to inhibit both apoptosis and DNA fragmentation also inhibited Ca2+/Mg2+ nuclease activity.     

Apoptosis in the chick wing bud and the permanence of FGF-2 rescue

Summary Two regions of programmed cell death that occur in the mesoderm of developing chick wing buds were studied in vitro. The opaque patch (OP) and posterior necrotic zone (PNZ) were examined for the presence of internucleosomal DNA degradation and for rescue by protein synthesis inhibition, two defining characteristics of apoptosis. Agarose gel electrophoresis showed that DNA from OP and PNZ tissue was cleaved into nucleosome size pieces and this cleavage was prevented by inhibition of protein synthesis with cycloheximide. Both regions showed rescue with cycloheximide as determined by the chromium release assay and examination of electron micrographs. Also, the permanence of basic fibroblast growth factor (FGF-2) rescue in the OP and PNZ was examined using the chromium release assay. While rescue in the OP was found to be permanent, rescue in the PNZ only delayed death while FGF-2 was present in the culture medium. This research shows that death in the OP and PNZ exhibits internucleosomal DNA fragmentation and is prevented by inhibition of protein synthesis with cycloheximide, biochemically characterizing this death as apoptosis. It also suggests that in vitro FGF-2 rescue is permanent in the OP but is merely a delay of cell death in the PNZ.     

Purification and characterization of Mycoplasma penetrans Ca2+/Mg2+-dependent endonuclease.

The major nuclease from Mycoplasma penetrans has been purified to homogeneity. The enzyme seems to be present as a membrane-associated precursor of 50 kDa and as a peripheral membrane monomeric polypeptide of 40 kDa that is easily removed by washing of cells with isotonic buffers and in the aqueous phase upon Triton partitioning of Triton X-114-solubilized protein. The 40-kDa nuclease was extracted from M. penetrans cells by Triton X-114 and phase fractionation and was further purified by chromatography on Superdex 75 and chelating Sepharose (Zn2+ form) columns. By gel filtration, the apparent molecular mass was 40 kDa. The purified enzyme exhibits both a nicking activity on superhelical and linear double-stranded DNA and a nuclease activity on RNA and single-stranded DNA. No exonuclease activity was found for this enzyme. This nuclease required both Mg2+ (optimum, 5 mM) and Ca2+ (optimum, 2 mM) for activity and exhibited a pH optimum between pH 7 and 8 for DNase activity. It was inhibited by Zn2+, Mn2+, heparin, sodium dodecyl sulfate, and chelator agents such EDTA and EGTA, but no effect was observed with ATP, 2-mercaptoethanol, N-ethylmaleimide, dithiothreitol, nonionic detergents, phenylmethylsulfonyl fluoride, and iodoacetamide. Nuclease activity was inhibited by diethylpyrocarbonate at both pH 6 and 8 and by pepstatin, suggesting the involvement of a histidine and an aspartate in the active site. When added to human lymphoblast nuclei, the purified M. penetrans endonuclease induced internucleosomal fragmentation of the chomatin into oligonucleosomal fragments. On the basis of this result, and taking into account the fact that M. penetrans has the capacity to invade eucaryotic cells, one can suggest, but not assert, that produced Ca2+/Mg2+-dependent endonuclease may alter the nucleic acid metabolism of host cells by DNA and/or RNA degradation and may act as a potential pathogenic determinant.     

Trypanosoma cruzi nucleoside diphosphate kinase 1 ( TcNDPK1) has a broad nuclease activity

Here, we present the characterization of a trypanosomatid nucleoside diphosphate kinase (TcNDPK1) exhibiting nuclease activity. This is the first identification of a NDPK with this property in trypanosomatid organisms. The recombinant TcNDPK1 protein cleaves not only linear DNA, but also supercoiled plasmid DNA. Additionally, TcNDPK1 is capable of degrading Trypanosoma cruzi genomic DNA. ATP or ADP did not affect the nuclease activity, while the absence of Mg2+ completely inhibits this activity. NDPK and nuclease activities were inhibited at the same temperature, suggesting the presence of related catalytic sites. Furthermore, phenogram analysis showed that TcNDPK1 is close to Drosophila melanogaster and human NDPKs. The unspecific nuclease activity could suggest a participation in cellular processes such as programmed cell death.     

Early-stage apoptosis is associated with DNA-damage-independent ATM phosphorylation and chromatin decondensation in NIH3T3 fibroblasts

Chromatin condensation and degradation of DNA into internucleosomal DNA fragments are key hallmarks of apoptosis. The phosphorylation of protein kinase ataxia telangiectasia mutated (ATM) and histone H2A.X was recently shown to occur concurrently with apoptotic DNA fragmentation. We have used immunofluorescence microscopy, Western blot analysis and alkali comet assays to show that phosphorylation of ATM in NIH3T3 fibroblasts occurs prior to apoptotic DNA fragmentation, nuclease degradation and phosphorylation of histone H2A.X in cells treated with low levels of either staurosporine (STS) or tumor necrosis factor-alpha mixed with cycloheximide (TNF-alpha/CHX). In extension to previous findings, ATM phosphorylation was associated with chromatin decondensation, i.e., by loss of dense foci of constitutive heterochromatin. These results suggest that chromatin is decondensed and that ATM is activated independently of DNA damage signaling pathways during the very early stages of apoptosis.