Dependent Endonuclease Research Articles

Peroxynitrite induces apoptosis in rat aortic smooth muscle cells: possible relation to vascular diseases.

An emerging body of evidence is accumulating to suggest that in vivo formation of free radicals in the vasculature, such as peroxynitrite (ONOO-), and programmed cell death (i.e., apoptosis) play important roles in vascular diseases such as atherosclerosis, hypertension, and restenosis. The present study was designed to determine whether primary rat aortic smooth muscle cells (SMCs) undergo apoptosis following treatment with ONOO-. Direct exposure of primary rat aortic SMCs to ONOO--induced apoptosis in a concentration-dependent manner, as confirmed by means of quantitative fluorescence staining and TUNEL assays. ONOO--induced apoptosis in rat aortic SMCs appears to involve activation of Ca2+-dependent endonucleases. Although the precise mechanisms by which peroxynitrite induces apoptosis in rat aortic SMCs need to be further investigated, the present, preliminary findings could be used to suggest that ONOO- formation in the vasculature may play roles in the processes of vascular diseases, such as atherosclerosis, hypertension, and restenosis, via adverse actions on blood vessels.

Regulation of DNaseY activity by actinin-alpha4 during apoptosis.

DNaseY, a Ca(2+)- and Mg(2+)-dependent endonuclease, has been implicated in apoptotic DNA degradation; however, the molecular mechanisms controlling its involvement in this process have not been fully elucidated. We have obtained evidence from yeast two-hybrid screening and coimmunoprecipitation experiments that DNaseY interacted physically with actinin-alpha4 and this interaction significantly enhanced its endonuclease activity. Accordingly, simultaneous overexpression of both proteins in PC12 cells dramatically increased the rate of apoptosis in response to teniposide' VM26. However, overexpression of DNaseY alone neither triggered apoptosis nor facilitated cell death in response to VM26 or serum deprivation. Instead, the overexpression of DNaseY increased the production of single-strand DNA breaks and evoked a profound upregulation of DNA repair pathways. Taken together, our results point to a novel regulatory mechanism of DNaseY activity and offer an explanation for why cells must first cleave key DNA repair and replication proteins before the successful execution of apoptosis.

Mechanism of Acetaminophen‐Induced Apoptosis in Cultured Cells: Roles of Caspase‐3, DNA Fragmentation Factor, and the Ca2+ and Mg2+ Endonuclease DNAS1L3

We have recently shown that acetaminophen induces many of the apoptotic traits in hepatoma cells and lymphocytes (Boulares et al. (2002d). In an effort to further investigate the mechanism by which non-metabolized acetaminophen induces apoptosis, we have now examined the roles of caspase-3, the DNA fragmentation factor, and the poly(ADP-ribose) polymerase-1-regulated Ca2+ and Mg2+-dependent endonuclease DNAS1L3 in the induction of such death process. This was achieved with the use of MCF-7 cells, a caspase-3-deficient breast adenocarcinoma cell line, thymocytes isolated from DFF45 (the inhibitory and chaperone subunit of the DNA fragmentation factor subunit, DFF40) deficient mice, and HeLa cells, a DNAS1L3-deficient cervical carcinoma cell line. MCF-7 exhibited a marked resistance to acetaminophen treatment. Ectopic expression of human caspase-3 significantly potentiated the cytotoxic effect of acetaminophen and promoted the release of cytochrome c into the cytosol of treated cells suggesting a direct role for caspase-3 in acetaminophen-induced apoptosis. Expression and cleavage of DFF45 were required but not sufficient for acetaminophen-induced internucleosomal DNA fragmentation. DFF45 gene knockout rendered thymocytes resistant against acetaminophen-induced generation of both large and internucleosomal DNA fragments. The treatment of HeLa cells with acetaminophen resulted in internuclesomal DNA fragmentation only after transfection of these cells with a plasmid encoding the DNAS1L3 gene suggesting that this endonuclease is required for acetaminophen-induced internucleosomal DNA fragmentation. DNAS1L3 expression potentiated the cytotoxic effect of acetaminophen in HeLa cells suggesting an active role in the death process induced by this drug. Altogether, these results demonstrate the specific roles of caspase-3, DNA fragmentation factor, and DNAS1L3 in the process of acetaminophen-induced apoptosis in cultured cells.

Aging increases DNase γ, an apoptosis-related endonuclease, in rat liver nuclei: effect of dietary restriction

Organ-specific endonuclease might play a role in the age-related increase in apoptosis in laboratory rodent tissues. In nuclear extracts from liver tissues of male F344 rats, the DNase activity gel system identified DNase γ, Ca2+/Mg2+-dependent endonuclease. The enzyme activity, which was measured at 3, 6, 16, and 24 months (mo) of age, was significantly increased between 16 and 24mo in control rats fed ad libitum (AL). The expression level of DNase γ-mRNA, estimated by a semi-quantitative reverse transcription-polymerase chain reaction method, was also increased at 24mo in group AL. The proportion of immunohistochemically DNase γ-positive cells, most of which were light-microscopically confined to apoptotic cells, was also significantly increased between 16 and 24mo. Dietary restriction, a powerful anti-aging intervention, which was achieved by providing 70% of the mean food intake in group AL from 6 weeks of age, inhibited the age-related increase in the enzyme activity and the proportion of immunostained cells; for the mRNA level, statistical significance was not obtained. The present study suggests that DNase γ is involved in an age-related increase in the apoptosis of rat liver, and that CR inhibits the increase as it minimized the age-related increase in the fraction of DNA-damaged hepatocytes susceptible to apoptosis.

Hydrogen peroxide induces apoptosis in cerebral vascular smooth muscle cells: possible relation to neurodegenerative diseases and strokes

Recently, reactive oxygen species (ROS) have been suggested as important mediators of brain damage in a number of disease states, including traumatic brain injury, neurodegenerative diseases and strokes. Apoptosis has been suggested to play an important role in neurodegenerative diseases, traumatic brain injury and strokes. The aim of this study was to determine whether or not cerebral vascular smooth muscle cells (CVSMCs) undergo apoptosis following treatment with hydrogen peroxide (H 2O 2). Herein, we demonstrate, for the first time, that H 2O 2 can induce apoptosis in a concentration-dependent manner in primary cultured CVSMCs, as measured by several morphological and biochemical criteria. H 2O 2-induced apoptosis may be initiated by stimulating Ca 2+-dependent endonuclease activity. The present new data suggest that apoptosis in cerebral VSMCs, induced by ROS, such as H 2O 2, could play important roles in neruodegenerative processes, traumatic brain injury and strokes.

Peroxynitrite induces apoptosis in canine cerebral vascular muscle cells: possible relation to neurodegenerative diseases and strokes

Considerable evidence is accumulating to suggest that in vivo formation of free radicals in the brain, such as peroxynitrite (ONOO −), and programmed cell death (i.e. apoptosis) play important roles in neurodegeneration and stroke. However, it is not known whether ONOO − can induce apoptosis in cerebral vascular smooth muscle cells (CVSMCs). The present study was designed to determine whether or not canine CVSMCs undergo apoptosis following treatment with ONOO −. Direct exposure of canine CVSMCs to ONOO − induced apoptosis in a concentration-dependent manner, as confirmed by means of fluorescence staining, TdT-mediated dUTP nick-end labeling and comet assays. Peroxynitrite treatment resulted in an elevation of [Ca 2+] i in the CVSMCs. Peroxynitrite-induced apoptosis may thus be brought about by activation of Ca 2+-dependent endonucleases. Although the precise mechanisms by which peroxynitrite induces apoptosis need to be further investigated, the present findings could be used to suggest that ONOO − formation in the brain may play important roles in neurodegenerative processes and strokes via detrimental actions on cerebral microvessels and blood flow.

Dykellic acid inhibits drug-induced caspase-3-like protease activation

Dykellic acid is a novel microbial metabolite isolated from the broth of Westerdykella multispora F50733. Investigations on the molecular function of dykellic acid revealed that this compound partially inhibits calcium influx, resulting in a decrease in Ca 2+-dependent endonuclease activation and DNA fragmentation induced by camptothecin. In our experiments, active caspase-3-like protease cleavage of procaspase-3, PARP, and cytosolic cytochrome c was inhibited by dykellic acid in a concentration-dependent manner when the apoptosis was induced by camptothecin as well as doxorubicin. We confirmed that dykellic acid did not bind to camptothecin using surface plasmon resonance analysis. These results suggest that dykellic acid inhibits drug-induced apoptosis via a caspase-3-like protease-suppressing mechanism. Our data provide important information on the mechanism of action of dykellic acid and indicate that this compound may be employed in the treatment of specific caspase-3-like protease-mediated diseases.

Ngl2p is a Ccr4p-like RNA nuclease essential for the final step in 3'-end processing of 5.8S rRNA in Saccharomyces cerevisiae.

Saccharomyces cerevisiae contains three nonessential genes (NGL1, NGL2, and NGL3) that encode proteins containing a domain with similarity to a Mg(2+)-dependent endonuclease motif present in the mRNA deadenylase Ccr4p. We have investigated a possible role of these proteins in rRNA processing, because for many of the pre-rRNA processing steps, the identity of the responsible nuclease remains elusive. Analysis of RNA isolated from cells in which the NGL2 gene has been inactivated (ngl2delta) demonstrates that correct 3'-end formation of 5.8S rRNA at site E is strictly dependent on Ngl2p. No role in pre-rRNA processing could be assigned to Ngl1p and Ngl3p. The 3'-extended 5.8S rRNA formed in the ngl2delta mutant is slightly shorter than the 6S precursor previously shown to accumulate upon combined deletion of the 3' --> 5' exonuclease-encoding REX1 and REX2 genes or upon depletion of the exosomal subunits Rrp40p or Rrp45p. Thus, our data add a further component to the set of nucleases required for correct 3'-end formation of yeast 5.8S rRNA.

Involvement of DNase γ in Apoptosis Associated with Myogenic Differentiation of C2C12 Cells

Nucleosomal DNA fragmentation is detected in myoblasts only when apoptosis is induced under differentiating conditions. However, the molecular mechanisms and the DNase responsible for the differentiation-dependent apoptotic DNA laddering are poorly understood. Here we show that a Ca(2+)/Mg(2+)-dependent endonuclease, DNase gamma, is induced in C2C12 myoblasts during myogenic differentiation and catalyzes apoptotic DNA fragmentation in differentiating myoblasts. A Ca(2+)/Mg(2+)-dependent, Zn(2+)-sensitive endonuclease activity appears in C2C12 myoblasts during myogenic differentiation. The enzymatic properties of the inducible DNase were found to be quite similar to those of DNase I family of DNases. Reverse transcriptase-PCR analysis revealed that the induction of DNase gamma, a member of the DNase I family of DNases, is correlated with the appearance of inducible DNase activity. The induction of DNase gamma occurs simultaneously with myogenin induction but precedes the up-regulation of p21. A high level of DNase gamma expression was also detected in differentiated myotubes but not in skeletal muscle fibers in which DNase X is highly expressed. The role of DNase gamma in myoblast apoptosis was evaluated in the following experiments. Proliferating myoblasts acquire DNA ladder producing ability by the ectopic expression of DNase gamma, but not DNase X, suggesting that the expression level of DNase gamma is the determinant of the differentiation-dependent apoptotic DNA laddering observed in myoblasts. DNA fragmentation during differentiation-induced apoptosis is strongly suppressed by the antisense-mediated down-regulation of DNase gamma. Importantly, the extent of DNA laddering is well correlated with the level of endogenous DNase gamma activity. Our data demonstrate that DNase gamma is the endonuclease responsible for DNA fragmentation in apoptosis associated with myogenic differentiation.

Embryonic extracts derived from the nematode Caenorhabditis elegans remove uracil from DNA by the sequential action of uracil-DNA glycosylase and AP (apurinic/apyrimidinic) endonuclease

DNA bases continuously undergo modifications in response to endogenous reactions such as oxidation, alkylation or deamination. The modified bases are primarily removed by DNA glycosylases, which cleave the N-glycosylic bond linking the base to the sugar, to generate an apurinic/apyrimidinic (AP) site, and this latter lesion is highly mutagenic. Previously, no study has demonstrated the processing of these lesions in the nematode Caenorhabditis elegans. Herein, we report the existence of uracil-DNA glycosylase and AP endonuclease activities in extracts derived from embryos of C. elegans. These enzyme activities were monitored using a defined 5'-end (32)P-labelled 42-bp synthetic oligonucleotide substrate bearing a single uracil residue opposite guanine at position 21. The embryonic extract rapidly cleaved the substrate in a time-dependent manner to produce a 20-mer product. The extract did not excise adenine or thymine opposite guanine, although uracil opposite either adenine or thymine was processed. Addition of the highly specific inhibitor of uracil-DNA glycosylase produced by Bacillus subtilis to the extract prevented the formation of the 20-mer product, indicating that removal of uracil is catalysed by uracil-DNA glycosylase. The data suggest that the 20-mer product was generated by a sequential reaction, i.e., removal of the uracil base followed by 5'-cleavage of the AP site. Further analysis revealed that product formation was dependent upon the presence of Mg(2+), suggesting that cleavage of the AP site, following uracil excision, is carried out by a Mg(2+)-dependent AP endonuclease. It would appear that these activities correspond to the first two steps of a putative base-excision-repair pathway in C. elegans.

Wogonin and fisetin induce apoptosis in human promyeloleukemic cells, accompanied by a decrease of reactive oxygen species, and activation of caspase 3 and Ca 2+-dependent endonuclease

Seven structurally related flavonoids including luteolin, nobiletin, wogonin, baicalein, apigenin, myricetin and fisetin were used to study their biological activities on the human leukemia cell line, HL-60. On MTT assay, wogonin, baicalein, apigenin, myricetin and fisetin showed obvious cytotoxic effects on HL-60 cells, with wogonin and fisetin being the most-potent apoptotic inducers among them. The cytotoxic effects of wogonin and fisetin were accompanied by the dose- and time-dependent appearance of characteristics of apoptosis including DNA fragmentation, apoptotic bodies and the sub-G1 ratio. Treatment with an apoptosis-inducing concentration of wogonin or fisetin causes rapid and transient induction of caspase 3/CPP32 activity, but not caspase 1 activity. Further, cleavage of poly(ADP-ribose) polymerase (PARP) and decrease of pro-caspase 3 protein were detected in wogonin- and fisetin-treated HL-60 cells. An increase in the pro-apoptotic protein, bax, and a decrease in the anti-apoptotic protein, Mcl-1, were detected in fisetin- and wogonin-treated HL-60 cells. However, Bcl-2, Bcl-XL, and Bad all remained unchanged in wogonin- and fisetin-treated HL-60 cells. In vitro chromatin digestion revealed that endonuclease activity was profoundly enhanced in wogonin- and fisetin-treated HL-60 cells, and the addition of ethylenediaminetetraacetic acid (EDTA) or ethyleneglycoltetraacetic acid (EGTA) into the reaction blocked endonuclease activation and at an optimum pH of 7.5. The caspase 3 inhibitor, Ac-DEVD-CHO, but not the caspase 1 inhibitor, Ac-YVAD-CHO, attenuated wogonin- and fisetin-induced DNA ladders, PARP cleavage, and endonuclease activation. Pretreatment of HL-60 cells with N-acetyl-cysteine or catalase efficiently inhibited H 2O 2 (200 μM)-induced apoptosis, but showed no inhibitory effect on wogonin- and fisetin-induced DNA ladders, caspase 3 activation, or bax protein induction. Decrease in endogenous ROS production was detected in wogonin- and fisetin-treated HL-60 cells by DCHF-DA assay. In conclusion, our experiments indicate that a decrease in intracellular peroxide level was involved in wogonin- and fisetin-induced apoptosis; activation of caspase 3 and endonuclease, induction of bax protein and suppression of Mcl-1 protein were detected in the process.

Regulation of DNAS1L3 Endonuclease Activity by Poly(ADP-ribosyl)ation during Etoposide-induced Apoptosis: ROLE OF POLY(ADP-RIBOSE) POLYMERASE-1 CLEAVAGE IN ENDONUCLEASE ACTIVATION

Several endonucleases are implicated in the internucleosomal DNA fragmentation associated with apoptosis. The human Ca2+- and Mg2+-dependent endonuclease DNAS1L3 is inhibited by poly(ADP-ribosyl)ation in vitro, and its activation during apoptosis shows a time course similar to that of the cleavage of poly(ADP-ribose) polymerase-1 (PARP-1). The role of the cleavage and consequent inactivation of PARP-1 by caspase-3 in the activation of DNAS1L3 has now been investigated further both in vitro and in vivo. In an in vitro system based on purified recombinant proteins and NAD, caspase-3 prevented the inhibition of DNAS1L3 endonuclease activity by wild-type PARP-1 but not that induced by a caspase-3-resistant PARP-1 mutant. The induction by etoposide of apoptosis in human osteosarcoma cells (which were shown not to express endogenous DNAS1L3) was accompanied by internucleosomal DNA fragmentation only after transfection of the cells with a plasmid encoding DNAS1L3. This DNA fragmentation in etoposide-treated cells was blocked by 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, an inhibitor of intracellular Ca2+ release. Expression of the endonuclease subunit of DNA fragmentation factor (DFF40) and cleavage of its inhibitor, DFF45, were not sufficient to cause internucleosomal DNA fragmentation in osteosarcoma cells during etoposide-induced apoptosis. Coexpression of caspase-3-resistant PARP-1 mutant with DNAS1L3 in osteosarcoma cells blocked etoposide-induced internucleosomal DNA fragmentation and resulted in persistent poly(ADP-ribosyl)ation of DNAS1L3; it did not, however, prevent the activation of caspase-3 and the consequent cleavage of endogenous PARP-1. These results indicate that PARP-1 cleavage during apoptosis is not simply required to prevent excessive depletion of NAD and ATP but is also necessary to release DNAS1L3 from poly(ADP-ribosyl)ation-mediated inhibition.

Crystallographic and Modeling Studies of RNase III Suggest a Mechanism for Double-Stranded RNA Cleavage

Background: Aquifex aeolicus Ribonuclease III (Aa-RNase III) belongs to the family of Mg 2+-dependent endonucleases that show specificity for double-stranded RNA (dsRNA). RNase III is conserved in all known bacteria and eukaryotes and has 1–2 copies of a 9-residue consensus sequence, known as the RNase III signature motif. The bacterial RNase III proteins are the simplest, consisting of two domains: an N-terminal endonuclease domain, followed by a double-stranded RNA binding domain (dsRBD). The three-dimensional structure of the dsRBD in Escherichia coli RNase III has been elucidated; no structural information is available for the endonuclease domain of any RNase III. Results: We present the crystal structures of the Aa-RNase III endonuclease domain in its ligand-free form and in complex with Mn 2+. The structures reveal a novel protein fold and suggest a mechanism for dsRNA cleavage. On the basis of structural, genetic, and biological data, we have constructed a hypothetical model of Aa-RNase III in complex with dsRNA and Mg 2+ ion, which provides the first glimpse of RNase III in action. Conclusions: The functional Aa-RNase III dimer is formed via mainly hydrophobic interactions, including a “ball-and-socket” junction that ensures accurate alignment of the two monomers. The fold of the polypeptide chain and its dimerization create a valley with two compound active centers at each end of the valley. The valley can accommodate a dsRNA substrate. Mn 2+ binding has significant impact on crystal packing, intermolecular interactions, thermal stability, and the formation of two RNA-cutting sites within each compound active center.

MECHANISMS OF THE INDUCTION OF APOPTOSIS IN HUMAN HEPATOMA CELLS BY TUMOUR NECROSIS FACTOR-α

Tumour necrosis factor alpha (TNF-α) at 20ng/ml induced apoptosis in human hepatoma cellsin vitro . The effect of TNF-α-induced apoptosis was exacerbated by the hypoxanthine-xanthine oxidase (HX/XO) system and cycloheximide (CHX), but alleviated by superoxide dismutase (SOD), suggesting that TNF-α-induced apoptosis may be due to oxidative stress, and independent of protein synthesis. TNF-α elevated free Ca2+concentration, triggered lipid peroxidation and decreased the expression of bcl-2 protein. The findings suggest that TNF-α-induced apoptosis may be involved in stimulating Ca2+-dependent endonuclease activity and increasing membrane lipid peroxidation. Bcl-2 may play a pivotal role in serving as a Ca2+regulator or antioxidant, preventing lipid peroxidation in the process.

Identification of four families of yCCR4- and Mg2+-dependent endonuclease-related proteins in higher eukaryotes, and characterization of orthologs of yCCR4 with a conserved leucine-rich repeat essential for hCAF1/hPOP2 binding.

BackgroundThe yeast yCCR4 factor belongs to the CCR4-NOT transcriptional regulatory complex, in which it interacts, through its leucine-rich repeat (LRR) motif with yPOP2. Recently, yCCR4 was shown to be a component of the major cytoplasmic mRNA deadenylase complex, and to contain a fold related to the Mg2+-dependent endonuclease core.ResultsHere, we report the identification of nineteen yCCR4-related proteins in eukaryotes (including yeast, plants and animals), which all contain the yCCR4 endonuclease-like fold, with highly conserved CCR4-specific residues. Phylogenetic and genomic analyses show that they form four distinct families, one of which contains the yCCR4 orthologs. The orthologs in animals possess a leucine-rich repeat domain. We show, using two-hybrid and far-Western assays, that the human member binds to the human yPOP2 homologs, i.e. hCAF1 and hPOP2, in a LRR-dependent manner.ConclusionsWe have identified the mammalian orthologs of yCCR4 and have shown that the human member binds to the human yPOP2 homologs, thus strongly suggesting conservation of the CCR4-NOT complex from yeast to human. All members of the four identified yCCR4-related protein families show stricking conservation of the endonuclease-like catalytic motifs of the yCCR4 C-terminal domain and therefore constitute a new family of potential deadenylases in mammals.

Sequence Analysis Identifies TTRAP, a Protein That Associates with CD40 and TNF Receptor-Associated Factors, as a Member of a Superfamily of Divalent Cation-Dependent Phosphodiesterases

CD40 is a member of the tumor necrosis factor (TNF) receptor family. CD40-mediated signal transduction involves the recruitment of several cytoplasmic proteins and induces expression of a large number of genes. TTRAP, a novel protein that interacts with the cytoplasmic domain of CD40 and with TNF-receptor associated factors (TRAFs), has been cloned and shown to inhibit nuclear factor-κB activation (NF-κB). By using various bioinformatics-based sequence and structure analyses of proteins involved in signaling by the TNF receptor family, we found that TTRAP is a member of a superfamily of Mg2+/Mn2+-dependent phosphodiesterases. More precisely, our results suggest that TTRAP is related to the human APE1, a Mg2+-dependent endonuclease. This potential novel function of TTRAP raises the intriguing possibility for a role of APE1-like DNA-repair endonucleases in TNF receptor family-mediated signaling and functions.

Zinc Inhibition of Caspase-3 Activation Does Not Protect HeLa Cells from Apoptotic Cell Death

Zinc is proposed to be antiapoptotic for it has been shown to inhibit late events of apoptotic pathways such as Ca2+/Mg2+-dependent endonuclease cleavage of chromatin DNA, poly-ADP ribose polymerase cleavage, and caspase-3 activity. Because caspase-3 is a critical executioner caspase in apoptosis, this study was undertaken to examine specifically a correlation between zinc inhibition of caspase-3 activation and apoptosis in HeLa cells. Cultured HeLa cells were exposed to 100 μM ZnCl2 for 1 h prior to 12 h treatment with 1.0 μM doxorubicin (DOX), an important anticancer agent that causes apoptosis in a wide variety of tumor cells. Western blot analysis of HeLa cells treated with DOX for 12 h revealed that DOX caused proteolytic activation of caspase-3 and zinc inhibited this activation. Interestingly, zinc did not inhibit DOX-induced apoptosis as measured by a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Furthermore, a microculture tetrazolium assay confirmed that cell death occurred in the presence of zinc. These results demonstrate that zinc specifically inhibits DOX-induced activation of caspase-3 in HeLa cells, but does not suppress DOX-induced apoptosis or otherwise cell death, thus suggesting DOX-induced caspase-3 activation may not play a major role in overall cell death and/or non-caspase-3 pathways are involved in DOX-induced apoptosis in HeLa cells.

Involvement of L-DNase II in Nuclear Degeneration during Chick Retina Development

During the development of the neural retina, 50% of the neurons die physiologically by apoptosis. In the chick embryo, the apoptotic wave starts at E8 and ends at E18, with a peak at E11. The onset of apoptosis is accompanied by the activation of several degradative enzymes. Among these, the activation of the endonucleases leads to the degradation of the genomic DNA of the cell which is thought to be the final event in apoptosis. Here, we have investigated the endonucleases activated during apoptosis associated with retinal development. We have found that Ca2+-Mg2+-dependent endonucleases, as well as acid endonucleases are activated. The results obtained in vitro using purified nuclei from chicken retina indicate that the endonuclease activity resulting from the activation of L-DNase II, an acid DNase is responsible for most of the DNA degradation observed in these cells.

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

Internucleosomal DNA fragmentation following the activation of endonucleases is the common end point of apoptosis. DNase I, a Ca(2+) / Mg(2+)-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.

Pharmacological interventions of cyanide-induced cytotoxicity and DNA damage in isolated rat thymocytes and their protective efficacy in vivo

Cyanide inhibits the mitochondrial respiratory chain enzyme cytochrome oxidase causing histotoxic hypoxia. It is primarily considered as a neurotoxin but its other toxic manifestations are also well documented. Cyanide-induced apoptosis in neuronal cells has also been demonstrated recently. At the same time we also reported that potassium cyanide (KCN) produces extensive cytotoxicity and DNA fragmentation in rat thymocytes. The DNA damage was sensitive to elevated levels of extracellular Ca 2+ and was attenuated by Zn 2+ (modulator of Ca 2+-dependent endonuclease), N-acetylcysteine (free radical scavenger) and diltiazem (Ca 2+ channel blocker). In a continuation of this work, in the present study we have shown that the cytotoxicity and DNA fragmentation induced by 5 mM KCN was preceded by loss of mitochondrial integrity (MTT assay and rhodamine-123 staining) and nuclear viability (propidium iodide uptake) which were mediated by generation of reactive oxygen species (DCHF-DA staining). The DNA damage was also accompanied by nuclear fragmentation (Hoechst 33342 staining), a phenomenon that characterises the ‘apoptotic’ type of cell death. The in vitro toxic insult of KCN was challenged by pre-treatment (0.5 h), simultaneous treatment or post-treatment (0.5–3 h) of various pharmacological agents viz., Trolox® (antioxidant), EGTA (Ca 2+ modulator) and aurintricarboxylic acid (ATA; Ca 2+/Mg 2+-dependent endonuclease inhibitor). In addition, Quercetin (antioxidant) was tested as simultaneous treatment alone and was found to be ineffective. On the basis of various biochemical indices and DNA fragmentation (quantitative and qualitative), simultaneous treatment of Trolox® was found to be the most effective in attenuating cyanide toxicity in vitro. This protection can be attributed to interventions in oxidative stress-mediated cell injury which is an early event preceding DNA damage. Both EGTA and ATA could not prevent this damage. Trolox® also increased the LD 50 of KCN in mice 2.5-fold as compared to 1.8- and 1.6-fold for EGTA and ATA, respectively.