methylnitronitrosoguanidine and Necrosis

Alkylating DNA-damage agents such as N-methyl-N'-nitro-N'-nitrosoguanidine (MNNG) trigger necroptosis, a newly defined form of programmed cell death (PCD) managed by receptor interacting protein kinases. This caspase-independent mode of cell death involves the sequential activation of poly(ADP-ribose) polymerase-1 (PARP-1), calpains, BAX and AIF, which redistributes from mitochondria to the nucleus to promote chromatinolysis. We have previously demonstrated that the BAX-mediated mitochondrial release of AIF is a critical step in MNNG-mediated necroptosis. However, the mechanism regulating BAX activation in this PCD is poorly understood. Employing mouse embryonic knockout cells, we reveal that BID controls BAX activation in AIF-mediated necroptosis. Indeed, BID is a link between calpains and BAX in this mode of cell death. Therefore, even if PARP-1 and calpains are activated after MNNG treatment, BID genetic ablation abolishes both BAX activation and necroptosis. These PCD defects are reversed by reintroducing the BID-wt cDNA into the BID(-/-) cells. We also demonstrate that, after MNNG treatment, BID is directly processed into tBID by calpains. In this way, calpain non-cleavable BID proteins (BID-G70A or BID-Δ68-71) are unable to promote BAX activation and necroptosis. Once processed, tBID localizes in the mitochondria of MNNG-treated cells, where it can facilitate BAX activation and PCD. Altogether, our data reveal that, as in caspase-dependent apoptosis, BH3-only proteins are key regulators of caspase-independent necroptosis.

Topics: Animals; Apoptosis; Apoptosis Inducing Factor; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; bcl-Associated Death Protein; BH3 Interacting Domain Death Agonist Protein; Calpain; Caspases; Cell Survival; DNA Damage; Embryo, Mammalian; Fibroblasts; Lentivirus; Membrane Proteins; Methylnitronitrosoguanidine; Mice; Models, Biological; Necrosis; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins; Transduction, Genetic

Poly(ADP-ribose) polymerase-1 (PARP-1) is widely involved in cell death responses. Depending on the degree of injury and on cell type, PARP activation may lead to autophagy, apoptosis or necrosis. In HEK293 cells exposed to the alkylating agent N-methyl-N'-nitro-N'-nitrosoguanine (MNNG), we show that PARP-1 activation triggers a necrotic cell death response. The massive poly(ADP-ribose) (PAR) synthesis following PARP-1 activation leads to the modulation of mTORC1 pathway. Shortly after MNNG exposure, NAD⁺ and ATP levels decrease, while AMP levels drastically increase. We characterized at the molecular level the consequences of these altered nucleotide levels. First, AMP-activated protein kinase (AMPK) is activated and the mTORC1 pathway is inhibited by the phosphorylation of Raptor, in an attempt to preserve cellular energy. Phosphorylation of the mTORC1 target S6 is decreased as well as the phosphorylation of the mTORC2 component Rictor on Thr1135. Finally, Akt phosphorylation on Ser473 is lost and then, cell death by necrosis occurs. Inhibition of PARP-1 with the potent PARP inhibitor AG14361 prevents all of these events. Moreover, the antioxidant N-acetyl-L-cysteine (NAC) can also abrogate all the signaling events caused by MNNG exposure suggesting that reactive oxygen species (ROS) production is involved in PARP-1 activation and modulation of mTOR signaling. In this study, we show that PARP-1 activation and PAR synthesis affect the energetic status of cells, inhibit the mTORC1 signaling pathway and possibly modulate the mTORC2 complex affecting cell fate. These results provide new evidence that cell death by necrosis is orchestrated by the balance between several signaling pathways, and that PARP-1 and PAR take part in these events.

Topics: Adenosine Monophosphate; Adenosine Triphosphate; Alkylating Agents; AMP-Activated Protein Kinases; Autophagy; Blotting, Western; Enzyme Activation; HEK293 Cells; HeLa Cells; Humans; Methylnitronitrosoguanidine; Microtubule-Associated Proteins; Models, Biological; NAD; Necrosis; Poly (ADP-Ribose) Polymerase-1; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerases; RNA Interference; Signal Transduction; TOR Serine-Threonine Kinases

Programmed necrosis induced by DNA alkylating agents, such as MNNG, is a caspase-independent mode of cell death mediated by apoptosis-inducing factor (AIF). After poly(ADP-ribose) polymerase 1, calpain, and Bax activation, AIF moves from the mitochondria to the nucleus where it induces chromatinolysis and cell death. The mechanisms underlying the nuclear action of AIF are, however, largely unknown. We show here that, through its C-terminal proline-rich binding domain (PBD, residues 543-559), AIF associates in the nucleus with histone H2AX. This interaction regulates chromatinolysis and programmed necrosis by generating an active DNA-degrading complex with cyclophilin A (CypA). Deletion or directed mutagenesis in the AIF C-terminal PBD abolishes AIF/H2AX interaction and AIF-mediated chromatinolysis. H2AX genetic ablation or CypA downregulation confers resistance to programmed necrosis. AIF fails to induce chromatinolysis in H2AX or CypA-deficient nuclei. We also establish that H2AX is phosphorylated at Ser139 after MNNG treatment and that this phosphorylation is critical for caspase-independent programmed necrosis. Overall, our data shed new light in the mechanisms regulating programmed necrosis, elucidate a key nuclear partner of AIF, and uncover an AIF apoptogenic motif.

Topics: Animals; Apoptosis Inducing Factor; bcl-2-Associated X Protein; Calpain; Caspases; Cell Line; Chromatin; Cyclophilin A; DNA Damage; Down-Regulation; Fibroblasts; Gene Deletion; Histones; Methylnitronitrosoguanidine; Mice; Models, Molecular; Necrosis; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases

Oxidative cell death contributes to neuronal cell death in many neurological diseases such as stroke, brain trauma, and Alzheimer's disease. In this study, we explored the involvement of poly(ADP-ribose)-polymerase (PARP) in oxidative stress-induced necroptosis. We showed that PJ34, a potent and specific inhibitor of PARP, can completely inhibit glutamate-induced necroptosis in HT-22 cells. This protective effect was still observed 8h after glutamate exposure followed by PJ34 treatment. These results suggest that PARP activation plays a critical role in glutamate-induced necroptosis. We also examined the interaction between PARP and a necroptosis inhibitor called necrostatin-1 (Nec-1). Previously, we showed that Nec-1 protects against glutamate-induced oxytosis by inhibiting the translocation of cellular apoptosis-inducing factor (AIF), a downstream target of PARP-1 activation. In this study, Nec-1 reduced PARP activity but had no effect on the expression of PARP-1 in cells treated with glutamate. Nec-1 also did not protect against cell death mediated by the PARP activator N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), although PJ34 did protect against MNNG-mediated cell death. These findings suggest that Nec-1 is not a direct PARP inhibitor and that its signaling target is located upstream of PARP.

Topics: Animals; Apoptosis; Apoptosis Inducing Factor; Cell Line, Transformed; Enzyme Inhibitors; Glutamic Acid; Imidazoles; Indoles; Methylnitronitrosoguanidine; Mice; Necrosis; Nerve Degeneration; Neurotoxins; Oxidative Stress; Phenanthrenes; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases

Methylating agents are first-line therapeutics for gliomas and malignant melanomas. They attack DNA at various sites, and both O(6)-methylguanine and N-methylated base adducts contribute to the killing response. The mechanism of cellular defense against these agents primarily involves O(6)-methylguanine-DNA methyltransferase (MGMT) and base excision repair (BER). Here, we determined whether a key protein involved in DNA double-strand break (DSB) recognition and signaling, nibrin (NBN alias NBS-1), plays a role in the cellular defense against methylating agents. Comparing NBN mutated fibroblasts and lymphoblastoid cells from patients suffering from Nijmegen breakage syndrome, we show that NBN mutants are clearly more sensitive to N-methyl-N'-nitro-N-nitrosoguanidine and temozolomide than the corresponding wild-type cells. Hypersensitivity was due to the induction of both apoptosis and necrosis. The mismatch repair proteins MSH2, MSH6, MLH1, and PMS2 were expressed at a similar level in the cell lines and BER was not affected by NBN mutation. Because MGMT expression abrogated the hypersensitivity of NBN mutated cells, we conclude that O(6)-methylguanine-derived lesions are responsible for triggering the response. Down-regulation of NBN in melanoma cells by small interfering RNA rendered them more sensitive to temozolomide, suggesting that NBN is a novel modulator of temozolomide sensitivity. Because NBN is part of the MRN complex, which recognizes DSBs, the data strongly indicate that MRN is critically involved in DSB processing after O(6)-methylguanine induction. The data provide first evidence that NBN is involved in the cellular defense against O(6)-methylguanine-inducing agents such as temozolomide and identify NBN as a critical target of methylating anticancer drug resistance.

Topics: Antineoplastic Agents, Alkylating; Apoptosis; Caspase 7; Cell Cycle Proteins; Cell Line, Transformed; Cell Line, Tumor; Dacarbazine; DNA Methylation; Drug Resistance, Neoplasm; Fibroblasts; Guanine; Homozygote; Humans; Melanoma; Methylnitronitrosoguanidine; Mutagens; Mutation; Necrosis; Nijmegen Breakage Syndrome; Nuclear Proteins; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Temozolomide; Uveal Neoplasms

DNA fragmentation and nuclear condensation are important nuclear changes in apoptosis. In this study we determined whether DNA fragmentation and nuclear condensation occur in astrocytes treated with 100-200 microM of the genotoxic agent M-nitroso-N-nitroguanidine (MNNG). Our study also investigated the roles of Ca(2+)-Mg(2+)-dependent endonuclease (CME) in the MNNG-induced nuclear changes. We found that MNNG induced profound ATP depletion as well as marked nuclear condensation and DNA fragmentation in the cells. Both the nuclear condensation and the DNA fragmentation were abolished by posttreatment of the cells with the CME inhibitor aurintricarboxylic acid (ATA). The ATA posttreatment also significantly, but only partially, decreased MNNG-induced cell death. In contrast, pretreatment plus cotreatment with ATA did not affect either MNNG-induced nuclear condensation or cell death. Our study further suggests that ATA does not decrease the cytotoxicity of MNNG by directly inhibiting poly(ADP-ribose) polymerases. Collectively, our observations suggest that MNNG can induce both DNA fragmentation and nuclear condensation in astrocytes by a CME-dependent mechanism, which partially contributes to the genotoxic agent-induced cell death. Published 2008 Wiley-Liss, Inc.

Topics: Animals; Apoptosis; Astrocytes; Aurintricarboxylic Acid; Cell Nucleus; Cells, Cultured; DNA Damage; DNA Fragmentation; Endonucleases; Enzyme Inhibitors; Immunohistochemistry; Methylnitronitrosoguanidine; Mice; Necrosis; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases

Topics: Alkylating Agents; Animals; Apoptosis; Cell Death; Cell Line, Tumor; Enzyme Inhibitors; HeLa Cells; Humans; Methylnitronitrosoguanidine; Mice; Mitochondria; Models, Biological; Necrosis; Neurons; Poly(ADP-ribose) Polymerase Inhibitors

The goal of the current study, conducted in freshly isolated thymocytes was (1) to investigate the possibility that the activation of poly(ADP-ribose) polymerase-1 (PARP-1) in an intact cell can be regulated by protein kinase C (PKC) mediated phosphorylation and (2) to examine the consequence of this regulatory mechanism in the context of cell death induced by the genotoxic agent. In cells stimulated by the PKC activating phorbol esters, DNA breakage was unaffected, PARP-1 was phosphorylated, 1-methyl-3-nitro-1-nitrosoguanidine-induced PARP activation and cell necrosis were suppressed, with all these effects attenuated by the PKC inhibitors GF109203X or Gö6976. Inhibition of cellular PARP activity by PKC-mediated phosphorylation may provide a plausible mechanism for the previously observed cytoprotective effects of PKC activators.

Topics: Animals; Apoptosis; Carbazoles; Cytoprotection; DNA Damage; Enzyme Activation; Indoles; Maleimides; Methylnitronitrosoguanidine; Mice; Necrosis; Phorbol Esters; Phosphorylation; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Protein Kinase C; Protein Kinase Inhibitors; Thymus Gland

Aqueous extracts of Nigella sativa (Ranunculaceae) (Ns), Teucrium polium (Labiatae) (Tp) and Trigonella foenum-graecum (Fabaceae) (Tf) have been traditionally used to treat inflammations, liver disorders, and arthritis. Experimentally, it has been demonstrated that these herbs possess antioxidant, anti-inflammatory and hepatoprotective properties. To evaluate their in vitro toxicological properties and potential antimutagenic effects aqueous extracts of the three plants were tested in primary rat hepatocyte cultures against N-methyl-N'-nitro-N-nitrosoguanidine. The extracts were applied before, during and after application of MNNG to discriminate between different mechanisms of action. Tp itself significantly increased apoptosis, but in the combined treatment with MNNG significantly reduced it. Post-treatment with Ns or combined treatment with Tf significantly reduced the percentages of necrotic cells. The three plant extracts themselves significantly increased the frequency of chromosomal aberrations. Summarizing, our results suggest that aqueous extracts of the three herbs have neither cytoprotective nor antimutagenic activity, instead there is evidence for a mutagenic potential.

Topics: Alkylating Agents; Animals; Antimutagenic Agents; Apoptosis; Cells, Cultured; Chromosome Aberrations; Cytoprotection; Dose-Response Relationship, Drug; Female; Hepatocytes; Methylnitronitrosoguanidine; Mutagenicity Tests; Necrosis; Nigella sativa; Plant Extracts; Plants, Medicinal; Rats; Rats, Inbred F344; Solvents; Teucrium; Trigonella; Water

Alkylating DNA damage induces a necrotic type of programmed cell death through the poly(ADP-ribose) polymerases (PARP) and apoptosis-inducing factor (AIF). Following PARP activation, AIF is released from mitochondria and translocates to the nucleus, where it causes chromatin condensation and DNA fragmentation. By employing a large panel of gene knockout cells, we identified and describe here two essential molecular links between PARP and AIF: calpains and Bax. Alkylating DNA damage initiated a p53-independent form of death involving PARP-1 but not PARP-2. Once activated, PARP-1 mediated mitochondrial AIF release and necrosis through a mechanism requiring calpains but not cathepsins or caspases. Importantly, single ablation of the proapoptotic Bcl-2 family member Bax, but not Bak, prevented both AIF release and alkylating DNA damage-induced death. Thus, Bax is indispensable for this type of necrosis. Our data also revealed that Bcl-2 regulates N-methyl-N'-nitro-N'-nitrosoguanidine-induced necrosis. Finally, we established the molecular ordering of PARP-1, calpains, Bax, and AIF activation, and we showed that AIF downregulation confers resistance to alkylating DNA damage-induced necrosis. Our data shed new light on the mechanisms regulating AIF-dependent necrosis and support the notion that, like apoptosis, necrosis could be a highly regulated cell death program.

Topics: Alkylating Agents; Alkylation; Animals; Apoptosis Inducing Factor; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Calpain; Caspases; Cell Death; Cell Nucleus; Cytosol; DNA; Enzyme Activation; Fibroblasts; Methylnitronitrosoguanidine; Mice; Mitochondria; Models, Biological; Necrosis; Poly(ADP-ribose) Polymerases; Protein Transport; Tumor Suppressor Protein p53

It has been previously reported that a short treatment of human cervix carcinoma HeLa cells with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induced apoptosis. We examined the action of MNNG on HeLa cells and compared it with that of cisplatin. MNNG damaged the integrity of the cell membrane and killed the cells within 3 hours. During this period no changes characteristic for apoptosis, such as cell shrinkage, condensation of nuclei, chromatin fragmentation or activation of caspases, could be detected. However, the exposure of HeLa cells to 50 micro M MNNG for 1 h resulted in dramatic DNA damage. The MNNG-induced disruption of cell membrane associated with cell death indicates that HeLa cells die by necrosis.

Topics: Antineoplastic Agents; Apoptosis; Cisplatin; Comet Assay; DNA Damage; Female; HeLa Cells; Humans; Kinetics; Methylnitronitrosoguanidine; Necrosis

A rat colonic adenocarcinoma was implanted subcutaneously (s.c.) in nude mice. After 7 days, the animals were divided into different groups. Two groups received subcutaneous injections twice daily with 3 or 6 micro g/kg body weight octreotide, galanin and serotonin. Three groups were respectively treated with 20, 30, and 40 micro g/kg body weight of the previously mentioned bioactive substances. Control group received only saline solution in the same fashion as treated animals. The treatment lasted for 5 days. The tumour volume and weight, the relative density of blood vessels, of tumour necrotic tissue, of apoptotic nuclei and of proliferating nuclei were measured. Apoptosis was detected by in situ labelling of nuclear DNA fragmentation according to TUNEL method, and proliferation by immunocytochemistry. Morphometry was done with the classical stereological point-counting method. Food consumption, animal weight, faeces weight and its water content were measured for 3 days before and after treatment. Triple therapy with 3 and 6 micro g/kg body weight had no effect on any of the parameters measured, except in reducing the relative volume density of tumour blood vessels. Treatment with 20, 30 and 40 micro g/kg body weight of the previously mentioned bioactive substances reduced the tumour volume, the relative volume density of blood vessels and increased the relative volume density of necrotic tissue and of apoptotic nuclei (in the 20 micro g group). However, there was no difference between treated mice and controls regarding the relative volume density of proliferating nuclei. There was no statistical difference between treated animals regarding food consumption, body weight, faeces weight and its water content before and during treatment. The present study confirms that triple therapy with octreotide, galanin and serotonin causes regression of a rat colon carcinoma. It further showed that optimum treatment dose is 20 micro g/kg body weight of each bioactive substance. Moreover, this therapy regime does not show apparent side effects in the experiments carried out on mice.

Topics: Adenocarcinoma; Animals; Apoptosis; Blood Vessels; Carcinogens; Colonic Neoplasms; Drug Therapy, Combination; Galanin; Methylnitronitrosoguanidine; Mice; Mice, Nude; Necrosis; Octreotide; Rats; Serotonin; Transplantation, Heterologous

In response to high levels of DNA damage, catalytic activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) triggers necrotic death because of rapid consumption of its substrate beta-nicotinamide adenine dinucleotide and consequent depletion of ATP. We examined whether there are other consequences of PARP activation that could contribute to cell death. Here, we show that PARP activation reaction in vitro becomes acidic with release of protons during hydrolysis of beta-nicotinamide adenine dinucleotide. In the cellular context, we show that Molt 3 cells respond to DNA damage by the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) with a dose-dependent acidification within 30 min. Whereas acidification by 0.15 pH units induced by 10 microM MNNG is reversed within 1 h, 100 , microinduced acidification by 0.5-0.6 pH units is persistent up to 7 h. Acidification is a general DNA damage response because H(2)O(2) exposure also acidifies Molt 3 cells, and MNNG causes acidification in Jurkat, U937, or HL-60 leukemia cells and in PARP(+/+) fibroblasts. Acidification is significantly decreased in the presence of PARP inhibitors or in PARP(-/-) fibroblasts, suggesting a major role for PARP activation in acidification. Inhibition of proton export through ATP-dependent Na(+)/H(+) exchanger is another major cause of acidification. Using the pH clamp method to either suppress or introduce changes in cellular pH, we show that brief acidification by 0.5-0.6 pH units may be a negative regulator of apoptosis while permitting necrotic death of cells with extensively damaged DNA.

Topics: Adenosine Triphosphate; Alkylating Agents; Apoptosis; Caspase 3; Caspases; Cell Death; Cells, Cultured; DNA Damage; Dose-Response Relationship, Drug; HL-60 Cells; Humans; Hydrogen-Ion Concentration; Immunoblotting; Jurkat Cells; Methylnitronitrosoguanidine; NAD; Necrosis; Poly(ADP-ribose) Polymerases; Time Factors; U937 Cells

Gastrointestinal cancer is the most important clinical target of gene therapy. Suicide gene therapy, such as with the herpes simplex virus type 1 thymidine kinase (HSV-TK) gene, has been shown to exert antitumor efficacy in various cancer models in vitro. We previously reported in situ gene transfer and gene therapy for gastric cancer induced by N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) in dogs. Here, we describe the sequential histopathological changes after suicide gene therapy of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced gastric cancer in rats. Gastric tumors were induced by MNNG in 38 / 73 (52%) of Wistar strain rats. The suicide gene therapy group (14 rats) was subjected to in situ gene transfer with a recombinant adenovirus vector carrying the HSV-TK gene driven by CAG promoter (Ad.CAGHSV-TK) in gastric tumor, followed by the antiviral drug ganciclovir (GCV). To observe the histopathological changes at various times after HSV-TK / GCV gene therapy, groups of animals were sacrificed at 3, 8, and 30 days after gene transfer. Apoptosis in the gastric tumors was detected by the TUNEL method to assess the efficacy of HSV-TK / GCV gene therapy, and it was marked in the 8- and 30-day treatment groups compared to the sham operation controls (P < 0.001). Various histopathological changes, degeneration of cancer tissue and fibrosis after necrosis and apoptosis were significantly greater in the 30-day treatment group. The HSV-TK gene was detectable in peripheral blood by PCR until 30 days after gene transfer. These results may be useful in devising a method of suicide gene therapy for humans.

Topics: Adenoviridae; Animals; Antiviral Agents; Apoptosis; Carcinogens; Cell Nucleus; Cytoplasm; Fibrosis; Ganciclovir; Gene Transfer Techniques; Genetic Therapy; Herpesvirus 1, Human; In Situ Nick-End Labeling; Male; Methylnitronitrosoguanidine; Necrosis; Polymerase Chain Reaction; Promoter Regions, Genetic; Rats; Rats, Wistar; Stomach Neoplasms; Thymidine Kinase; Time Factors

DNA polymerase beta (beta-pol), which is involved in base excision repair, was investigated for its role in protection of cells against various genotoxic agents and cytostatic drugs using beta-pol knockout mouse fibroblasts. We show that cells lacking beta-pol are highly sensitive to induction of apoptosis and chromosomal breakage by methylating agents, such as N-methyl-N'-nitro-N-nitrosoguanidine and methyl methanesulfonate and the cross-linking antineoplastic drugs mitomycin C and mafosfamide. The cross-sensitivity between the agents observed suggests that beta-pol is involved in repair not only of DNA methylation lesions but also of other kinds of DNA damage induced by various cytostatic drugs. Cells deficient in beta-pol were not hypersensitive to cisplatin, melphalan, benzo(a)pyrene diol epoxide, chloroethylnitrosourea, or UV light. Because both established and primary beta-pol knockout fibroblasts displayed the hypersensitive phenotype, which, moreover, was complemented by transfection with a beta-pol expression vector, the alkylating agent hypersensitivity can clearly be attributed to the beta-pol deficiency. The results demonstrate that beta-pol-driven base excision repair is highly important for protection of cells against cell killing due to apoptosis and induced chromosomal breakage and suggest that incompletely repaired DNA damage causes chromosomal changes and may act as a trigger of DNA damage-induced apoptosis.

Topics: Alkylating Agents; Animals; Apoptosis; Chromosome Aberrations; DNA Damage; DNA Polymerase beta; DNA Repair; Methyl Methanesulfonate; Methylnitronitrosoguanidine; Mice; Mice, Knockout; Necrosis

Bcl-xL is the predominant anti-apoptotic Bcl-2 family member in the liver. Suppression of cell death promotes carcinogenesis and contributes to resistance to radiation and chemotherapeutic agents.. Direct effects of Bcl-xL protein on apoptosis and necrosis were investigated in rat hepatoma cells. Rat hepatoma cell line McA-RH8994 cells were transfected with expression plasmids containing a whole coding sequence of rat bcl-xL cDNA of sense orientation. Stable transfectant cell lines expressing bcl-xL cDNA (designated as RH8994/Bcl-xL-S), or control plasmid DNA (designated as RH8994/pT) were established.. Cellular amounts of Bcl-xL in RH8994/Bcl-xL-S cells were demonstrated to be more than 20-fold that of RH8994/pT and parental cells. Three independent clones of RH8994/Bcl-xL-S were isolated and their susceptibility to various cell death stimuli was compared with that of the control cells. Transforming growth factor-beta1 and tumour necrosis factor-alpha induced apoptosis dose dependently in these cells, but the 50% cytotoxicity concentrations of these factors in RH8994/Bcl-xL-S cells were more than 10-fold higher than those in RH8994/pT and parental cells. Similarly, RH8994/Bcl-xL-S cells were shown to be significantly less susceptible to necrotic cell death induced by a calcium ionophore, A23187; a mutagen, N-methyl-N'-nitro-N-nitrosoguanidine; and UV-irradiation when compared with the control cells.. Over-expression of Bcl-xL was shown to provide protection against apoptotic and necrotic cell death in rat hepatoma cells.

Topics: Animals; Apoptosis; bcl-X Protein; Calcimycin; Cell Death; Gene Expression; Ionophores; Liver Neoplasms, Experimental; Methylnitronitrosoguanidine; Mutagens; Necrosis; Proto-Oncogene Proteins c-bcl-2; Rats; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Ultraviolet Rays

Apoptotic and necrotic cell death are well characterized and are influenced by intracellular ATP levels. Poly(ADP-ribose) polymerase (PARP), a nuclear enzyme activated by DNA strand breaks, physiologically participates in DNA repair. Overactivation of PARP after cellular insults can lead to cell death caused by depletion of the enzyme's substrate beta-nicotinamide adenine dinucleotide and of ATP. In this study, we have differentially elicited apoptosis or necrosis in mouse fibroblasts. Fibroblasts from PARP-deficient (PARP(-/-)) mice are protected from necrotic cell death and ATP depletion but not from apoptotic death. These findings, together with cell death patterns in PARP(-/-) animals receiving other types of insults, indicate that PARP activation is an active trigger of necrosis, whereas other mechanisms mediate apoptosis.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Cell Death; Enzyme Activation; fas Receptor; Intracellular Fluid; Methylnitronitrosoguanidine; Mice; Mice, Knockout; Necrosis; Poly(ADP-ribose) Polymerases

2-Chloro-4-methylthiobutanoic acid (CMBA, a mutagen from Japanese salted fish) at 15-500 mg/kg body weight induced several-fold increase in replicative DNA synthesis (RDS) (P < 0.05) after 80 min and 17 h, equivocal unscheduled DNA synthesis (UDS) after 80 min and necrosis 80 min after its administration in the stomach pyloric mucosa of F344 and ACI male rats. A positive control, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 50 mg/kg body weight), induced RDS, UDS and erosion. However, the negative control L-methionine (500 mg/kg body weight) did not display any effect. The results suggest possible tumor-initiating and -promoting activity of CMBA but at a lower potency than that of MNNG.

Topics: Animals; Biological Assay; Butyrates; Carcinogens; Disease Susceptibility; DNA; DNA Damage; DNA Repair; Gastric Fundus; Gastric Mucosa; Male; Methylnitronitrosoguanidine; Mutagens; Necrosis; Pylorus; Rats; Rats, Inbred ACI; Rats, Inbred F344; Sulfhydryl Compounds

N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces fetal asymmetric limb malformations with exposure of pregnant mice to 50 mg/kg on day 11 of gestation. Hindlimbs were more frequently malformed than forelimbs, and a fourfold greater incidence of postaxial ectrodactyly was found in left forelimbs than in right forelimbs, and a two fold excess in left hindlimbs compared to right hindlimbs. The level of cell death and mitotic index were measured in forelimbs and hindlimbs from treated and control embryos at 1, 4, 18, 24, 48, and 72 hr after exposure to ascertain if these parameters could be correlated with the differential teratogenic susceptibility of the limbs. An increase in necrotic index was first detected in treated limbs at 4 hr, increased at 18 hr, peaked at 24 hr, and began declining at 48 hr to reach the control baseline at 72 hr. At 24 hr, the correlation between the level of cell death and susceptibility of malformation was the strongest, with the left hindlimb having a necrotic index of 58%, the right hindlimb 47%, the left forelimb 30% and the right forelimb 12%. In both forelimbs and hindlimbs, MNNG treatment initially depressed mitotic activity followed by an elevation at 48 hr relative to controls. The magnitude of the depression, extent of the elevation, and overall pattern of mitotic activity could not be uniformly related to limb defects. These results indicate that the amount of cell death in limb buds at 24 hr after MNNG exposure may predict target organ susceptibility. Depressions in mitotic activity and alterations in the pattern of mitosis were also observed which were not as clearly correlated with the incidence of malformations as was the amount of cell death.

Topics: Animals; Cell Survival; Extremities; Female; Fetus; Limb Deformities, Congenital; Methylnitronitrosoguanidine; Mice; Mice, Inbred ICR; Mitosis; Necrosis; Pregnancy; Teratogens

MNNG is a strong topically acting carcinogen. Various single oral doses (150, 120, 60 mg/kg) were investigated regarding their effect on the mitotic activity of epithelial tissues in male Sprague-Dawley rats. MNNG inhibits cell proliferation in the forestomach, jejunum and esophagus. Necroses were observed in the forestomach and in the liver (150 mg/kg). Independent of any tissue lesions MNNG produces an elevated mitotic activity of the hepatocytes and in the adrenal cortex. These proliferative effects are not observed when the structurally related noncarcinogenic compounds guanidine carbonate (GC) and guanidine nitrate (GN) are used.

Topics: Administration, Oral; Adrenal Cortex; Animals; Body Weight; Cell Division; Esophagus; Guanidines; Jejunum; Liver; Male; Methylnitronitrosoguanidine; Mitosis; Mitotic Index; Necrosis; Rats; Rats, Inbred Strains; Stomach