The influence of thymine and 5-bromouracil on the sensitivity ofEscherichia coli to alkylation,UV and X-irradiation

The influence is followed of an alkylating agent (triethylene-melamine) UV and X-irradiation on the survival ofEscherichia coli 15 T∋ells grown in a minimal medium containing enzymatic hydrolysate of caseine. Thymine-less death of a considerable number of cells was observed in a culture grown in this medium. A conclusive difference in the sensitivity to the lethal agents used was found between a culture grown in a thymine-less medium and bacteria grown in a medium containing excess (20 μg/ml) of thymine. The culture grown with a sufficient thymine concentration was more sensitive to alkylation and X-rays, whereas bacteria surviving conditions of thymine-less death were more resistant to the above agents. However, such cells were more sensitive to UV-irradiation. The differences found are discussed from the point of view of DNA concentrations found in the individual cultures.     

Reversible arrest of Chinese hamster V79 cells in G2 by dibutytyl AMP.

Mouse L cells 929 were cloned in supplemented Eagle's minimal medium enriched with lactalbumin and yeast extract and buffered with HEPES. Multiplication was followed photographically in single clones from the 8-cell stage through 6–7 days. Addition of the folic acid analogue methotrexate (amethopterin) in 5 × 10^?6 M concentration slowed growth only after two cell generations; 10^?4 M uridine had no effect on growth except when combined with methotrexate. The two agents together blocked cell division quickly and symptoms of thymine-less death developed in few days. The cells could be rescued before 48 h by removal of the inhibitors, or by addition of folic acid or thymidine. The combination of methotrexate with uridine blocks DNA synthesis in Tetrahymena by inhibition of thymidylate synthesis and of thymidine uptake from the complex medium. Apparently the same mechanisms operate in L cells grown in a complex medium containing thymidine.     

dUTP pyrophosphatase is an essential enzyme in Saccharomyces cerevisiae.

dUTP pyrophosphatase (dUTPase; EC 3.6.1.23) catalyses the hydrolysis of dUTP to dUMP and PPi and thereby prevents the incorporation of uracil into DNA during replication. Although it is widely believed that dUTPase is essential for cell viability because of this role, direct evidence supporting this assumption has not been presented for any eukaryotic system. We have analysed the role of dUTPase (DUT1) in the life cycle of yeast. Using gene disruption and tetrad analysis, we find that DUT1 is necessary for the viability of S. cerevisiae; however, under certain conditions dut1 null mutants survive if supplied with exogenous thymidylate (dTMP). Analyses with isogenic uracil-DNA-glycosylase (UNG1) deficient or proficient strains indicate that in the absence of dUTPase, cell death results from the incorporation of uracil into DNA and the attempted repair of this damage by UNG1-mediated excision repair. However, in dut1 ung1 double mutants, starvation for dTMP causes dividing cells to arrest and die in all phases of the cell cycle. This latter effect suggests that the extensive stable substitution of uracil for thymine in DNA leads to a general failure in macromolecular synthesis. These results are in general agreement with previous models in thymine-less death that implicate dUTP metabolism. They also suggest an alternative approach for chemotherapeutic drug design.     

Mechanistic studies of dUTPases

The essential enzyme dUTPase is responsible for preventive DNA repair via exclusion of uracil. Lack or inhibition of the enzyme induces thymine-less cell death in cells performing active DNA synthesis, serving therefore as an important chemotherapeutic target. In the present work, employing differential circular dichroism spectroscopy, we show that D. mel. dUTPase, a recently described eukaryotic model, has a similar affinity of binding towards alpha,beta-imino-dUTP as compared to the prokaryotic E. coli enzyme. However, in contrast to the prokaryotic dUTPase, the nucleotide exerts significant protection against tryptic digestion at a specific tryptic site 20 A far from the active site in the fly enzyme. This result indicates that binding of the nucleotide in the active site induces an allosteric conformational change within the central threefold channel of the homotrimer exclusively in the eukaryotic enzyme. Nucleotide binding induced allosterism in the D. mel. dUTPase, but not in the E. coli enzyme, might be associated with the altered hydropathy of subunit interfaces in these two proteins.     

Limited TTP supply affects telomere length regulation in a telomerase-independent fashion

An adequate supply of nucleotides is essential for DNA replication and DNA repair. Moreover, inhibition of TTP synthesis can cause cell death by a poorly characterized mechanism called thymine-less death. In the yeast Saccharomyces cerevisiae, the genes encoding thymidylate synthetase (CDC21) and thymidylate kinase (CDC8) are both essential for de novo TTP synthesis. The effects of temperature-sensitive mutations in these genes have been characterized and, curiously, the phenotypes displayed by cells harboring them include shortened telomeric repeat tracts. This finding raised the possibility that the enzyme telomerase is very sensitive to TTP-pools. We tested this possibility in vivo by assessing telomerase-dependent extension in situations of lowered TTP supply. The results show that the above-mentioned short telomere phenotype is not a consequence of an inability of telomerase to elongate telomeres when TTP synthesis is impaired. Moreover, this telomere shortening was abolished in cells harboring a mutation in DNA polymerase α. Previously, this same mutation was shown to affect the coordination between conventional replication and telomerase-mediated extension. These results thus re-emphasize the importance of the interplay between conventional replication and telomerase-mediated addition of telomeric repeats in telomere replication.     

Novel Developments in the Use of Antimetabolites

Antimetabolites are the most widely used and most efficacious group of anticancer drugs. Antimetabolites are also the oldest rationally designed anticancer drugs, targeted against RNA and DNA, and can, therefore, be considered as the first generation of targeted drugs. Unfortunately, resistance often develops, leading to the design of new antimetabolites, which either have a novel mechanism of action, bypass resistance or in combination enhance the effect of other drugs, such as another antimetabolite, other DNA, or protein kinase targeted anticancer drugs. Several novel antimetabolites are in clinical development. The cytidine-analog fluorocyclopentenylcytosine (RX-3117) is active in gemcitabine-resistant tumors and is activated by uridine-cytidine-kinase, can be incorporated into RNA and DNA and can downregulate DNA-methyltransferase-1. TAS-114 is a new generation dUTPase inhibitor. dUTPase normally prevents incorporation of dUTP and of the 5FU-nucleotide FdUTP into DNA. However, inhibition of dUTPase will enhance their incorporation, thereby increasing thymine-less cell-death. The formulation TAS-102 (trifluorothymidine and thymidine-phosphorylase-inhibitor) acts by incorporation into DNA and has shown efficacy in tumors progressing on 5FU therapy. Gemcitabine and cytarabine prodrugs were tested in model systems and have entered clinical evaluation. The elaidic-acid prodrugs of gemcitabine (CP-4126, CO101) and cytarabine (elacytarabine) failed in randomized Phase III studies. Two other gemcitabine prodrugs LY2334737 (gemcitabine with a valproic acid at the 5′-position) and NUC1031 (a 5′-arylphosphoamidate prodrug, with a side-chain at the 5′-phosphate) are in early clinical development.

In summary, several novel antimetabolites show promise in clinical development, either because of a novel mechanism of action, or clever combination or by innovative prodrug design.     

Altered active site flexibility and a structural metal-binding site in eukaryotic dUTPase: kinetic characterization, folding, and crystallographic studies of the homotrimeric Drosophila enzyme

dUTPase is responsible for preventive DNA repair via exclusion of uracil. Developmental regulation of the Drosophila enzyme is suggested to be involved in thymine-less apoptosis. Here we show that in addition to conserved dUTPase sequence motifs, the gene of Drosophila enzyme codes for a unique Ala-Pro-rich segment. Kinetic and structural analyses of the recombinant protein and a truncation mutant show that the Ala-Pro segment is flexible and has no regulatory role in vitro. The homotrimer enzyme unfolds reversibly as a trimeric entity with a melting temperature of 54 degrees C, 23 degrees C lower than Escherichia coli dUTPase. In contrast to the bacterial enzyme, Mg(2+) binding modulates conformation of fly dUTPase, as identified by spectroscopy and by increment in melting temperature. A single well folded, but inactive, homotrimeric core domain is generated through three distinct steps of limited trypsinolysis. In fly, but not in bacterial dUTPase, binding of the product dUMP induces protection against proteolysis at the tryptic site reflecting formation of the catalytically competent closed conformer. Crystallographic analysis argues for the presence of a stable monomer of Drosophila dUTPase in crystal phase. The significant differences between prototypes of eukaryotic and prokaryotic dUTPases with respect to conformational flexibility of the active site, substrate specificity, metal ion binding, and oligomerization in the crystal phase are consistent with alteration of the catalytic mechanism and hydropathy of subunit interfaces.     

Reactive oxygen species induce different cell death mechanisms in cultured neurons

Apoptosis is characterized by chromatin condensation, phosphatidylserine translocation, and caspase activation. Neuronal apoptotic death involves the participation of reactive oxygen species (ROS), which have also been implicated in necrotic cell death. In this study we evaluated the role of different ROS in neuronal death. Superoxide anion was produced by incubating cells with xanthine and xanthine oxidase plus catalase, singlet oxygen was generated with rose Bengal and luminic stimuli, and hydrogen peroxide was induced with the glucose and glucose oxidase. Cultured cerebellar granule neurons died with the characteristics of apoptotic death in the presence of superoxide anion or singlet oxygen. These two conditions induced caspase activation, nuclear condensation, phosphatidylserine translocation, and a decrease in intracellular calcium levels. On the other hand, hydrogen peroxide led to a necrosis-like cell death that did not induce caspase activation, phosphatidylserine translocation, or changes in calcium levels. Cell death produced by both singlet oxygen and superoxide anion, but not hydrogen peroxide, was partially reduced by an increase in intracellular calcium levels. These results suggest that formation of specific ROS can lead to different molecular cell death mechanisms (necrosis and apoptosis) and that ROS formed under different conditions could act as initiators or executioners on neuronal death.     

Neuronal death is an active, caspase-dependent process after moderate but not severe DNA damage

Mild insults to neurons caused by ischemia or glutamate induce apoptosis, whereas severe insults induce non apoptotic death, such as necrosis. The molecular targets that are damaged by these insults and ultimately induce cell death are not fully established. To determine if DNA damage can induce apoptotic or non apoptotic death depending on the severity, neurons were treated with up to 128 Gy of ionizing radiation. Such treatment induced a dose-related increase in DNA single-strand breaks but no immediate membrane disruption or lipid peroxidation. Following moderate doses of < or = 32 Gy, neuronal death had many characteristics of apoptosis including nuclear fragmentation and DNA laddering. Nuclear fragmentation and membrane breakdown after moderate DNA damage could be blocked by inhibition of active protein synthesis with cycloheximide and by inhibition of caspases. In contrast, cell death after doses of > 32 Gy was not blocked by cycloheximide or caspase inhibitors, and membrane breakdown occurred relatively early in the cell death process. These data suggest that cell death after high dose irradiation and severe DNA damage can occur by non apoptotic mechanisms and that blocking apoptotic pathways may not prevent death after severe damage.     

Neurotrophic Factor Effects on Oxidative Stress–Induced Neuronal Death

Neurotrophic factors have been shown to potentiate necrotic neuronal death in cortical cultures. In this study we characterized the death induced by various oxidative insults and tested the effects of neurotrophic factors on that death. Treatment with fibroblast growth factor-2, neurotrophin-4, or insulin-like growth factor-1 potentiated neuronal cell death induced by iron-citrate (Fe) or buthionine sulfoximine (BSO), but not ethacrynic acid (EA). Neuronal death induced by each insult was blocked by the free radical scavenger, trolox. An analysis of the death indicated that Fe and BSO induced necrotic cell death, while EA induced apoptotic cell death. BSO and EA caused decreased cellular glutathione levels, whereas Fe had no effect on glutathione levels. Neurotrophic factors had no effect on the changes in glutathione. The results indicate that oxidative insults can induce either apoptotic or necrotic death and that the effects of neurotrophic factors are dependent on the type of cell death.     

The release of DNA into the plasma of mice following hepatic cell death by apoptosis and necrosis

The goal of these investigations was to measure levels of DNA in the plasma of mice following administration of hepatotoxic agents to induce apoptotic or necrotic cell death and determine any differences in the release of this marker depending upon death pathway. For this purpose, the effects of varying doses of anti-Fas, acetaminophen (APAP) or carbon tetrachloride (CCl₄) were assessed in normal mice. Plasma DNA was measured fluorometrically by the dye PicoGreen while lactate dehydrogenase (LDH) and caspase 3, other molecules released with cell injury or death, were measured by enzymatic assays. Histology was used to assess the occurrence of apoptosis or necrosis. Results of these experiments indicate that increased blood DNA levels occurred with all three agents and were highest with anti-Fas and CCl₄; caspase 3 levels were much higher with anti-Fas than the other agents. Histological examination confirmed the predominance of apoptotic death with anti-Fas and necrotic death with APAP and CCl₄. These results indicate that increased blood DNA is common in hepatotoxic injury and is a feature of both apoptotic and necrotic death.     

A novel caspase-1/toll-like receptor 4-independent pathway of cell death induced by cytosolic Shigella in infected macrophages

Shigella-induced macrophage cell death is an important step in the induction of acute inflammatory responses that ultimately lead to bacillary dysentery. Cell death was previously reported to be dependent upon the activation of caspase-1 via interaction with IpaB secreted by intracellular Shigella, but in this study, we show that Shigella infection of macrophages can also induce cell death independent of caspase-1 or IpaB activity. Time-lapse imaging and electron microscopic analyses indicated that caspase-1-dependent and -independent cell death is morphologically indistinguishable and that both resemble necrosis. Analyses of Shigella mutants or Escherichia coli using co-infection with Listeria suggested that a component common to Gram-negative bacteria is involved in inducing caspase-1-independent cell death. Further studies revealed that translocation of bacterial lipid A into the cytosol of macrophages potentially mediates cell death. Notably, cell death induced by cytosolic bacteria was TLR4-independent. These results identify a novel cell death pathway induced by intracellular Gram-negative bacteria that may play a role in microbial-host interactions and inflammatory responses.     

Fumonisin blunts nitric oxide-induced and nitroprusside-induced cardiomyocyte death.

The objective of this study was to determine whether nitric oxide (NO)-induced cell death in cardiomyocytes was operative through de novo synthesis of ceramide by determining whether the ceramide synthase inhibitor fumonisin blocked NO-mediated cell death. Neonatal mouse cardiomyocytes in culture were pretreated with fumonisin B1 (FB1). FB1 is a competitive inhibitor of sphinganine N-acyl transferase, also known as ceramide synthase (EC 2.3.1.24). Cell viability was assessed by the (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, which is based on the ability of viable cells to reduce MTT. Treatment with the NO donor nitroso-glutathione (NO-GSH) for 24h produced a significant (p<0.05) concentration-dependent reduction in OD(570) or an increase in cell death. Sodium nitroprusside (SNP) treatment for 24h produced a significant (p<0.001) concentration-dependent reduction in OD(570) and an increase in cardiomyocyte cell death but the effects of SNP were greater than those of NO-GSH. FB1 significantly (p<0.05) reduced cell death induced by either SNP or NO-GSH. The SNP (0.1mM) increase in cell death of 36.9+/-2.8% was significantly (p<0.05) reduced to 24.7+/-1.8% by FB1 (10 microM). The effect of FB1 was not mediated through inhibition of the cell death effects of H(2)O(2), which is produced by SNP, as FB1 did not prevent H(2)O(2)-induced cell death. Confirmation of the ability of ceramide to produce cell death was demonstrated by the cell-permeable ceramide analogue, C(2)-ceramide (100 and 200 microM), which induced, respectively, 23.4+/-11.3 and 78.0+/-3.7% increases in cell death. The cell death effects of SNP and NO-GSH are likely independent of cGMP signal transduction pathways, which are activated by either SNP or NO-GSH, as there was no significant concentration-dependent change in cardiomyocyte viability after treatment with the cell-permeable analogue dibutyryl-GMP. These data show that FB1 blunts SNP- and NO-induced cardiomyocyte death and raise the novel possibility of preventing some of SNP- or NO-induced cardiomyocyte cell death by ceramide synthase inhibition.     

Radiosensitizing and cytocidal effects of misonidazole: evidence for separate modes of action

Hypoxic BP-8 murine sarcoma cells were exposed to misonidazole and/or radiation and the kinetics and extent of cell death were evaluated with the [125I]iododeoxyuridine-prelabeling assay. Cell death after treatment with lethal doses of misonidazole was rapid and essentially complete within 2 or 3 days after drug exposure. In contrast, radiation death became apparent only after a delay period of 4 days and was complete by Day 10 after irradiation. Radiosensitization by short exposures to sublethal doses of misonidazole affected only the delayed component of cell death, that is, the radiation component of death. In experiments involving sequential radiation and drug treatment, prior irradiation of cells did not enhance the direct cytocidal effects of misonidazole, as evidenced by the fact that the early component of cell death was equal in control and preirradiated cells. However, postirradiation treatment with misonidazole did enhance the delayed radiation component of cell death. These results suggest that radiosensitization and direct killing by misonidazole are two distinct phenomena mediated by different cellular mechanisms, and radiosensitization by misonidazole represents a two-component effect composed of true dose modification and dose additive damage interactions, but these additive effects must occur at a site different from the cellular structure responsible for direct drug-induced cell death.     

Mitochondrial dysfunction is involved in P2X7 receptor-mediated neuronal cell death

J. Neurochem. (2012) 122, 1118-1128. ABSTRACT: P2X7 receptor (P2X7R) is known to be a 'death receptor' in immune cells, but its functional expression in non-immune cells such as neurons is controversial. Here, we examined the involvement of P2X7R activation and mitochondrial dysfunction in ATP-induced neuronal death in cultured cortical neurons. In P2X7R- and pannexin-1-expressing neuron cultures, 5 or more mM ATP or 0.1 or more mM BzATP induced neuronal death including apoptosis, and cell death was prevented by oxATP, P2X7R-selective antagonists. ATP-treated neurons exhibited Ca(2+) entry and YO-PRO-1 uptake, the former being inhibited by oxATP and A438079, and the latter by oxATP and carbenoxolone, while P2X7R antagonism with oxATP, but not pannexin-1 blocking with carbenoxolone, prevented the ATP-induced neuronal death. The ATP treatment induced reactive oxygen species generation through activation of NADPH oxidase and activated poly(ADP-ribose) polymerase, but both of them made no or negligible contribution to the neuronal death. Rhodamine123 efflux from neuronal mitochondria was increased by the ATP-treatment and was inhibited by oxATP, and a mitochondrial permeability transition pore inhibitor, cyclosporine A, significantly decreased the ATP-induced neuronal death. In ATP-treated neurons, the cleavage of pro-caspase-3 was increased, and caspase inhibitors, Q-VD-OPh and Z-DEVD-FMK, inhibited the neuronal death. The cleavage of apoptosis-inducing factor was increased, and calpain inhibitors, MDL28170 and PD151746, inhibited the neuronal death. These findings suggested that P2X7R was functionally expressed by cortical neuron cultures, and its activation-triggered Ca(2+) entry and mitochondrial dysfunction played important roles in the ATP-induced neuronal death.     

The release of DNA into the plasma of mice following hepatic cell death by apoptosis and necrosis

The goal of these investigations was to measure levels of DNA in the plasma of mice following administration of hepatotoxic agents to induce apoptotic or necrotic cell death and determine any differences in the release of this marker depending upon death pathway. For this purpose, the effects of varying doses of anti-Fas, acetaminophen (APAP) or carbon tetrachloride (CCl₄) were assessed in normal mice. Plasma DNA was measured fluorometrically by the dye PicoGreen while lactate dehydrogenase (LDH) and caspase 3, other molecules released with cell injury or death, were measured by enzymatic assays. Histology was used to assess the occurrence of apoptosis or necrosis. Results of these experiments indicate that increased blood DNA levels occurred with all three agents and were highest with anti-Fas and CCl₄; caspase 3 levels were much higher with anti-Fas than the other agents. Histological examination confirmed the predominance of apoptotic death with anti-Fas and necrotic death with APAP and CCl₄. These results indicate that increased blood DNA is common in hepatotoxic injury and is a feature of both apoptotic and necrotic death.     

Protease Activity of Procaspase-8 Is Essential for Cell Survival by Inhibiting Both Apoptotic and Nonapoptotic Cell Death Dependent on Receptor-interacting Protein Kinase 1 (RIP1) and RIP3

Caspase-8 has an important role as an initiator caspase during death receptor-mediated apoptosis. Moreover, it has been reported to contribute to the regulation of cell fate in various types of cells including T-cells. In this report, we show that caspase-8 has an essential role in cell survival in mouse T-lymphoma-derived L5178Y cells. The knockdown of caspase-8 expression decreased the growth rate and increased cell death, both of which were induced by the absence of protease activity of procaspase-8. The cell death was associated with reactive oxygen species (ROS) accumulation, caspase activation, and autophagosome formation. The cell death was inhibited completely by treatment with ROS scavengers, but only partly by treatment with caspase inhibitors, expression of Bcl-xL, and knockdown of caspase-3 or Atg-7 which completely inhibits apoptosis or autophagosome formation, respectively, indicating that apoptosis and autophagy-associated cell death are induced simultaneously by the knockdown of caspase-8 expression. Further analysis indicated that RIP1 and RIP3 regulate this multiple cell death, because the cell death as well as ROS production was completely inhibited by not only treatment with the RIP1 inhibitor necrostatin-1, but also by knockdown of RIP3. Thus, in the absence of protease activity of procaspase-8, RIP1 and RIP3 simultaneously induce not only nonapoptotic cell death conceivably including autophagic cell death and necroptosis but also apoptosis through ROS production in mouse T-lymphoma cells.     

Expression of c-Myc is related to host cell death following <Emphasis Type="Italic">Salmonella typhimurium</Emphasis> infection in macrophage

It has been known that ornithine decarboxylase (ODC) induced by the binding of c-Myc to odc gene is closely linked to cell death. Here, we investigated the relationship between their expressions and cell death in macrophage cells following treatment with Salmonella typhimurium or lipopolysaccharide (LPS). ODC expression was increased by bacteria or LPS and repressed by inhibitors against mitogen-activated protein kinases (MAPKs) in Toll-like receptor 4 (TLR4) signaling pathway. In contrast, c-Myc protein level was increased after treatment with bacteria, but not by treatment with LPS or heat-killed bacteria although both bacteria and LPS increased the levels of c-myc mRNA to a similar extent. c-Myc protein level is dependent upon bacterial invasion because treatment with cytochalasin D (CCD), inhibitors of endocytosis, decreased c-Myc protein level. The cell death induced by bacteria was significantly decreased after treatment of CCD or c-Myc inhibitor, indicating that cell death by S. typhimurium infection is related to c-Myc, but not ODC. Consistent with this conclusion, treatment with bacteria mutated to host invasion did not increase c-Myc protein level and cell death rate. Taken together, it is suggested that induction of c-Myc by live bacterial infection is directly related to host cell death.     

Induction of microsomal epoxide hydrolase by sulfur amino acid deprivation via the pathway of C-Jun N-terminal kinase and its extracellular exposure during cell death

Microsomal epoxide hydrolase (mEH), an epoxide detoxifying enzyme and putative cell surface autoantigen, is inducible by xenobiotics and by certain pathophysiological conditions (e.g., tumorigenesis and protein-calorie malnutrition). The present study was designed to determine mEH expression in H4IIE cells during cell death initiated by sulfur amino acid deprivation (SAAD) and to identify the signaling pathway for the enzyme induction. SAAD induced cell death at 48-72 h with translocation of Bax to mitochondria and increased mitochondrial permeability with cytochrome c release, both of which were prevented by SB203580 or by dominant-negative JNK1 [JNK1(-)] stable transfection. Caspase-3 activity was only marginally increased by SAAD. Neither genomic DNA fragmentation nor poly(ADP-ribose) polymerase cleavage was observed during SAAD-induced cell death. Thus, SAAD induced cell death independent of caspase activation. This was supported by the observation that benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a general caspase inhibitor, did not prevent cell death. The levels of mEH mRNA and protein were notably increased in cells under SAAD for 48-72 h. The induction of mEH occurred in parallel with cell death. Whereas SAAD-induced cell death resulted from both JNK1 and p38 kinase activation, mEH induction was decreased only by JNK1(-) transfection. Immunocytochemistry revealed that mEH protein was intensely stained in dying cells, cellular fragments and cell debris. Furthermore, the number of cells positive for surface mEH substantially increased by SAAD, as evidenced by flow cytometry analysis. These results demonstrated that SAAD induced nonapoptotic cell death with Bax translocation to mitochondria and mitochondrial cytochrome c release, but not through caspase-3 activation, and that mEH was induced by SAAD via the pathway of JNK1, but not ERK1/2 or p38 kinase, in parallel with cell death.