A cellular defense pathway regulating transcription through poly(ADP-ribosyl)ation in response to DNA damage

DNA damage is known to trigger key cellular defense pathways such as those involved in DNA repair. Here we provide evidence for a previously unrecognized pathway regulating transcription in response to DNA damage and show that this regulation is mediated by the abundant nuclear enzyme poly(ADP-ribose) polymerase. We found that poly(ADP-ribose) polymerase reduced the rate of transcription elongation by RNA polymerase II, suggesting that poly(ADP-ribose) polymerase negatively regulates transcription, possibly through the formation of poly(ADP-ribose) polymerase–RNA complexes. In damaged cells, poly(ADP-ribose) polymerase binds to DNA breaks and automodifies itself in the presence of NAD+, resulting in poly(ADP-ribose) polymerase inactivation. We found that automodification of poly(ADP-ribose) polymerase in response to DNA damage resulted in the up-regulation of transcription, presumably because automodified poly(ADP-ribose) polymerase molecules were released from transcripts, thereby relieving the block on transcription. Because agents that damage DNA damage RNA as well, up-regulation of RNA synthesis in response to DNA damage may provide cells with a mechanism to compensate for the loss of damaged transcripts and may be critical for cell survival after exposure to DNA-damaging agents.

Futile transmembrane NH4+ cycling: A cellular hypothesis to explain ammonium toxicity in plants

Most higher plants develop severe toxicity symptoms when grown on ammonium (NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document}) as the sole nitrogen source. Recently, NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document} toxicity has been implicated as a cause of forest decline and even species extinction. Although mechanisms underlying NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document} toxicity have been extensively sought, the primary events conferring it at the cellular level are not understood. Using a high-precision positron tracing technique, we here present a cell-physiological characterization of NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document} acquisition in two major cereals, barley (Hordeum vulgare), known to be susceptible to toxicity, and rice (Oryza sativa), known for its exceptional tolerance to even high levels of NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document}. We show that, at high external NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document} concentration ([NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document}]o), barley root cells experience a breakdown in the regulation of NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document} influx, leading to the accumulation of excessive amounts of NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document} in the cytosol. Measurements of NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document} efflux, combined with a thermodynamic analysis of the transmembrane electrochemical potential for NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document}, reveal that, at elevated [NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document}]o, barley cells engage a high-capacity NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document}-efflux system that supports outward NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document} fluxes against a sizable gradient. Ammonium efflux is shown to constitute as much as 80% of primary influx, resulting in a never-before-documented futile cycling of nitrogen across the plasma membrane of root cells. This futile cycling carries a high energetic cost (we record a 40% increase in root respiration) that is independent of N metabolism and is accompanied by a decline in growth. In rice, by contrast, a cellular defense strategy has evolved that is characterized by an energetically neutral, near-Nernstian, equilibration of NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document} at high [NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document}]o. Thus our study has characterized the primary events in NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document} nutrition at the cellular level that may constitute the fundamental cause of NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document} toxicity in plants.

Evidence for a protective role of metallothionein-1 in focal cerebral ischemia

Metallothioneins (MTs) are a family of metal binding proteins that have been proposed to participate in a cellular defense against zinc toxicity and free radicals. In the present study, we investigated whether increased expression of MT in MT-1 isoform-overexpressing transgenic mice (MT-TG) affords protection against mild focal cerebral ischemia and reperfusion. Transient focal ischemia was induced in control (wild type) and MT-TG mice by occluding the right middle cerebral artery for 45 min. Upon reperfusion, cerebral edema slowly developed and peaked at 24 hr as shown by T2-weighted MRI. The volume of affected tissue was on the average 42% smaller in MT-TG mice compared with control mice at 6, 9, 24, and 72 hr and 14 days postreperfusion (P < 0.01). In addition, functional studies showed that 3 weeks after reperfusion MT-TG mice showed a significantly better motor performance compared with control mice (P = 0.011). Although cortical baseline levels of MT-1 mRNA were similar in control and MT-TG mice, there was an increase in MT-1 mRNA levels in the ischemic cortex of MT-TG mice to 7.5 times baseline levels compared with an increase to 2.3 times baseline levels in control mice 24 hr after reperfusion. In addition, MT-TG mice showed an increased MT immunoreactivity in astrocytes, vascular endothelial cells, and neurons 24 hr after reperfusion whereas in control mice MT immunoreactivity was restricted mainly to astrocytes and decreased in the infarcted tissue. These results provide evidence that increased expression of MT-1 protects against focal cerebral ischemia and reperfusion.

p19Arf induces p53-dependent apoptosis during Abelson virus-mediated pre-B cell transformation

The Ink4a/Arf locus encodes p16Ink4a and p19Arf and is among the most frequently mutated tumor suppressor loci in human cancer. In mice, many of these effects appear to be mediated by interactions between p19Arf and the p53 tumor-suppressor protein. Because Tp53 mutations are a common feature of the multistep pre-B cell transformation process mediated by Abelson murine leukemia virus (Ab-MLV), we examined the possibility that proteins encoded by the Ink4a/Arf locus also play a role in Abelson virus transformation. Analyses of primary transformants revealed that both p16Ink4a and p19Arf are expressed in many of the cells as they emerge from the apoptotic crisis that characterizes the transformation process. Analyses of primary transformants from Ink4a/Arf null mice revealed that these cells bypassed crisis. Because expression of p19Arf but not p16 Ink4a induced apoptosis in Ab-MLV-transformed pre-B cells, p19Arf appears to be responsible for these events. Consistent with the link between p19Arf and p53, Ink4a/Arf expression correlates with or precedes the emergence of cells expressing mutant p53. These data demonstrate that p19Arf is an important part of the cellular defense mounted against transforming signals from the Abl oncoprotein and provide direct evidence that the p19Arf–p53 regulatory loop plays an important role in lymphoma induction.

ATP-dependent uptake of natural product cytotoxic drugs by membrane vesicles establishes MRP as a broad specificity transporter.

MRP is a recently isolated ATP-binding cassette family transporter. We previously reported transfection studies that established that MRP confers multidrug resistance [Kruh, G. D., Chan, A., Myers, K., Gaughan, K., Miki, T. & Aaronson, S. A. (1994) Cancer Res. 54, 1649-1652] and that expression of MRP is associated with enhanced cellular efflux of lipophilic cytotoxic agents [Breuninger, L. M., Paul, S., Gaughan, K., Miki, T., Chan, A., Aaronson, S. A. & Kruh, G. D. (1995) Cancer Res. 55, 5342-5347]. To examine the biochemical mechanism by which MRP confers multidrug resistance, drug uptake experiments were performed using inside-out membrane vesicles prepared from NIH 3T3 cells transfected with an MRP expression vector. ATP-dependent transport was observed for several lipophilic cytotoxic agents including daunorubicin, etoposide, and vincristine, as well as for the glutathione conjugate leukotriene C4 (LTC4). However, only marginally increased uptake was observed for vinblastine and Taxol. Drug uptake was osmotically sensitive and saturable with regard to substrate concentration, with Km values of 6.3 microM, 4.4 microM, 4.2 microM, 35 nM, and 38 microM, for daunorubicin, etoposide, vincristine, LTC4, and ATP, respectively. The broad substrate specificity of MRP was confirmed by the observation that daunorubicin transport was competitively inhibited by reduced and oxidized glutathione, the glutathione conjugates S-(p-azidophenacyl)-glutathione (APA-SG) and S-(2,4-dinitrophenyl)glutathione (DNP-SG), arsenate, and the LTD4 antagonist MK571. This study establishes that MRP pumps unaltered lipophilic cytotoxic drugs, and suggests that this activity is an important mechanism by which the transporter confers multidrug resistance. The present study also indicates that the substrate specificity of MRP is overlapping but distinct from that of P-glycoprotein, and includes both the neutral or mildly cationic natural product cytotoxic drugs and the anionic products of glutathione conjugation. The widespread expression of MRP in tissues, combined with its ability to transport both lipophilic xenobiotics and the products of phase II detoxification, indicates that the transporter represents a widespread and remarkably versatile cellular defense mechanism.

Reduced stress defense in heme oxygenase 1-deficient cells

Stressed mammalian cells up-regulate heme oxygenase 1 (Hmox1; EC 1.14.99.3), which catabolizes heme to biliverdin, carbon monoxide, and free iron. To assess the potential role of Hmox1 in cellular antioxidant defense, we analyzed the responses of cells from mice lacking functional Hmox1 to oxidative challenges. Cultured Hmox1−/− embryonic fibroblasts demonstrated high oxygen free radical production when exposed to hemin, hydrogen peroxide, paraquat, or cadmium chloride, and they were hypersensitive to cytotoxicity caused by hemin and hydrogen peroxide. Furthermore, young adult Hmox1−/− mice were vulnerable to mortality and hepatic necrosis when challenged with endotoxin. Our in vitro and in vivo results provide genetic evidence that up-regulation of Hmox1 serves as an adaptive mechanism to protect cells from oxidative damage during stress.

An important role of an inducible RNA-dependent RNA polymerase in plant antiviral defense

Plants contain RNA-dependent RNA polymerase (RdRP) activities that synthesize short cRNAs by using cellular or viral RNAs as templates. During studies of salicylic acid (SA)-induced resistance to viral pathogens, we recently found that the activity of a tobacco RdRP was increased in virus-infected or SA-treated plants. Biologically active SA analogs capable of activating plant defense response also induced the RdRP activity, whereas biologically inactive analogs did not. A tobacco RdRP gene, NtRDRP1, was isolated and found to be induced both by virus infection and by treatment with SA or its biologically active analogs. Tobacco lines deficient in the inducible RDRP activity were obtained by expressing antisense RNA for the NtRDRP1 gene in transgenic plants. When infected by tobacco mosaic virus, these transgenic plants accumulated significantly higher levels of viral RNA and developed more severe disease symptoms than wild-type plants. After infection by a strain of potato virus X that does not spread in wild-type tobacco plants, the transgenic NtRDRP1 antisense plants accumulated virus and developed symptoms not only locally in inoculated leaves but also systemically in upper uninoculated leaves. These results strongly suggest that inducible RdRP activity plays an important role in plant antiviral defense.

Defense Responses to Tetrapyrrole-Induced Oxidative Stress in Transgenic Plants with Reduced Uroporphyrinogen Decarboxylase or Coproporphyrinogen Oxidase Activity1

We analyzed the antioxidative defense responses of transgenic tobacco (Nicotiana tabacum) plants expressing antisense RNA for uroporphyrinogen decarboxylase or coproporphyrinogen oxidase. These plants are characterized by necrotic leaf lesions resulting from the accumulation of potentially photosensitizing tetrapyrroles. Compared with control plants, the transformants had increased levels of antioxidant mRNAs, particularly those encoding superoxide dismutase (SOD), catalase, and glutathione peroxidase. These elevated transcript levels correlated with increased activities of cytosolic Cu/Zn-SOD and mitochondrial Mn-SOD. Total catalase activity decreased in the older leaves of the transformants to levels lower than in the wild-type plants, reflecting an enhanced turnover of this photosensitive enzyme. Most of the enzymes of the Halliwell-Asada pathway displayed increased activities in transgenic plants. Despite the elevated enzyme activities, the limited capacity of the antioxidative system was apparent from decreased levels of ascorbate and glutathione, as well as from necrotic leaf lesions and growth retardation. Our data demonstrate the induction of the enzymatic detoxifying defense system in several compartments, suggesting a photosensitization of the entire cell. It is proposed that the tetrapyrroles that initially accumulate in the plastids leak out into other cellular compartments, thereby necessitating the local detoxification of reactive oxygen species.

Inhibition of ascorbate peroxidase by salicylic acid and 2,6-dichloroisonicotinic acid, two inducers of plant defense responses.

In recent years, it has become apparent that salicylic acid (SA) plays an important role in plant defense responses to pathogen attack. Previous studies have suggested that one of SA's mechanisms of action is the inhibition of catalase, resulting in elevated levels of H2O2, which activate defense-related genes. Here we demonstrate that SA also inhibits ascorbate peroxoidase (APX), the other key enzyme for scavenging H2O2. The synthetic inducer of defense responses, 2,6-dichloroisonicotinic acid (INA), was also found to be an effective inhibitor of APX. In the presence of 750 microM ascorbic acid (AsA), substrate-dependent IC50 values of 78 microM and 95 microM were obtained for SA and INA, respectively. Furthermore, the ability of SA analogues to block APX activity correlated with their ability to induce defense-related genes in tobacco and enhance resistance to tobacco mosaic virus. Inhibition of APX by SA appears to be reversible, thus differing from the time-dependent, irreversible inactivation by suicide substrates such as p-aminophenol. In contrast to APX, the guaiacol-utilizing peroxidases, which participate in the synthesis and crosslinking of cell wall components as part of the defense response, are not inhibited by SA or INA. The inhibition of both catalase and APX, but not guaiacol peroxidases, supports the hypothesis that SA-induced defense responses are mediated, in part, through elevated H2O2 levels or coupled perturbations of the cellular redox state.

Induction, modification, and transduction of the salicylic acid signal in plant defense responses.

Studies in our laboratory as well as others strongly suggest that salicylic acid (SA) plays an important signaling role in plant defense against pathogens. We have found that increases in endogenous SA levels correlates with both resistance of tobacco to infection with tobacco mosaic virus and induction of defense-related genes such as that encoding pathogenesis-related protein 1 (PR-1). Some of this newly synthesized SA was conjugated to glucose to form SA beta-glucoside. A cell wall-associated beta-glucosidase activity that releases SA from this glucoside has been identified, suggesting that SA beta-glucoside serves as an inactive storage form of SA. By purifying a soluble SA-binding protein and isolating its encoding cDNA from tobacco, we have been able to further characterize the mechanism of SA signaling. This protein is a catalase, and binding of SA and its biologically active analogues inhibited catalase's ability to convert H2O2 to O2 and H2O. The resulting elevated levels of cellular H2O2 appeared to induce PR-1 gene expression, perhaps by acting as a second messenger. Additionally, transgenic tobacco expressing an antisense copy of the catalase gene and exhibiting depressed levels of catalase also showed constitutive expression of PR-1 genes. To further dissect the SA signaling pathway, we have tested several abiotic inducers of PR gene expression and disease resistance for their ability to stimulate SA production. Levels of SA and its glucoside rose following application of all of the inducers except 2,6-dichloroisonicotinic acid. 2,6-Dichloroisonicotinic acid was found to bind catalase directly and inhibit its enzymatic activity. Thus, it appears that many compounds that induce PR gene expression and disease resistance in plants inactivate catalases directly or indirectly.

Involvement of small GTP-binding proteins in defense signal-transduction pathways of higher plants.

Small GTP-binding proteins play a critical role in the regulation of a range of cellular processes--including growth, differentiation, and intracellular transportation. Previously, we isolated a gene, rgp1, encoding a small GTP-binding protein, by differential screening of a rice cDNA library with probe DNAs from rice tissues treated with or without 5-azacytidine, a powerful inhibitor of DNA methylation. To determine the physiological role of rgp1, the coding region was introduced into tobacco plants. Transformants, with rgp1 in either sense or antisense orientations, showed distinct phenotypic changes with reduced apical dominance, dwarfism, and abnormal flower development. These abnormal phenotypes appeared to be associated with the higher levels of endogenous cytokinins that were 6-fold those of wild-type plants. In addition, the transgenic plants produced salicylic acid and salicylic acid-beta-glucoside in an unusual response to wounding, thus conferring increased resistance to tobacco mosaic virus infection. In normal plants, the wound- and pathogen-induced signal-transduction pathways are considered to function independently. However, the wound induction of salicylic acid in the transgenic plants suggests that expression of rgp1 somehow interfered with the normal signaling pathways and resulted in cross-signaling between these distinct transduction systems. The results imply that the defense signal-transduction system consists of a complicated and finely tuned network of several regulatory factors, including cytokinins, salicylic acid, and small GTP-binding proteins.

Molecular mechanisms of defense by rhizobacteria against root disease.

Genetic resistance in plants to root diseases is rare, and agriculture depends instead on practices such as crop rotation and soil fumigation to control these diseases. "Induced suppression" is a natural phenomenon whereby a soil due to microbiological changes converts from conducive to suppressive to a soilborne pathogen during prolonged monoculture of the susceptible host. Our studies have focused on the wheat root disease "take-all," caused by the fungus Gaeumannomyces graminis var. tritici, and the role of bacteria in the wheat rhizosphere (rhizobacteria) in a well-documented induced suppression (take-all decline) that occurs in response to the disease and continued monoculture of wheat. The results summarized herein show that antibiotic production plays a significant role in both plant defense by and ecological competence of rhizobacteria. Production of phenazine and phloroglucinol antibiotics, as examples, account for most of the natural defense provided by fluorescent Pseudomonas strains isolated from among the diversity of rhizobacteria associated with take-all decline. There appear to be at least three levels of regulation of genes for antibiotic biosynthesis: environmental sensing, global regulation that ties antibiotic production to cellular metabolism, and regulatory loci linked to genes for pathway enzymes. Plant defense by rhizobacteria producing antibiotics on roots and as cohabitants with pathogens in infected tissues is analogous to defense by the plant's production of phytoalexins, even to the extent that an enzyme of the same chalcone/stilbene synthase family used to produce phytoalexins is used to produce 2,4-diacetylphloroglucinol. The defense strategy favored by selection pressure imposed on plants by soilborne pathogens may well be the ability of plants to support and respond to rhizosphere microorganisms antagonistic to these pathogens.