UV-induced DNA damage promotes resistance to the biotrophic pathogen Hyaloperonospora parasitica in Arabidopsis

Plant innate immunity to pathogenic microorganisms is activated in response to recognition of extracellular or intracellular pathogen molecules by transmembrane receptors or resistance proteins, respectively. The defense signaling pathways share components with those involved in plant responses to UV radiation, which can induce expression of plant genes important for pathogen resistance. Such intriguing links suggest that UV treatment might activate resistance to pathogens in normally susceptible host plants. Here, we demonstrate that pre-inoculative UV (254 nm) irradiation of Arabidopsis (Arabidopsis thaliana) susceptible to infection by the biotrophic oomycete Hyaloperonospora parasitica, the causative agent of downy mildew, induces dose- and time-dependent resistance to the pathogen detectable up to 7 d after UV exposure. Limiting repair of UV photoproducts by postirradiation incubation in the dark, or mutational inactivation of cyclobutane pyrimidine dimer photolyase, (6-4) photoproduct photolyase, or nucleotide excision repair increased the magnitude of UV-induced pathogen resistance. In the absence of treatment with 254-nm UV, plant nucleotide excision repair mutants also defective for cyclobutane pyrimidine dimer or (6-4) photoproduct photolyase displayed resistance to H. parasitica, partially attributable to short wavelength UV-B (280–320 nm) radiation emitted by incubator lights. These results indicate UV irradiation can initiate the development of resistance to H. parasitica in plants normally susceptible to the pathogen and point to a key role for UV-induced DNA damage. They also suggest UV treatment can circumvent the requirement for recognition of H. parasitica molecules by Arabidopsis proteins to activate an immune response.

Repair and tolerance of DNA damage in higher plants

DNA repair mechanisms constitute an essential cellular response to DNA damage arising either from metabolic processes or from environmental sources such as ultraviolet radiation. Repair of these lesions may be via direct reversal, or by processes such as nucleotide excision repair (NER), a coordinated pathway in which lesions and the surrounding nucleotides are excised and replaced via DNA resynthesis. The importance of repair is illustrated by human disease states such as xeroderma pigmentosum and Cockayne's syndrome which result from defects in the NER system arising from mutations in XP- genes or XP- and CS- genes respectively Little detail is known of DNA damage repair processes in plants, despite the economic and ecological importance of these organisms. This study aimed to expand our knowledge of the process of NER in plants, largely via a polymerase chain reaction (PCR)-based approach involving amplification, cloning and characterisation of plant genomic DNA and cDNA. Homologues of the NER components XPF/RAD1 and XPD/RAD3 were isolated as both genomic and complete cDNA sequences from the model dicotyledonous plant Arabidopsis thaliana. The sequence of the 3'-untranslated region of atXPD was also determined. Comparison of genomic and cDNA sequences allowed a detailed analysis of gene structures, including details of intron/exon processing. Variable transcript processing to produce three distinct transcripts was found in the case of atXPF. In an attempt to validate the proposed homologous function of these cDNAs, assays to test complementation of resistance to ultraviolet radiation in the relevant yeast mutants were performed. Despite extensive amino acid sequence conservation, neither plant cDNA was able to restore UV-resistance. As the yeast RAD3 gene product is also involved in vivo in transcription, and so is required for viability, the atXPD cDNA was tested in a complementation assay for this function in an appropriate yeast mutant. The plant cDNA was found to substantially increase the viability of the yeast mutant. The structural and functional significance of these results is discussed comparatively with reference to yeast, human and other known homologues. Other putative NER homologues were identified in A. thaliana database sequences, including those of ERCC1/RAD10 and XPG/ERCC5/RAD2, and are now the subjects of ongoing investigations. This study also describes preliminary investigations of putative REVS and RAD30 translesion synthesis genes from A. thaliana.

Influence of age and DNA damage on expression of damage-processing genes

The effect of DNA damaging agents and age on expression of damage-processing genes was examined in plants and mice. Treatment with these agents increased expression of some genes. The effect of gene expression in the absence of treatment decreased with age, suggesting links between ageing and genetic instability.

Consumption of a low glycaemic index diet in late life extends lifespan of Balb/c mice with differential effects on DNA damage

Background : Caloric restriction is known to extend the lifespan of all organisms in which it has been tested. Consequently, current research is investigating the role of various foods to improve health and lifespan. The role of various diets has received less attention however, and in some cases may have more capacity to improve health and longevity than specific foods alone. We examined the benefits to longevity of a low glycaemic index (GI) diet in aged Balb/c mice and examined markers of oxidative stress and subsequent effects on telomere dynamics.

Results : In an aged population of mice, a low GI diet extended average lifespan by 12%, improved glucose tolerance and had impressive effects on amelioration of oxidative damage to DNA in white blood cells. Telomere length in quadriceps muscle showed no improvement in the dieted group, nor was telomerase reactivated.

Conclusion : The beneficial effects of a low GI diet are evident from the current study and although the impact to telomere dynamics late in life is minimal, we expect that earlier intervention with a low GI diet would provide significant improvement in health and longevity with associated effects to telomere homeostasis.

DNA damage in rats after treatment with methylphenidate.

Methylphenidate (MPH) is a widely prescribed psychostimulant for the treatment of attention-deficit hyperactivity disorder (ADHD). Recently, some studies have addressed the genotoxic potential of the MPH, but the results have been contradictory. Hence, the present study aimed to investigate the index of cerebral and peripheral DNA damage in young and adult rats after acute and chronic MPH exposure.

Analysis of 177Lu-octreotate therapy-induced DNA damage in peripheral blood lymphocytes of patients with neuroendocrine tumors

Ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) can lead to cell death, genome instability and carcinogenesis. Immunofluorescence detection of phosphorylated histone variant H2AX (γ-H2AX) is a reliable and sensitive technique to monitor external beam IR-induced DSBs in peripheral blood lymphocytes (PBL). Here, we investigated whether γ-H2AX could be used as an in vivo marker to assess normal tissue toxicity after extended internal irradiation with (177)Lu-DOTA-octreotate peptide receptor radionuclide therapy (LuTate PRRT) of neuroendocrine tumors.

YM155 down-regulates survivin and XIAP, modulates autophagy and induces autophagy-dependent DNA damage in breast cancer cells

The aim of this study was to determine the potency and molecular mechanism of action of YM155, a first-in-class survivin inhibitor that is currently under phase I/II clinical investigations, in various drug-resistant breast cancers including the oestrogen receptor positive (ER(+) ) tamoxifen-resistant breast cancer and the caspase-3-deficient breast cancer.

Associations between DNA damage, DNA base excision repair gene variability and Alzheimer's disease risk

BACKGROUND: Increased oxidative damage to DNA is one of the pathways involved in Alzheimer's disease (AD). Insufficient base excision repair (BER) is in part responsible for increased oxidative DNA damage. The aim of the present study was to assess the effect of polymorphic variants of BER-involved genes and the peripheral markers of DNA damage and repair in patients with AD. MATERIAL AND METHODS: Comet assays and TaqMan probes were used to assess DNA damage, BER efxFB01;ciency and polymorphic variants of 12 BER genes in blood samples from 105 AD patients and 130 controls. The DNA repair efficacy (DRE) was calculated according to a specific equation. RESULTS: The levels of endogenous and oxidative DNA damages were higher in AD patients than controls. The polymorphic variants of XRCC1 c.580C>T XRCC1 c.1196A>G and OGG1 c.977C>G are associated with increased DNA damage in AD. CONCLUSION: Our results show that oxidative stress and disturbances in DRE are particularly responsible for the elevated DNA lesions in AD. The results suggest that oxidative stress and disruption in DNA repair may contribute to increased DNA damage in AD patients and risk of this disease. In addition, disturbances in DRE may be associated with polymorphisms of OGG1 and XRCC1.

Regulation of redox and DNA repair genes by arsenic : low dose protection against oxidative stress?

We have evaluated the molecular responses of human epithelial cells to low dose arsenic to ascertain how target cells may respond to physiologically relevant concentrations of arsenic. Data gathered in numerous experiments in different cell types all point to the same conclusion: low dose arsenic induces what appears to be a protective response against subsequent exposure to oxidative stress or DNA damage, whereas higher doses often provoke synergistic toxicity. In particular, exposure to low, sub-toxic doses of arsenite, As(III), causes coordinate up-regulation of multiple redox and redox-related genes including thioredoxin (Trx) and glutathione reductase (GR). Glutathione peroxidase (GPx) is down-regulated in fibroblasts, but up-regulated in keratinocytes, as is glutathione S-transferase (GST). The maximum effect on these redox genes occurs after 24 h exposure to 5–10 mM As(III). This is 10-fold higher than the maximum As(III) concentrations required for induction of DNA repair genes, but within the dose region where DNA repair genes are co-ordinately down-regulated. These changes in gene regulation are brought about in part by changes in DNA binding activity of the transcription factors activating protein-1 (AP-1), nuclear factor kappa-B, and cAMP response element binding protein (CREB). Although sub-acute exposure to micromolar As(III) up-regulates transcription factor binding, chronic exposure to submicromolar As(III) causes persistent down-regulation of this response. Similar long-term exposure to micromolar concentrations of arsenate in drinking water results in a decrease in skin tumour formation in dimethylbenzanthracene (DMBA)/phorbol 12-tetradecanoate 13-acetate (TPA) treated mice. Altered response patterns after long exposure to As(III) may play a significant role in As(III) toxicology in ways that may not be predicted by experimental protocols using short-term exposures.

DNA repair deficiencies increase the resistance of Arabidopsis Thaliana to Hyaloperonospora Parasitica

We have found that UV-C treatment of Arabidopsis thaliana induces resistance to the biotrophic pathogen Hyaloperonospora parasitica, and our data suggest UV induced DNA photoproducts are involved (see accompanying abstract by K.G. McKenzie et al.). To address the potential role of DNA damage, we have examined the effect of mutations in nucleotide excision repair (uvr1-1), photoreactivation of cyclobutane pyrimidine dimers (uvr2-1) or flavonoid production (tt5) on the resistance of Arabidopsis to the pathogen with or without pre-inoculation treatment with UV-C. In the mutant backgrounds, UV-C induced pathogen resistance (as measured by decreased conidiophore formation) to the same degree as in the wildtype plants, but much lower UV doses were required (e.g., 100 Jm-2 in the mutant vs. 400 Jm-2 in the wildtype). This is the result expected if damage to DNA rather than a non DNA target is involved. Interestingly, in the absence of UV-C, the tt5 mutation alone resulted in a slight increase in resistance. However, when coupled with uvr1-1, resistance was enhanced to an even greater extent. Remarkably, the tt5 uvr1-1 uvr2-1 triple mutant was completely resistant to the pathogen. Since tt5 mutants are sensitive to reactive oxygen species, which can cause DNA damage susceptible to nucleotide excision repair, our results suggest that in addition to UV photoproducts, an accumulation of endogenous oxidative DNA damage may also trigger resistance to the pathogen. We are currently examining pathogen resistance in other DNA repair deficient mutants, and quantifying UV-C-induced DNA damage in Arabidopsis in order to assess the relationship between damage levels and the extent of resistance.

A potential copper-regulatory role for cytosolic expression of the DNA repair protein XRCC5

Copper (Cu) has a critical role in the generation of oxidative stress during neurodegeneration and cancer. Reactive oxygen species generated through abnormal elevation or deficiency of Cu can lead to lipid, protein, and DNA damage. Oxidation of DNA can induce strand breaks and is associated with altered cell fate including transformation or death. DNA repair is mediated through the action of the multimeric DNA-PK repair complex. The components of this complex are the Ku autoantigens, XRCC5 and XRCC6 (Ku80 and Ku70, respectively). How this repair complex responds to perturbed Cu homeostasis and Cu-mediated oxidative stress has not been investigated. We previously reported that XRCC5 expression is altered in response to cellular Cu levels, with low Cu inhibiting XRCC5 expression and high Cu levels enhancing expression. In this study we further investigated the interaction between XRCC5 and Cu. We report that cytosolic XRCC5 is increased in response to Cu, but not zinc, iron, or nickel, and the level of cytosolic XRCC5 correlates with protection against oxidative damage to DNA. These observations were made in both HeLa cells and fibroblasts. Cytosolic XRCC5 interacted with the Cu chaperone and detoxification protein human Atox1 homologue (HAH), and down regulation of XRCC5 expression using siRNA led to enhanced HAH expression when cells were exposed to Cu. XRCC5 could also be purified from cytosolic extracts using a Cu-loaded column. These findings provide further evidence that cytosolic XRCC5 has a key role in protection against DNA oxidation from Cu, through either direct sequestration or signaling through other Cu-detoxification molecules. Our findings have important implications for the development of therapeutic treatments targeting Cu in neurodegeneration and/or cancer.

Arsenic, mode of action at biologically plausible low doses : what are the implications for low dose cancer risk?

Arsenic is an established human carcinogen. However, there has been much controversy about the shape of the arsenic response curve, particularly at low doses. This controversy has been exacerbated by the fact that the  mechanism(s) of arsenic carcinogenesis are still unclear and because there are few satisfactory animal models for arsenic-induced carcinogenesis. Recent epidemiological studies have shown that the relative risk for cancer among populations exposed to ≤60 ppb As in their drinking water is often lower than the risk for the unexposed control population. We have found that treatment of human keratinocyte and fibroblast cells with 0.1 to 1 μM arsenite (As^III) also produces a low dose protective effect against oxidative stress and DNA damage. This response includes increased transcription, protein levels and enzyme activity of several base excision repair genes, including DNA polymerase β and DNA ligase I. At higher concentrations (> 10 μM), As induces down-regulation of DNA repair, oxidative DNA damage and apoptosis. This low dose adaptive (protective) response by a toxic agent is known as hormesis and is characteristic of many agents that induce oxidative stress. A mechanistic model for arsenic carcinogenesis based on these data would predict that the low dose risk for carcinogenesis should be sub-linear. The threshold dose where toxicity outweighs protection is hard to predict based on in vitro dose response data, but might be estimated if one could determine the form (metabolite) and concentration of arsenic responsible for changes in gene regulation in the target tissues.

Effects of purified herbal extract of Saliva miltiorrhiza on ischemic rat myocardium after acute myocardial infarction

In the current study, we compared purified Salvia miltiorrhiza extract (PSME) with Angiotensin-converting enzyme inhibitor, Ramipril, in in vitro experiments and also in vivo using animal model of myocardial infarction. PSME was found to have a significantly higher trolox equivalent antioxidant capacity which indicated a great capacity for scavenging free radicals. PSME could also prevent pyrogallo red bleaching and DNA damage.

After 2 weeks treatment with PSME or Ramipril, survival rates of rats with experimental myocardial infarction were marginally increased (68.2% and 71.4%) compared with saline (61.5%). The ratios of infarct size to left ventricular size in both PSME-and Ramipril-treated rats were significantly less than that in the saline-treated group. Activity of cardiac antioxidant enzyme superoxide dismutase (SOD) was significant higher while level of Thiobarbituric acid-reactive substances (TBARs) was lower in the PSME treated group. Purified and standardized Chinese herb could provide an alternative regimen for the prevention of ischemic heart disease.

Insights into exazaphosphorine resistance and possible approaches to its circumvention

The oxazaphosphorines cyclophosphamide, ifosfamide and trofosfamide remain a clinically useful class of anticancer drugs with substantial antitumour activity against a variety of solid tumors and hematological malignancies. A major limitation to their use is tumour resistance, which is due to multiple mechanisms that include increased DNA repair, increased cellular thiol levels, glutathione S-transferase and aldehyde dehydrogenase activities, and altered cell-death response to DNA damage. These mechanisms have been recently re-examined with the aid of sensitive analytical techniques, high-throughput proteomic and genomic approaches, and powerful pharmacogenetic tools. Oxazaphosphorine resistance, together with dose-limiting toxicity (mainly neutropenia and neurotoxicity), significantly hinders chemotherapy in patients, and hence, there is compelling need to find ways to overcome it. Four major approaches are currently being explored in preclinical models, some also in patients: combination with agents that modulate cellular response and disposition of oxazaphosphorines; antisense oligonucleotides directed against specific target genes; introduction of an activating gene (CYP3A4) into tumor tissue; and modification of dosing regimens. Of these approaches, antisense oligonucleotides and gene therapy are perhaps more speculative, requiring detailed safety and efficacy studies in preclinical models and in patients. A fifth approach is the design of novel oxazaphosphorines that have favourable pharmacokinetic and pharmacodynamic properties and are less vulnerable to resistance. Oxazaphosphorines not requiring hepatic CYP-mediated activation (for example, NSC 613060 and mafosfamide) or having additional targets (for example, glufosfamide that also targets glucose transport) have been synthesized and are being evaluated for safety and efficacy. Characterization of the molecular targets associated with oxazaphosphorine resistance may lead to a deeper understanding of the factors critical to the optimal use of these agents in chemotherapy and may allow the development of strategies to overcome resistance.