Eva Siles

ROS-induced DNA damage and PARP-1 are required for optimal induction of starvation-induced autophagy

In response to nutrient stress, cells start an autophagy program that can lead to adaptation or death. The mechanisms underlying the signaling from starvation to the initiation of autophagy are not fully understood. In the current study we show that the absence or inactivation of PARP-1 strongly delays starvation-induced autophagy. We have found that DNA damage is an early event of starvation-induced autophagy as measured by γ-H2AX accumulation and comet assay, with PARP-1 knockout cells displaying a reduction in both parameters. During starvation, ROS-induced DNA damage activates PARP-1, leading to ATP depletion (an early event after nutrient deprivation). The absence of PARP-1 blunted AMPK activation and prevented the complete loss of mTOR activity, leading to a delay in autophagy. PARP-1 depletion favors apoptosis in starved cells, suggesting a pro-survival role of autophagy and PARP-1 activation after nutrient deprivation. In vivo results show that neonates of PARP-1 mutant mice subjected to acute starvation, also display deficient liver autophagy, implying a physiological role for PARP-1 in starvation-induced autophagy. Thus, the PARP signaling pathway is a key regulator of the initial steps of autophagy commitment following starvation.

Relationship between p53 status and radiosensitivity in human tumour cell lines

We examined the relationship between p53 levels before and after irradiation, radiation-induced cell cycle delays, apoptotic cell death and radiosensitivity in a panel of eight human tumour cell lines. The cell lines differed widely in their clonogenic survival after radiation, (surviving fraction at 2 Gy: SF2=0.18-0.82). Constitutive p53 protein levels varied from 2.2 +/- 0.4 to 6.3 +/- 0.3 optical density units (OD) per 10(6) cells. p53 after irradiation (6 Gy) also varied between the cell lines, ranging from no induction to a 1.6-fold increase in p53 levels 4 h after treatment. p53 function was also assessed by G1 cell cycle arrest after irradiation. The cellular response to radiation, measured as G0/G1 arrest, and the induction of apoptosis were in good agreement. However, a trace amount of DNA ladder formation was found in two cell lines lacking G1 arrest. Overall cellular radiosensitivity correlated well with the level of radiation-induced G1 arrest (correlation coefficient r=0.856; P=0.0067), with p53 constitutive levels (r=0.874, P=0.0046), and with p53 protein fold induction (r=-0.882, P=0.0038). Our data suggest that (1) the constitutive p53 level, (2) G1 arrest after irradiation, or (3) the p53 protein response to radiation may be good predictive tests for radiosensitivity in some cell types.

Age-related changes of the nitric oxide system in the rat brain

This work examines the age-related changes of the NO pathway in the central nervous system (CNS), analyzing nitric oxide synthase (NOS) isoform expression, the level of nitrotyrosine-modified proteins, and the NOS activity in the cerebral cortex, decorticated brain (basal ganglia, thalamus, hypothalamus, tegtum and tegmentum) and cerebellum of young, adult and aged rats. Our data demonstrate that the different NOS isoforms are not uniformly expressed across the CNS. In this sense, the nNOS and eNOS isoenzymes are expressed mainly in the cerebellum and decorticated brain, respectively, while the iNOS isoenzyme shows the highest level in cerebellum. Concerning age, in the cerebral cortex nNOS significantly increased its expression only in adult animals; meanwhile, in the cerebellum the eNOS expression decreased whereas iNOS increased in adult and aged rats. No age-related changes in any isoform were found in decorticated brain. NOS activity, determined by nitrate plus nitrite quantification, registered the highest levels in the cerebellum, where the significant increase detected with aging was probably related to iNOS activity. The number of nitrotyrosine-modified immunoreactive bands differed among regions; thus, the highest number was detected in the decorticated brain while the cerebellum showed the least number of bands. Finally, bulk protein nitration increased in cerebral cortex only in adult animal. No changes were found in the decorticated brain, and the decrease detected in the cerebellum of aged animals was not significant. According to these results, the NO pathway is differently modified with age in the three CNS regions analyzed.

Tyrosol, a main phenol present in extra virgin olive oil, increases lifespan and stress resistance in Caenorhabditis elegans.

Extra virgin olive oil (EVOO) consumption has been traditionally related to a higher longevity in the human population. EVOO effects on health are often attributed to its unique mixture of phenolic compounds with tyrosol and hydroxityrosol being the most biologically active. Although these compounds have been extensively studied in terms of their antioxidant potential and its role in different pathologies, their actual connection with longevity remains unexplored. This study utilized the nematode Caenorhabditis elegans to investigate the possible effects of tyrosol in metazoan longevity. Significant lifespan extension was observed at one specific tyrosol concentration, which also induced a higher resistance to thermal and oxidative stress and delayed the appearance of a biomarker of ageing. We also report that, although tyrosol was efficiently taken up by these nematodes, it did not induce changes in development, body length or reproduction. In addition, lifespan experiments with several mutant strains revealed that components of the heat shock response (HSF-1) and the insulin pathway (DAF-2 and DAF-16) might be implicated in mediating tyrosol effects in lifespan, while caloric restriction and sirtuins do not seem to mediate its effects. Together, our results point to hormesis as a possible mechanism to explain the effects of tyrosol on longevity in C. elegans.

Glutathione S-transferase isoenzymatic response to aging in rat cerebral cortex and cerebellum

Aging is associated with increased oxidant generation. One mechanism involved in the defense of oxidative products is the family of glutathione transferases (GST). We have analyzed the activity, distribution and expression of GSTP1 and GSTA4 isoenzymes in the cerebral cortex and cerebellum of young, adult and aged rats. The total GST activity, measured with the universal substrate 1-chloro-2,4-dinitrobenzene (CDNB), increased only with the maturation process; however GSTA4 activity, using the specific substrate 4-hydroxynonenal (HNE), did show an age-dependent increase in both brain regions. Cellular location of GSTA4 in astrocytes was not changed except for young cerebral cortex and adult/aged cerebellum that also showed immunoreactivity in layer III pyramidal neurons and Bergman radial glia, respectively. Distribution of GSTP1 was similar among groups and only an increased number of positive oligodendrocytes was found in the Purkinje and granular layer of adult/aged cerebellum. The GSTA4 and GSTP1 expression increased from young to adult/aged brain and GSTA4 even augmented in the aged cerebral cortex. These results suggest a GST isoenzymatic response with aging, but above all with the maturation process.

Aging affects but does not eliminate the enzymatic antioxidative response to hypoxia/reoxygenation in cerebral cortex.

The effect of aging on basal and hypoxia/reoxygenation levels of both oxidative stress (protein carbonyl and TBARS) and antioxidative-enzyme activity (Cu/Zn-SOD; Mn-SOD; Catalase, CAT; Se-independent and Se-dependent glutathione peroxidase, GPX; glutathione transferase, GST and glutathione reductase, GR) has been studied in the cerebral cortex of adult and old rats. Oxidative stress markers increased with aging and show an age-dependent post-hypoxic response. Moreover, aging caused either no change (GST, GR and CAT) or an increase (Se-GPX, Cu/Zn-SOD, Mn-SOD) in the basal activity of the enzymes analysed. Only Se-independent GPX activity decreases. However, we detected an age-dependent response of SODs to the hypoxic injury. The early and sustained Cu/Zn-SOD activity rise in adult animals became late and weak in aged animals. Meanwhile, aging slowed the Mn-SOD post-hypoxic response although this activity was consistently higher in aged rats. Aging eliminated the post-hypoxic CAT response, but, perhaps offset by increased GPX activity, did not affect the GST response and slightly reduced post-hypoxic GR activity. In conclusion, aging rise basal ROS production, does not diminish or even increase the antioxidative-enzyme activity, and may slow but does not usually eliminate the enzymatic antioxidant response to the increased post-hypoxic ROS generation.

PARP-1 modulates deferoxamine-induced HIF-1α accumulation through the regulation of nitric oxide and oxidative stress

Poly(ADP‐ribose) polymerase‐1 (PARP‐1) is a nuclear protein that, once activated by genotoxic agents, modulates the activity of several nuclear proteins including itself. Previous studies have established that PARP‐1 inhibition may provide benefit in the treatment of different diseases, particularly those involving a hypoxic situation, in which an increased oxidative and nitrosative stress occurs. One of the most important transcription factors involved in the response to the hypoxic situation is the hypoxia‐inducible factor‐1 (HIF‐1). The activity of HIF‐1 is determined by the accumulation of its α subunit which is regulated, in part, by oxidative stress (ROS) and nitric oxide (NO), both of them highly dependent on PARP‐1. Besides, HIF‐1α can be induced by iron chelators such as deferoxamine (DFO). In this sense, the therapeutical use of DFO to strengthen the post‐hypoxic response has recently been proposed. Taking into account the increasing interest and potential clinical applications of PARP inhibition and DFO treatment, we have evaluated the impact of PARP‐1 on HIF‐1α accumulation induced by treatment with DFO. Our results show that, in DFO treated cells, PARP‐1 gene deletion or inhibition decreases HIF‐1α accumulation. This lower HIF‐1α stabilization is parallel to a decreased inducible NO synthase induction and NO production, a higher response of some antioxidant enzymes (particularly glutathione peroxidase and glutathione reductase) and a lower ROS level. Taken together, these results suggest that the absence of PARP‐1 modulates HIF‐1 accumulation by reducing both NO and oxidative stress. J. Cell. Biochem. 104: 2248–2260, 2008.

Poly(ADP-ribose) polymerase-1 modulation of in vivo response of brain hypoxia-inducible factor-1 to hypoxia/reoxygenation is mediated by nitric oxide and factor inhibiting HIF

Poly(ADP‐ribose) polymerase‐1 (PARP‐1) is a nuclear protein that once activated by genotoxic agents, modulates its own activity and that of several other nuclear proteins. The absence or pharmacological inhibition of this protein has been proven to be beneficial in the treatment of different diseases involving a hypoxic situation. We previously reported that PARP‐1 modulates the hypoxia‐inducible factor‐1 (HIF‐1) response in vitro, but this effect has not yet been demonstrated in vivo. The brain is especially susceptible to hypoxic injury, and the present study demonstrates that PARP‐1 plays a major role in the post‐hypoxic response of HIF‐1α in the cerebral cortex. Immediate post‐hypoxic HIF‐1α accumulation was higher in the presence of PARP‐1, and this differential response was mediated by nitric oxide and to a lesser extent, reactive oxygen species. PARP‐1 was also found to induce a more rapid but less sustained HIF‐1 transcriptional activity by up‐regulating the factor inhibiting HIF. The implication of PARP‐1 in these results was further demonstrated by pharmacologically inhibiting PARP in wild‐type mice. In conclusion, our data suggest that PARP‐1 has an important regulatory role in the in vivo response of brain HIF‐1 to hypoxia/reoxygenation.

The nitric oxide system response to hypoxia/reoxygenation in the aged cerebral cortex.

Aged individuals are more susceptible to hypoxic insults, but little is known about the response of the nitric oxide (NO) system to hypoxia in the senescent brain. We have analysed the effect of aging on the hypobaric hypoxia/reoxygenation NO synthase (NOS) expression and activity in the cerebral cortex. In aged animals, the absence of significant changes in NOx and activity indicates a weaker response of the systems involving NO production in this pathological situation. The nNOS protein levels remained invariable and similar in both age groups after hypoxia, although in aged animals the mRNA did not change and was consistently lower than in adults. Both eNOS mRNA and protein increased shortly after hypoxia. However, although eNOS protein levels were quite similar in both age groups, the increase appeared later and was less persistent in aged animals. Real-time RT-PCR revealed a similar basal inducible NOS (iNOS) mRNA expression that responded late in reoxygenation, mainly in aged rats. However, neither iNOS protein nor activity was detected in any age group. Altogether our results indicate that aging attenuates the response of the NO system to a hypoxic injury, particularly at eNOS level, the activity of which is crucial for maintaining vascular homeostasis.

Upregulation of endothelial nitric oxide synthase maintains nitric oxide production in the cerebellum of thioacetamide cirrhotic rats

This study examines the expression and cellular distribution pattern of nitric oxide synthase (NOS) isoforms, nitrotyrosine-derived complexes, and the nitric oxide (NO) production in the cerebellum of rats with cirrhosis induced by thioacetamide (TAA). The results showed local changes in the tissue distribution pattern of the NOS isoforms and nitrated proteins in the cerebellum of these animals. Particularly, eNOS immunoreactivity in perivascular glial cells of the white matter was detected only in TAA-treated animals. In addition, although neither neuronal NOS (nNOS) nor inducible NOS (iNOS) cerebellar protein levels appeared to be affected, the endothelial NOS (eNOS) isoform significantly increased its expression, and NO production slightly augmented in TAA-treated rats. These NOS/NO changes may contribute differently to the evolution of the hepatic disease either by maintaining the guanosine monophosphate-NO signal transduction pathways and the physiological cerebellar functions or by inducing oxidative stress and cell damage. This model gives rise to the hypothesis that the upregulation of the eNOS maintains the physiological production of NO, while the iNOS is silenced and the nNOS remains unchanged. The differential NOS-distribution and expression pattern may be one of the mechanisms involved to balance cerebellar NO production in order to minimize TAA toxic injury. These data help elucidate the role of the NOS/NO system in the development and progress of hepatic encephalopathy associated with TAA cirrhosis.