David Martín-Oliva

PARP-1 is involved in autophagy induced by DNA damage

Autophagy is a lysosome-dependent degradative pathway frequently activated in tumor cells treated with chemotherapy or radiation. PARP-1 has been implicated in different pathways leading to cell death and its inhibition potentiates chemotherapy-induced cell death. Whether PARP-1 participates in the cell’s decision to commit to autophagy following DNA damage is still not known. To address this issue PARP-1 wild type and deficient cells have been treated with a dose of doxorubicin that induces autophagy. Electron microscopy examination and GFP-LC3 transfection revealed autophagic vesicles and increased expression of genes involved in autophagy (bnip-3, cathepsin b and l and beclin-1) in wild type cells treated with doxo but not in parp-1-/- cells or cells treated with a PARP inhibitor. Mechanistically the lack of autophagic features in PARP-1 deficient/PARP inhibited cells is attributed to prevention of ATP and NAD+ depletion and to the activation of the key autophagy regulator mTOR. Pharmacological or genetical inhibition of autophagy results in increased cell death, suggesting a protective role of autophagy induced by doxorubicin. These results suggest that autophagy might be cytoprotective during the response to DNA damage and suggest that PARP-1 activation is involved in the cell’s decision to undergo autophagy.

Interaction between ATM and PARP-1 in response to DNA damage and sensitization of ATM deficient cells through PARP inhibition

ATM and PARP-1 are two of the most important players in the cells response to DNA damage. PARP-1 and ATM recognize and bound to both single and double strand DNA breaks in response to different triggers. Here we report that ATM and PARP-1 form a molecular complex in vivo in undamaged cells and this association increases after γ-irradiation. ATM is also modified by PARP-1 during DNA damage. We have also evaluated the impact of PARP-1 absence or inhibition on ATM-kinase activity and have found that while PARP-1 deficient cells display a defective ATM-kinase activity and reduced γ-H2AX foci formation in response to γ-irradiation, PARP inhibition on itself is able to activate ATM-kinase. PARP inhibition induced γ H2AX foci accumulation, in an ATM-dependent manner. Inhibition of PARP also induces DNA double strand breaks which were dependent on the presence of ATM. As consequence ATM deficient cells display an increased sensitivity to PARP inhibition. In summary our results show that while PARP-1 is needed in the response of ATM to gamma irradiation, the inhibition of PARP induces DNA double strand breaks (which are resolved in and ATM-dependent pathway) and activates ATM kinase.

Embryonic and postnatal development of microglial cells in the mouse retina

Macrophage/microglial cells in the mouse retina during embryonic and postnatal development were studied by immunocytochemistry with Iba1, F4/80, anti‐CD45, and anti‐CD68 antibodies and by tomato lectin histochemistry. These cells were already present in the retina of embryos aged 11.5 days (E11.5) in association with cell death. At E12.5 some macrophage/microglial cells also appeared in peripheral regions of the retina with no apparent relationship with cell death. Immediately before birth microglial cells were present in the neuroblastic, inner plexiform (IPL), and ganglion cell (GCL) layers, and their distribution suggested that they entered the retina from the ciliary margin and the vitreous. The density of retinal microglial cells strongly decreased at birth, increased during the first postnatal week as a consequence of the entry of microglial precursors into the retina from the vitreous, and subsequently decreased owing to the cessation of microglial entry and the increase in retina size. The mature topographical distribution pattern of microglia emerged during postnatal development of the retina, apparently by radial migration of microglial cells from the vitreal surface in a vitreal‐to‐scleral direction. Whereas microglial cells were only seen in the GCL and IPL at birth, they progressively appeared in more scleral layers at increasing postnatal ages. Thus, microglial cells were present within all layers of the retina except the outer nuclear layer at the beginning of the second postnatal week. Once microglial cells reached their definitive location, they progressively ramified. J. Comp. Neurol. 506:224–239, 2008.

Modulation of Transcription by PARP-1: Consequences in Carcinogenesis and Inflammation

Post-translational modification of proteins by poly(ADP-ribosyl)ation is involved in the regulation of a number of biological functions. While an 18 member superfamily of poly(ADP-ribose) polymerases (PARP)s has been described PARP-1 accounts for more than 90% of the poly(ADP-ribosyl)ating capacity of the cells. PARP-1 act as a DNA nick sensor and is activated by DNA breaks to cleave NAD(+) into nicotinamide and ADP-ribose to synthesize long branching poly(ADP-ribose) polymers (PAR) covalently attached to nuclear acceptor proteins. Whereas activation of PARP-1 by mild genotoxic stimuli facilitate DNA repair and cell survival, severe DNA damage triggers different pathways of cell death including PARP-mediated cell death through the translocation of apoptosis inducing factor (AIF) from the mitochondria to the nucleus. PAR and PARP-1 have also been described as having a function in transcriptional regulation through their ability to modify chromatin-associated proteins and as a cofactor of different transcription factors, most notably NF-kappaB and AP-1. Pharmacological inhibition or genetic ablation of PARP-1 not only provided remarkable protection from tissue injury in various oxidative stress-related disease models but it result in a clear benefit in the treatment of cancer by different mechanisms including selective killing of homologous recombination-deficient tumor cells, down regulation of tumor-related gene expression and decrease in the apoptotic threshold in the co-treatment with chemo and radiotherapy. We will summarize in this review the current findings and concepts for the role of PARP-1 and poly(ADP-ribosyl)ation in the regulation of transcription, oxidative stress and carcinogenesis.

Inhibition of Poly(ADP-Ribose) Polymerase Modulates Tumor-Related Gene Expression, Including Hypoxia-Inducible Factor-1 Activation, during Skin Carcinogenesis

Poly(ADP-ribose) polymerase (PARP)-1, an enzyme that catalyzes the attachment of ADP ribose to target proteins, acts as a component of enhancer/promoter regulatory complexes. In the present study, we show that pharmacologic inhibition of PARP-1 with 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinolinone (DPQ) results in a strong delay in tumor formation and in a dramatic reduction in tumor size and multiplicity during 7,12-dimethylbenz(a)anthracene plus 12-O-tetradecanoylphorbol-13-acetate-induced skin carcinogenesis. This observation was parallel with a reduction in the skin inflammatory infiltrate in DPQ-treated mice and tumor vasculogenesis. Inhibition of PARP also affected activator protein-1 (AP-1) activation but not nuclear factor-kappaB (NF-kappaB). Using cDNA expression array analysis, a substantial difference in key tumor-related gene expression was found between chemically induced mice treated or not with PARP inhibitor and also between wild-type and parp-1 knockout mice. Most important differences were found in gene expression for Nfkbiz, S100a9, Hif-1alpha, and other genes involved in carcinogenesis and inflammation. These results were corroborated by real-time PCR. Moreover, the transcriptional activity of hypoxia-inducible factor-1alpha (HIF-1alpha) was compromised by PARP inhibition or in PARP-1-deficient cells, as measured by gene reporter assays and the expression of key target genes for HIF-1alpha. Tumor vasculature was also strongly inhibited in PARP-1-deficient mice and by DPQ. In summary, this study shows that inhibition of PARP on itself is able to control tumor growth, and PARP inhibition or genetic deletion of PARP-1 prevents from tumor promotion through their ability to cooperate with the activation AP-1, NF-kappaB, and HIF-1alpha.

PARP inhibition sensitizes p53-deficient breast cancer cells to doxorubicin-induced apoptosis

p53 deficiency confers resistance to doxo (doxorubicin), a clinically active and widely used antitumour anthracycline antibiotic. The purpose of the present study was to investigate the reversal mechanism of doxo resistance by the potent PARP [poly(ADP-ribose) polymerase] inhibitor ANI (4-amino-1,8-naphthalimide) in the p53-deficient breast cancer cell lines EVSA-T and MDA-MB-231. The effects of ANI, in comparison with doxo alone, on doxo-induced apoptosis, were investigated in matched pairs of EVSA-T or MDA-MB-231 with or without ANI co-treatment. Doxo elicited PARP activation as determined by Western blotting and immunofluorescence of poly(ADP-ribose), and ANI enhanced the cytotoxic activity of doxo 2.3 times and in a caspase-dependent manner. The long-term cytotoxic effect was studied by a colony-forming assay. Using this assay, ANI also significantly potentiates the long-term cytotoxic effect with respect to treatment with doxo alone. Decrease in mitochondrial potential together with an increase in cytochrome c release, association of Bax with the mitochondria and caspase 3 activation were also observed in the presence of ANI. Therefore PARP inhibition may represent a novel way of selectively targeting p53-deficient breast cancer cells. The underlying mechanism is probably a potentiation of unrepaired DNA damage, shifting from DNA repair to apoptosis due to the effective inhibition of PARP activity.

Microglia and neuronal cell death

Microglia, the brains innate immune cell type, are cells of mesodermal origin that populate the central nervous system (CNS) during development. Undifferentiated microglia, also called ameboid microglia, have the ability to proliferate, phagocytose apoptotic cells and migrate long distances toward their final destinations throughout all CNS regions, where they acquire a mature ramified morphological phenotype. Recent studies indicate that ameboid microglial cells not only have a scavenger role during development but can also promote the death of some neuronal populations. In the mature CNS, adult microglia have highly motile processes to scan their territorial domains, and they display a panoply of effects on neurons that range from sustaining their survival and differentiation contributing to their elimination. Hence, the fine tuning of these effects results in protection of the nervous tissue, whereas perturbations in the microglial response, such as the exacerbation of microglial activation or lack of microglial response, generate adverse situations for the organization and function of the CNS. This review discusses some aspects of the relationship between microglial cells and neuronal death/survival both during normal development and during the response to injury in adulthood.

Microglial response to light-induced photoreceptor degeneration in the mouse retina

The microglial response elicited by degeneration of retinal photoreceptor cells was characterized in BALB/c mice exposed to bright light for 7 hours and then kept in complete darkness for survival times ranging from 0 hours to 10 days. Photodegeneration resulted in extensive cell death in the retina, mainly in the outer nuclear layer (ONL), where the photoreceptor nuclei are located. Specific immunolabeling of microglial cells with anti‐CD11b, anti‐CD45, anti‐F4/80, anti‐SRA, and anti‐CD68 antibodies revealed that microglial cells were activated in light‐exposed retinas. They migrated to the ONL, changed their morphology, becoming rounded cells with short and thick processes, and, finally, showed immunophenotypic changes. Specifically, retinal microglia began to strongly express antigens recognized by anti‐CD11b, anti‐CD45, and anti‐F4/80, coincident with cell degeneration. In contrast, upregulation of the antigen recognized by anti‐SRA was not detected by immunocytochemistry until 6 hours after light exposure. Differences were also observed at 10 days after light exposure: CD11b, CD45, and F4/80 continued to be strongly expressed in retinal microglia, whereas the expression of CD68 and SRA had decreased to near‐normal values. Therefore, microglia did not return to their original state after photodegeneration and continued to show a degree of activation. The accumulation of activated microglial cells in affected regions simultaneously with photoreceptor degeneration suggests that they play some role in photodegeneration. J. Comp. Neurol. 518:477–492, 2010.

Early and late skin reactions to radiotherapy for breast cancer and their correlation with radiation-induced DNA damage in lymphocytes

IntroductionRadiotherapy outcomes might be further improved by a greater understanding of the individual variations in normal tissue reactions that determine tolerance. Most published studies on radiation toxicity have been performed retrospectively. Our prospective study was launched in 1996 to measure the in vitro radiosensitivity of peripheral blood lymphocytes before treatment with radical radiotherapy in patients with breast cancer, and to assess the early and the late radiation skin side effects in the same group of patients. We prospectively recruited consecutive breast cancer patients receiving radiation therapy after breast surgery. To evaluate whether early and late side effects of radiotherapy can be predicted by the assay, a study was conducted of the association between the results of in vitro radiosensitivity tests and acute and late adverse radiation effects.MethodsIntrinsic molecular radiosensitivity was measured by using an initial radiation-induced DNA damage assay on lymphocytes obtained from breast cancer patients before radiotherapy. Acute reactions were assessed in 108 of these patients on the last treatment day. Late morbidity was assessed after 7 years of follow-up in some of these patients. The Radiation Therapy Oncology Group (RTOG) morbidity score system was used for both assessments.ResultsRadiosensitivity values obtained using the in vitro test showed no relation with the acute or late adverse skin reactions observed. There was no evidence of a relation between acute and late normal tissue reactions assessed in the same patients. A positive relation was found between the treatment volume and both early and late side effects.ConclusionAfter radiation treatment, a number of cells containing major changes can have a long survival and disappear very slowly, becoming a chronic focus of immunological system stimulation. This stimulation can produce, in a stochastic manner, late radiation-related adverse effects of varying severity. Further research is warranted to identify the major determinants of normal tissue radiation response to make it possible to individualize treatments and improve the outcome of radiotherapy in cancer patients.

Inhibition of poly adenosine diphosphate‐ribose polymerase decreases hepatocellular carcinoma growth by modulation of tumor‐related gene expression

Hepatocellular carcinoma (HCC) is associated with a poor prognosis due to a lack of effective treatment options. In HCC a significant role is played by DNA damage and the inflammatory response. Poly (ADP‐ribose) polymerase‐1 (PARP‐1) is an important protein that regulates both these mechanisms. The objective of this study was to examine the effect of pharmacology PARP‐1 inhibition on the reduction of tumor volume of HCC xenograft and on the hepatocarcinogenesis induced by diethyl‐nitrosamine (DEN). Pharmacologic PARP‐1 inhibition with DPQ greatly reduces tumor xenograft volume with regard to a nontreated xenograft (394 mm3 versus 2,942 mm3, P < 0.05). This observation was paralleled by reductions in xenograft mitosis (P = 0.02) and tumor vasculogenesis (P = 0.007, confirmed by in vitro angiogenesis study), as well as by an increase in the number of apoptotic cells in DPQ‐treated mice (P = 0.04). A substantial difference in key tumor‐related gene expression (transformed 3T3 cell double minute 2 [MDM2], FLT1 [vascular endothelial growth factor receptor‐1, VEGFR1], epidermal growth factor receptor [EPAS1]/hypoxia‐inducible factor 2 [HIF2A], EGLN1 [PHD2], epidermal growth factor receptor [EGFR], MYC, JUND, SPP1 [OPN], hepatocyte growth factor [HGF]) was found between the control tumor xenografts and the PARP inhibitor‐treated xenografts (data confirmed in HCC cell lines using PARP inhibitors and PARP‐1 small interfering RNA [siRNA]). Furthermore, the results obtained in mice treated with DEN to induce hepatocarcinogenesis showed, after treatment with a PARP inhibitor (DPQ), a significant reduction both in preneoplastic foci and in the expression of preneoplastic markers and proinflammatory genes (Gstm3, Vegf, Spp1 [Opn], IL6, IL1b, and Tnf), bromodeoxyuridine incorporation, and NF‐κB activation in the initial steps of carcinogenesis (P < 0.05). Conclusion: This study shows that PARP inhibition is capable of controlling HCC growth and preventing tumor vasculogenesis by regulating the activation of different genes involved in tumor progression. (HEPATOLOGY 2010;51:255–266.)