Francisco Javier Oliver

Poly(ADP-ribose) signaling in cell death

Poly(ADP-ribosyl)ation (PARylation) is a reversible protein modification carried out by the concerted actions of poly(ADP-ribose) polymerase (PARP) enzymes and poly(ADP-ribose) (PAR) decomposing enzymes such as PAR glycohydrolase (PARG) and ADP-ribosyl hydrolase 3 (ARH3). Reversible PARylation is a pleiotropic regulator of various cellular functions but uncontrolled PARP activation may also lead to cell death. The cellular demise pathway mediated by PARylation in oxidatively stressed cells has been described almost thirty years ago. However, the underlying molecular mechanisms have only begun to emerge relatively recently. PARylation has been implicated in necroptosis, autophagic cell death but its role in extrinsic and intrinsic apoptosis appears to be less predominant and depends largely on the cellular model used. Currently, three major pathways have been made responsible for PARP-mediated necroptotic cell death: (1) compromised cellular energetics mainly due to depletion of NAD, the substrate of PARPs; (2) PAR mediated translocation of apoptosis inducing factor (AIF) from mitochondria to nucleus (parthanatos) and (3) a mostly elusive crosstalk between PARylation and cell death/survival kinases and phosphatases. Here we review how these PARP-mediated necroptotic pathways are intertwined, how PARylation may contribute to extrinsic and intrinsic apoptosis and discuss recent developments on the role of PARylation in autophagy and autophagic cell death.

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.

Assessing the Use of p16INK4a Promoter Gene Methylation in Serum for Detection of Bladder Cancer

Objective: This study was undertaken to investigate whether hypermethylation in p16 INK4a gene promoter could serve as plasma biomarker of bladder cancer. Methods and Patients: We examined the p16 INK4a status using methylation-specific PCR in 86 cancer patients and 49 controls (31 healthy people and 18 patients with benign urological diseases). Results: The p16 INK4a methylation was found in 22% of the serum samples and in 26% of the bladder cancer biopsies; one of them with carcinoma in situ. The presence of hypermethylated p16 INK4a in serum seems to be a product from tumour cells because a strong statistical association was found between both matched DNA signals (p < 0:0001). Using the control group, the presence of methylated p16 INK4a in the serum of individuals with suspicion of bladder cancer was found to be associated with the tumour presence (p ¼ 0:0009). Aberrant p16 INK4a methylation was also observed in one non-cancer patient, which is undergoing further assessment. Conclusions: According with our results, methylation of p16 INK4a promoter may be involved in the bladder cancer genesis and the presence of p16 INK4a methylated in serum of these patients could be useful in the cancer diagnosis with values of sensitivity, specificity and positive predictive value of 0.226, 0.950 and 0.98, respectively. These figures support the use of methylated p16 INK4a as a new class of tumour marker in bladder cancer.

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.)

Human umbilical cord stromal stem cell express CD10 and exert contractile properties

BACKGROUND It has been demonstrated that human umbilical cord stromal stem cells (UCSSCs) are bio-equivalent to bone marrow mesenchymal stem cells. However, little is known about their tissue origin or in vivo functions, and data on their expansion properties are limited due to early senescence in the culture methods described to date. METHODS UC sections and cultured UCSSCs were analyzed with a panel of 12 antibodies. UCSSCs were grown in low-FCS containing medium at 5% or 21% oxygen and were assayed for their clonogenic properties, karyotype stability, expression of specific cellular markers, and multi-lineage potential. UCSSC contractile properties were evaluated by using collagen gel contraction assays under cytokine stimulus. RESULTS Immunohistochemistry studies showed that the UCSSCs were derived from the Whartons jelly and not from the vascular smooth muscle sheath of the blood vessels. UCSSC growth properties were increased in a 5% oxygen atmosphere in comparison to normoxic culture conditions. In both culture conditions, UCSSCs were CD14-, CD34-, and CD45-negative while expressing high levels of CD73, CD90 and CD105 and maintaining their differentiation potentialities. UCSSCs expressed alpha smooth muscle actin and behaved as functional myofibroblasts when cellular contraction was challenged with appropriate stimuli. CONCLUSIONS UCSCs are mesenchymal stem cells that reside in the perivascular area of Whartons jelly and are phenotypically and functionally related to myofibroblasts.

Inhibition of poly (ADP-ribose) polymerase-1 enhances doxorubicin activity against liver cancer cells

The purpose of this study was to investigate whether PARP-1 inhibition sensitizes human liver cancer cell lines to doxorubicin treatment. Both the addition of PARP-1 inhibitor (ANI) and depletion by means of stable siRNA significantly enhanced the growth inhibition induced by the DNA damage agents used. This effect was associated with an accumulation of unrepaired DNA, with a reduction in EGFR and Bcl-xL gene expression as well as with positive annexin-V staining. These results provide novel evidence of the direct role of PARP-1 in tumour chemoresistance in relation to its effects on the transcription of key genes involved in tumour survival.

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 importance of bystander effects in radiation therapy in melanoma skin-cancer cells and umbilical-cord stromal stem cells

PURPOSE To examine direct and bystander radiation-induced effects in normal umbilical-cord stromal stem cell (HCSSC) lines and in human cancer cells. MATERIALS AND METHODS The UCSSC lines used in this study were obtained in our laboratory. Two cell lines (UCSSC 35 and UCSSC 37) and two human melanoma skin-cancer cells (A375 and G361) were exposed to ionizing radiation to measure acute radiation-dosage cell-survival curves and radiation-induced bystander cell-death response. Normal cells, although extremely sensitive to ionizing radiation, were resistant to the bystander effect whilst tumor cells were sensitive to irradiated cell-conditioned media, showing a dose-response relationship that became saturated at relatively low doses. We applied a biophysical model to describe bystander cell-death through the binding of a ligand to the cells. This model allowed us to calculate the maximum cell death (χ(max)) produced by the bystander effect together with its association constant (K(By)) in terms of dose equivalence (Gy). The values obtained for K(By) in A375 and G361 cells were 0.23 and 0.29 Gy, respectively. CONCLUSION Our findings help to understand how anticancer therapy could have an additional decisive effect in that the response of sub-lethally hit tumor cells to damage might be required for therapy to be successful because the survival of cells communicating with irradiated cells is reduced.

Dioxin receptor regulates aldehyde dehydrogenase to block melanoma tumorigenesis and metastasis

BackgroundThe dioxin (AhR) receptor can have oncogenic or tumor suppressor activities depending on the phenotype of the target cell. We have shown that AhR knockdown promotes melanoma primary tumorigenesis and lung metastasis in the mouse and that human metastatic melanomas had reduced AhR levels with respect to benign nevi.MethodsMouse melanoma B16F10 cells were engineered by retroviral transduction to stably downregulate AhR expression, Aldh1a1 expression or both. They were characterized for Aldh1a1 activity, stem cell markers and migration and invasion in vitro. Their tumorigenicity in vivo was analyzed using xenografts and lung metastasis assays as well as in vivo imaging.ResultsDepletion of aldehyde dehydrogenase 1a1 (Aldh1a1) impairs the pro-tumorigenic and pro-metastatic advantage of melanoma cells lacking AhR expression (sh-AhR). Thus, Aldh1a1 knockdown in sh-AhR cells (sh-AhR + sh-Aldh1a1) diminished their migration and invasion potentials and blocked tumor growth and metastasis to the lungs in immunocompetent AhR+/+ recipient mice. However, Aldh1a1 downmodulation in AhR-expressing B16F10 cells did not significantly affect tumor growth in vivo. Aldh1a1 knockdown reduced the high levels of CD133+/CD29+/CD44+ cells, melanosphere size and the expression of the pluripotency marker Sox2 in sh-AhR cells. Interestingly, Sox2 increased Aldh1a1 expression in sh-AhR but not in sh-AhR + sh-Aldh1a1 cells, suggesting that Aldh1a1 and Sox2 may be co-regulated in melanoma cells. In vivo imaging revealed that mice inoculated with AhR + Aldh1a1 knockdown cells had reduced tumor burden and enhanced survival than those receiving Aldh1a1-expressing sh-AhR cells.ConclusionsAldh1a1 overactivation in an AhR-deficient background enhances melanoma progression. Since AhR may antagonize the protumoral effects of Aldh1a1, the AhRlow-Aldh1a1high phenotype could be indicative of bad outcome in melanoma.