José Mariano Ruiz de Almodóvar

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.

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.

PARP-1 regulates metastatic melanoma through modulation of vimentin-induced malignant transformation

PARP inhibition can induce anti-neoplastic effects when used as monotherapy or in combination with chemo- or radiotherapy in various tumor settings; however, the basis for the anti-metastasic activities resulting from PARP inhibition remains unknown. PARP inhibitors may also act as modulators of tumor angiogenesis. Proteomic analysis of endothelial cells revealed that vimentin, an intermediary filament involved in angiogenesis and a specific hallmark of EndoMT (endothelial to mesenchymal transition) transformation, was down-regulated following loss of PARP-1 function in endothelial cells. VE-cadherin, an endothelial marker of vascular normalization, was up-regulated in HUVEC treated with PARP inhibitors or following PARP-1 silencing; vimentin over-expression was sufficient to drive to an EndoMT phenotype. In melanoma cells, PARP inhibition reduced pro-metastatic markers, including vasculogenic mimicry. We also demonstrated that vimentin expression was sufficient to induce increased mesenchymal/pro-metastasic phenotypic changes in melanoma cells, including ILK/GSK3-β-dependent E-cadherin down-regulation, Snail1 activation and increased cell motility and migration. In a murine model of metastatic melanoma, PARP inhibition counteracted the ability of melanoma cells to metastasize to the lung. These results suggest that inhibition of PARP interferes with key metastasis-promoting processes, leading to suppression of invasion and colonization of distal organs by aggressive metastatic cells.

ESR1 gene promoter region methylation in free circulating DNA and its correlation with estrogen receptor protein expression in tumor tissue in breast cancer patients

BackgroundTumor expression of estrogen receptor (ER) is an important marker of prognosis, and is predictive of response to endocrine therapy in breast cancer. Several studies have observed that epigenetic events, such methylation of cytosines and deacetylation of histones, are involved in the complex mechanisms that regulate promoter transcription. However, the exact interplay of these factors in transcription activity is not well understood. In this study, we explored the relationship between ER expression status in tumor tissue samples and the methylation of the 5′ CpG promoter region of the estrogen receptor gene (ESR1) isolated from free circulating DNA (fcDNA) in plasma samples from breast cancer patients.MethodsPatients (n = 110) with non-metastatic breast cancer had analyses performed of ER expression (luminal phenotype in tumor tissue, by immunohistochemistry method), and the ESR1-DNA methylation status (fcDNA in plasma, by quantitative methylation specific PCR technique).ResultsOur results showed a significant association between presence of methylated ESR1 in patients with breast cancer and ER negative status in the tumor tissue (p = 0.0179). There was a trend towards a higher probability of ESR1-methylation in those phenotypes with poor prognosis i.e. 80% of triple negative patients, 60% of HER2 patients, compared to 28% and 5.9% of patients with better prognosis such as luminal A and luminal B, respectively.ConclusionSilencing, by methylation, of the promoter region of the ESR1 affects the expression of the estrogen receptor protein in tumors of breast cancer patients; high methylation of ESR1-DNA is associated with estrogen receptor negative status which, in turn, may be implicated in the patient’s resistance to hormonal treatment in breast cancer. As such, epigenetic markers in plasma may be of interest as new targets for anticancer therapy, especially with respect to endocrine treatment.

Individualisation of radiotherapy in breast cancer patients: possible usefulness of a DNA damage assay to measure normal cell radiosensitivity

PURPOSE The purpose of this study was to determine whether the distribution of sensitivities in breast cancer patients, measured using a DNA damage assay on lymphocytes, is likely to provide sufficient discrimination to enable the reliable identification of patients with abnormal sensitivities. MATERIAL AND METHODS Radiosensitivity (x) was assessed in 226 samples of lymphocytes from unselected women with breast cancer and was quantified as the initial number of DNA double-strand breaks (dsb) induced per Gy and per DNA unit (200 Mbp). RESULTS The existence of an inter-individual variation in the parameter (x) is described through the range (0.40-4.72 dsb/Gy/DNA unit) of values found, which have been fitted to the mathematical model defined by the log-normal distribution (mu = 0.42+/-0.03; sigma = 0.52+/-0.03; R(2)=0.9475). A total of 189 patients received radiotherapy after surgical treatment. Among them, we have detected 15 patients who developed severe skin reactions and we have compared their radiosensitivity values with the rest of patients treated. CONCLUSIONS Our results suggest that DNA initial damage measured on lymphocytes offers an approach to predict the acute response of human normal tissues prior to radiotherapy. Values of x higher than 3.20 dsb/Gy/DNA unit theoretically should correspond to the highly radio-sensitive patients. Using the experimental results, we have calculated the strength of the test by means of the area under the receiver operator characteristic curves (A(Z)) to determine whether the radiosensitivity assay can discriminate between patients according to their radiation response. The value found (A(Z)=0.675+/-0.072) is indicative of a fair-poor discriminating capacity of the test to identify the patients with higher risk of developing a severe acute reaction during the radiotherapy treatment.

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.

Prediction of normal tissue response and individualization of doses in radiotherapy.

In this work we estimate the therapeutic gain that could be obtained using a radiotherapy programme in which doses were based on a radiosensitivity test that was able to predict the final response of normal tissues to radiation for each particular patient. To date, no good radiosensitivity assay has been demonstrated and by way of example we use an assay based on initial DNA damage. The individualized programme we propose is based on an increase in the dose delivered to patients showing a resistant behaviour to radiation and on the adoption of alternative programmes or a careful monitoring of those patients in whom an excessive reaction is expected. To quantify the results produced by the individualization programme, both analytical and Monte Carlo simulation methods are used. The increase in tumour control probability obtained by means of this individualization strongly depends on the dose-response curve for the particular tumour. In certain cases, this enhancement can be marked, and 40% of the patients considered in this work could attain more than 10% increase in tumour control probability. The quantitative estimations in this study indicate the need to seek a predictive assay of radiosensitivity, for both normal and tumour tissue response, in order to develop individualized treatment protocols.

Capillary electrophoresis of DNA damage after irradiation: apoptosis and necrosis.

Apoptosis is a type of cellular death but also directly regulates tumorigenesis through different gene expression. This phenomenon is often used as end-point in studies of radio- and chemosensitivity of cancer cells. Restriction DNA fragments have been separated quickly, efficiently and successfully by capillary gel electrophoresis (CGE). In this study CGE has been applied to distinguish between the discrete pattern of degraded DNA produced by apoptosis and randomized DNA breaks produced by ionizing radiation. The influence of different variables has been discussed and an example of fast separation by CGE of the apoptotic fragments produced by UV light treatment is shown.

Direct and bystander radiation effects: a biophysical model and clinical perspectives.

In planning treatment for each new patient, radiation oncologists pay attention to the aspects that they control. Thus their attention is usually focused on volume and dose. The dilemma for the physician is how to protract the treatment in a way that maximizes control of the tumor and minimizes normal tissue injury. The initial radiation-induced damage to DNA may be a biological indicator of the quantity of energy transferred to the DNA. However, until now the biophysical models proposed cannot explain either the early or the late adverse effects of radiation, and a more general theory appears to be required. The bystander component of tumor cell death after radiotherapy measured in many experimental works highlights the importance of confirming these observations in a clinical situation.

Non-homologous end-joining defect in fanconi anemia hematopoietic cells exposed to ionizing radiation

Abstract Casado, J. A., Núñez, M. I., Segovia, J. C., Ruiz de Almodóvar, J. M. and Bueren, J. A. Non-homologous End-Joining Defect in Fanconi Anemia Hematopoietic Cells Exposed to Ionizing Radiation. Radiat. Res. 164, 635–641 (2005). Fanconi anemia is a genetically heterogeneous recessive disease characterized mainly by bone marrow failure and cancer predisposition. Although it is accepted that Fanconi cells are highly sensitive to DNA crosslinking agents, their response to ionizing radiation is still unclear. Using pulsed-field gel electrophoresis, we have observed that radiation generates a similar number of DNA double-strand breaks in normal and Fanconi cells from three (FA-A, FA-C and FA-F) of the 11 complementation groups identified. Nonsynchronized as well as nonproliferating Fanconi anemia cells showed an evident defect in rejoining the double-strand breaks generated by ionizing radiation, indicating defective non-homologous end-joining repair. At the cellular level, no difference in the radiosensitivity of normal and FA-A lymphoblast cells was noted, and a modest increase in the radiosensitivity of Fanca−/− hematopoietic progenitor cells was observed compared to Fanca+/+ cells. Finally, when animals were exposed to a fractionated total-body irradiation of 5 Gy, a similar hematopoietic syndrome was observed in wild-type and Fanca−/− mice. Taken together, our observations suggest that Fanconi cells, in particular those having nonfunctional Fanconi proteins upstream of FANCD2, have a defect in the non-homologous end-joining repair of double-strand breaks produced by ionizing radiation, and that compensatory mechanisms of DNA repair and/or stem cell regeneration should limit the impact of this defect in irradiated organisms.