Genomic instability and molecular signaling pathways involving DNA damage responses and DNA repair in human diseases

Abstract

The proposal includes two sub-projects:Sub-project-01: Oxidative stress is associated with the pathogenesis of human diseases, such as Alzheimer's disease (AD) and Diabetes Mellitus (DM). Despite the efforts to identify risk factors and protective mechanisms, the molecular basis of these diseases is still poorly elucidated. Considering the hypothesis that oxidative stress and alterations in DNA repair processes are important factors for the development of AD and type 1 diabetes mellitus (T1DM), the aims of the project are the following: 1) To analyze the transcriptional expression data (from the microarray method) to detect genes with significantly altered expression in peripheral blood mononuclear cells (PBMCs) from AD patients compared with healthy individuals (the results will be compared with the data already obtained for the T1DM group); 2) To investigate the expression of specific genes and proteins (selected based on the results obtained after performing the microarray experiments for both comparisons: DA versus controls and T1DM versus controls); 3) To study the expression of microRNAs (microarrays)in AD patients compared with healthy individuals (the results will be compared with the data already obtained for the T1DM group) aiming to seek their association with messenger RNAs, mainly for genes related to oxidative stress/DNA repair; 4) To validate microRNA-mRNA interactions via the luciferase assay; 5) To study the expression of some key proteins of the TP53 pathway in DA. The data to be generated may provide information to understand the role of oxidative stress and DNA repair in the pathogenesis of AD and T1DM, with the possibility of detecting altered biological processes in each disease, as well as common dysregulated pathways shared by both diseases.Sub-project-02: Glioblastoma multiforme (GBM) is one of the most lethal tumors and their resistance to conventional treatments is a major challenge to be overcome. The present proposal aims the application of molecular inhibition (DNA repair genes and transcription factors) to influence the responses of GBM cells to Temozolomide (TMZ) or irradiation, in order to increase cell lethality. The objectives are the following: 1) To assess the effects of PARP-1 inhibition by NU1025 in TMZ-resistant GBM cell lines (LN18 and T98G, which are proficient and deficient for PTEN, respectively); 2) To study the influence of PTEN (which also participates in the HR pathway) in cellular responses by siRNA PTEN in LN18 cells; 3) To analyze the participation of MGMT repair (using the inhibitor O6-BG) in GBM cell lines that are proficient for MGMT. The effects of combined treatments (TMZ + NU1025) will be evaluated by various methods at cellular level (clonogenic survival, cell cycle kinetics, induction of DNA damage by ³ - H2AX and apoptosis) and molecular analyses (gene expression profiles, and gene silencing by siRNA). Furthermore, on the basis of literature data and our previous results, we aim to inhibit E2F transcription factors as a strategy to increase the lethality of GBM cells, using a chemical inhibitor (HLM006474), which targets E2F family proteins. This inhibitor will be tested in GBM cells (U87MG and U343MG-a) exposed to gamma-rays, and cellular responses will be assessed by several methods performed at cellular and molecular levels (including the microarray method). Furthermore, the genetic background of these cells will also be considered for data interpretation, and some genes will be sequenced for GBM cell lines. (AU)