The Role Of p53 And SP1 In DNMTs Gene Regulation
Vanessa SOUSA, Instituto Nacional de Câncer (INCA), Brazil
SIMÃO T. 2
, COSTA N. 1
, PINTO L. 1,2
1 Programa de Carcinogênese Molecular, Centro de Pesquisa (CPQ), Instituto Nacional de Câncer (INCA), Brazil
2 Laboratório de Toxicologia e Biologia Molecular, Departamento de Bioquímica, Instituto de Biologia Roberto Alcantara Gomes (IBRAG), Universidade do Estado do Rio de Janeiro (UERJ), Brazil
TP53 mutations are the most frequent alterations found in tumors and the loss of its encoded protein activity can lead to genetic instability. Epigenetic alterations have recently been recognized as key events in tumors development. DNA methylation is an epigenetic mechanism characterized by the methylation of cytosines in CpG dinucleotides and catalyzed by the DNA methyltransferases (DNMTs). The aim of this project is to evaluate the role of p53 and SP1 (transcription factor overexpressed in a wide range of tumor and p53 partner in the regulation of some genes) in DNMTs gene regulation. The possible gene regulation of DNMTs by p53 may be an important indicator of the interaction between genetic and epigenetic mechanisms involved in tumors development. DNMTs mRNA (qRT-PCR) and protein expression (Western blotting) were evaluated in the esophageal squamous cell carcinoma cell lines TE-1 (TP53 temperature sensitive mutant: 32°C - WT p53 and 37°C - mutant p53) and TE-13 (p53 null) and in the colorectal carcinoma cell lines HCT116 p53 + / + and HCT116 p53 - /-. DNMTs and SP1 expression levels were higher in cell lines presenting null or mutant p53. TP53 silencing led to DNMTs increased expression in cells expressing mutant p53 whereas in p53 competent cells, the opposite effect was observed. Transfection with a p53 expression vector and treatment with the DNA alkylating agent methyl methanesulfonate resulted in augmentation of DNMTs levels only in p53-competent cells. SP1 silencing led to a reduced DNMTs expression in p53-deficient cells and increased in p53 wild-type cells. Conversely, transfection of SP1 expression vector resulted in the opposite effect. Together, these data show that p53 and SP1 play a role in DNMTs gene regulation, in a dose-dependent manner, and further experiments are needed to understand the mechanisms by which this regulation occurs. Financial support: CNPq, CAPES and MS.