Dnmt1 gene Transcriptional Regulation by Oxidative Stress Caused by Anchorage Blockade Assays in melanocytes

Abstract

Melanoma represents only 4% of skin cancer types but it has high rates of mortality, especially in advanced cases where there are not effective treatments. Like other cancer types, genetic predisposition, exposure to environmental factors and epigenetic changes can contribute to melanoma development. DNA methylation is one of the most important eukaryotic epigenetic mechanisms and in normal cells promotes silencing of expression of certain genes and maintaining genome stability. Changes in these patterns can lead to cancer cells formation. DNA methyltransferases (Dnmts) are a group of enzymes that act in this process. Different melanoma cell lines were established by our group after submitting a immortalized murine melanocyte cell line (melan-a) to cycles of anchorage blockade. Our group has already showed increased global DNA methylation as well increased dnmt1 (DNA methyltransfase 1) protein levels few hours after melan-a anchorage blockade. Additionally, this increase seems to be related with the high amounts of superoxide anion (O2-) produced during this process. Moreover, we showed that treating melan-a cells during anchorage blockade with Ras inhibitor (FTI) or Erk inhibitor (UO126) abrogates superoxide anion increase, DNA global hypermethylation and Dnmt1 overexpression obeserved. RAS/MEK/ERK signaling pathway seems to be regulated by and regulates superoxide anion production and its activation could be responsible for the high dnmt1 protein level and changes in global DNA methylation during the loss of cell adhesion. Therefore, this work's aim is to uncover the ways in which the O2- modulates dnmt1 gene transcription during the lockout condition anchor. For this, we are going to verify which transcription factors interact with Dnmt1 gene and if they are essential for the transcriptional activity of its promoter. We will also check which pathways are inhibited after O2- depletion or Ras inhibition. This study is very relevant once could elucidate the molecular mechanisms involved in the early stages of melanoma progression. Additionally, may provide a useful data to new drugs development, which could act directly in this pathway.