Investigating dynamics of checkpoint activation in response to DNA alkylation damage in breast epithelial cells

Abstract

N-nitroso-N-methylurea (MNU) is an SN1 type DNA methylating agent which forms the modified base O6-methylguanine. Two major pathways involved in mediating DNA damage response in mammalian cells are ATM through Chk2 for double-strand breaks and ATR through Chk1 for single-strand breaks or stalled replication forks. We investigated the dynamics of checkpoint activation in breast adenocarcinoma-derived MCF7 cells at different concentrations of MNU and found a correlation between degradation of the suicidal repair enzyme O6-methylguanine DNA methyltransferase (MGMT) and activation of the DNA damage signaling pathways. ATM phosphorylation was detected at all MNU doses; however phosphorylation of effector kinases, Chk1 and Chk2, was observed only at lower MNU concentrations. Interestingly, at higher doses of MNU, activation of caspase 9 occurred in absence of Chk1 and Chk2 phosphorylation. MCF10A cells of normal breast epithelial origin and MGMT-depleted MCF7 cells showed activation of DNA damage checkpoint kinases at a much lower MNU concentration in comparison with MCF7 cells. ATM, Chk2 and Chk1 were phosphorylated at the same time point after induction of MNU damage; however for damage induced by the DNA ethylating agent N-nitroso-N-ethylurea (NEU), ATM and Chk2 phosphorylation preceded Chk1 phosphorylation. Interestingly, Chk1 phosphorylation induced after NEU damage was independent of ATM and DNA-PK activation. We have also established a 3-dimensional basement membrane culture of MCF10A cells to investigate early tumorigenic effects of MNU-induced damage during different stages of development of human mammary acini.