Abstract
O6-Methylguanine produced in DNA can pair with thymine during DNA replication, thus leading to a G-to-A transition mutation. To prevent such outcomes, cells harboring O6-methylguanine-containing mispair undergo apoptosis that requires the function of mismatch repair (MMR) protein complex. To identify the genes involved in the induction of apoptosis, we performed gene-trap mutagenesis and isolated a clone of mouse cells exhibiting an increased resistance to the killing effect of an alkylating agent, N-methyl-N-nitrosourea (MNU). The mutant carries an insertion in the Hmga2 gene, which belongs to a gene family encoding the high-mobility group A non-histone chromatin proteins. To elucidate the function of HMGA proteins in the apoptosis pathway, we introduced siRNAs for HMGA1 and/or HMGA2 into human HeLa MR cells defective in O6-methylguanine-DNA methyltransferase. HMGA1- and HMGA2-single knockdown cells showed an increased resistance to MNU, and HMGA1/HMGA2-double knockdown cells exhibited further increased tolerance compared to the control. The phosphorylation of ATR and CHK1, the appearance of a sub-G1 population, and caspase-9 activation were suppressed in the knockdown cells, although the formation of mismatch recognition complex was unaffected. These results suggest that HMGA family proteins function at the step following the damage recognition in the process of apoptosis triggered by O6-methylguanine.
Highlights
The modification of DNA bases occurs spontaneously during normal cell growth, and the rate of formation of modified bases increases considerably when cells are exposed to certain chemical agents
We further showed that HMGA1, another high-mobility group A (HMGA) family protein, functions in the induction of apoptosis in response to O6-meG
HeLa MR cells transfected with siRNA specific for either HMGA1 or HMGA2 acquired certain degrees of resistance to MNU, and double-knockdown cells treated with both siHMGA1 and siHMGA2 exhibited further resistance to the agent, implying that these two proteins cooperate in the process of apoptosis triggered by MNU
Summary
The modification of DNA bases occurs spontaneously during normal cell growth, and the rate of formation of modified bases increases considerably when cells are exposed to certain chemical agents. In cells treated with MNU, the activation of the DNA damage response, which involves the phosphorylation of checkpoint kinases, such as ATR, CHK1, ATM, and CHK2, have been shown to take place in an MMR protein-dependent manner[9,10,11]. Gene-trap mutagenesis is a useful technique for identifying genes that function in various cellular processes By applying this method to a mouse-derived cell line, we previously isolated the new genes Mapo[1] and Mapo[2], which are involved in the O6-meG-induced apoptosis[12,15]. HMGA2 overexpression is involved in doxorubicin-induced G2-M cell cycle delay and induces persistent phosphorylation of H2AX by modulating the activation of ATM24 These data suggest possible roles of HMGA proteins in the signaling pathway in response to DNA damage
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