Abstract
O6-Methylguanine, one of alkylated DNA bases, is especially mutagenic. Cells containing this lesion are eliminated by induction of apoptosis, associated with the function of mismatch repair (MMR) proteins. A retrovirus-mediated gene-trap mutagenesis was used to isolate new genes related to the induction of apoptosis, triggered by the treatment with an alkylating agent, N-methyl-N-nitrosourea (MNU). This report describes the identification of a novel gene, MAPO2 (O6-methylguanine-induced apoptosis 2), which is originally annotated as C1orf201. The MAPO2 gene is conserved among a wide variety of multicellular organisms and encodes a protein containing characteristic PxPxxY repeats. To elucidate the function of the gene product in the apoptosis pathway, a human cell line derived from HeLa MR cells, in which the MAPO2 gene was stably knocked down by expressing specific miRNA, was constructed. The knockdown cells grew at the same rate as HeLa MR, thus indicating that MAPO2 played no role in the cellular growth. After exposure to MNU, HeLa MR cells and the knockdown cells underwent cell cycle arrest at G2/M phase, however, the production of the sub-G1 population in the knockdown cells was significantly suppressed in comparison to that in HeLa MR cells. Moreover, the activation of BAK and caspase-3, and depolarization of mitochondrial membrane, hallmarks for the induction of apoptosis, were also suppressed in the knockdown cells. These results suggest that the MAPO2 gene product might positively contribute to the induction of apoptosis triggered by O6-methylguanine.
Highlights
SN1 type alkylating agents, such as N-methyl-N-nitrosourea (MNU) and N-methyl-N9-nitro-N-nitorosoguanidine (MNNG), produce various methylated bases, most of which are believed to be efficiently repaired by base excision repair (BER) [1,2]
O6-methylguanine that is not repaired by methylguanine-DNA methyltransferase (MGMT) has chance to pair with thymine, and such a mispair is recognized by a mismatch repair (MMR) protein complex, composed of MSH2, MSH6, MLH1 and PMS2 [9,10,11]
Isolation of a mouse cell line defective in the Mapo2 gene Retrovirus-mediated gene-trap mutagenesis was performed to identify new genes functioning in the process of apoptosis triggered by O6-methylguanine, as described previously [22]
Summary
SN1 type alkylating agents, such as N-methyl-N-nitrosourea (MNU) and N-methyl-N9-nitro-N-nitorosoguanidine (MNNG), produce various methylated bases, most of which are believed to be efficiently repaired by base excision repair (BER) [1,2]. Mice with mutations in both the Mgmt gene and one of the mismatch repair genes, such as Mlh, are as resistant to MNU as are wild-type mice, in terms of survival, but do develop numerous tumors after receiving MNU [16]. These findings clearly indicate that the MMR proteins-dependent apoptosis contributes to the suppression of cells predisposed to form tumors [16,17]
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