Department of Physiological Science and Molecular Biology, Fukuoka Dental College, Fukuoka, Japan.
PLoS One. 2012;7(9):e44817. doi: 10.1371/journal.pone.0044817. Epub 2012 Sep 24.
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.
O⁶-甲基鸟嘌呤是一种烷基化 DNA 碱基，具有很强的诱变性。含有这种损伤的细胞通过诱导细胞凋亡而被清除，这与错配修复（MMR）蛋白的功能有关。我们采用逆转录病毒介导的基因捕获诱变技术，分离与烷化剂 N-甲基-N-亚硝脲（MNU）诱导的细胞凋亡相关的新基因。本研究报告描述了一种新基因 MAPO2（O⁶-甲基鸟嘌呤诱导的凋亡 2）的鉴定，该基因最初被注释为 C1orf201。MAPO2 基因在多种真核生物中保守，编码一种含有特征性 PxPxxY 重复序列的蛋白质。为了阐明该基因产物在凋亡途径中的功能，构建了一个源自 HeLa MR 细胞的人细胞系，该细胞系通过表达特定的 miRNA 稳定敲低了 MAPO2 基因。与 HeLa MR 细胞相比，敲低细胞的生长速度相同，表明 MAPO2 在细胞生长中没有作用。用 MNU 处理后，HeLa MR 细胞和敲低细胞在 G₂/M 期停滞，但敲低细胞中出现亚 G₁ 期细胞的比例明显低于 HeLa MR 细胞。此外，敲低细胞中 BAK 和 caspase-3 的激活以及线粒体膜去极化等凋亡诱导的标志性事件也受到抑制。这些结果表明，MAPO2 基因产物可能正向促进 O⁶-甲基鸟嘌呤诱导的细胞凋亡。
