The collapse of the mechanisms which stably maintain M phase chromosome and its biological effect

邻苯基苯酚(OPP)及其肝脏代谢物苯基对苯二酚(PHQ)是广谱杀菌剂和抗菌剂。在Ames系统中，OPP和PHQ检测为阴性，而在饮食中给药的大鼠膀胱肿瘤形成方面呈阳性，显示出非遗传毒性致癌物的属性。研究还表明，OPP和PHQ在体内和体外不结合或切割DNA，而是在OPP处理的大鼠中观察到剂量依赖性的蛋白质结合。然而，OPP和PHQ会产生包括非整倍体在内的染色体异常。因此，需要阐明非遗传毒性致癌物发挥作用的步骤。在这项研究中，我们使用了酿酒酵母和培养的人类细胞的杂合性缺失(LOH)分析来确定OPP和PHQ的生物学效应。OPP和PHQ引起LOH的原因是一种功能性染色体丢失：非整倍体。PHQ在体外结合并干扰微管蛋白的解聚。我们进一步证明，由于Swe1(Wee1同源)的稳定，PHQ可以将细胞周期停滞在G2/M转变中，这可能导致了CDc28(CDK1/CDC2同源)的失活。此外，Hog1(p38MAPK同源物)被PHQ强效磷酸化，从而稳定Swe1。另一方面，Chk1和Rad53没有被PHQ磷酸化，表明Mec1/Tel1 DNA损伤检查点不起作用。Swe1和hog1的突变取消了PHQ诱导的G2/M期停滞，并对PHQ致死性和非整倍体形成产生了抗性。这些结果表明，由Hog1-Swe1途径激活的PHQ诱导的G2/M转换检查点在非整倍体的形成中发挥了作用。我们认为OPP和PHQ激活了MAPK通路，使细胞周期停滞在G2/M期，导致细胞异倍体。

Ortho-phenyl phenol (OPP) and its hepatic metabolite, phenyl hydroquinone (PHQ), are broad-spectrum fungicides and antibacterial agents. OPP and PHQ tested negative in an Ames system and positive with respect to the formation of tumors in the urinary bladder in rats when administered in diet, showing attributes of a non-genotoxic carcinogen. It has also been demonstrated that OPP and PHQ do not bind or cleave DNA in vivo or in vitro, rather dose-dependent protein binding in OPP-treated rats was observed. OPP and PHQ, however, generate chromosomal aberrations including aneuploidy. Thus, the steps by which non-genotoxic carcinogens exert their effects need to be elucidated. In this study, we used an assay of loss of heterozygosity (LOH) in Saccharomyces cerevisiae and cultured human cells to determine the biological effects of OPP and PHQ. LOH was found to be induced by OPP and PHQ because of a functional chromosome loss : aneuploidy. PHQ bound to and interfered with the depolymerization of tubulin in vitro. We further demonstrate that PHQ can arrest the cell cycle at the G2/M transition as a result of the stabilization of Swe1 (Wee1 homolog), probably leading to inactivation of the Cdc28 (Cdk1/Cdc2 homolog). Furthermore, Hog1 (p38 MAPK homolog) was robustly phosphorylated by PHQ, which can stabilize Swe1. On the other hand, Chk1 and Rad53 were not phosphorylated by PHQ, indicating that Mec1/Tel1 DNA damage checkpoint was not functional. Mutation of swe1 and hog1 abolished the PHQ-induced arrest at the G2/M transition and became resistant to PHQ lethality and aneuploidy formation. These results suggest that PHQ-induced G2/M transition checkpoint which is activated by the Hog1-Swe1 pathway plays a role in the formation of aneuploidy. We argue that OPP and PHQ activate MAPK pathway arrested cell cycle at G2/M transition and caused aneuploidy.