Induction of C-anaphase and diplochromosome through dysregulation of spindle assembly checkpoint by sodium arsenite in human fibroblasts.

Cytogenetic alterations induced by arsenite are associated with its carcinogenic activity. Cytogenetic analysis revealed first that arsenite induced c-anaphases in a time- and dose-dependent manner in human fibroblasts (HFW). With additional incubation of arsenite-arrested mitotic cells in drug-free medium for 0-48 h, approximately 35% exited from mitosis without cell division. This was confirmed by the appearance of tetraploid metaphase, mainly diplochromosomes, in the subsequent cell division. Treatment of HFW cells with both nocodazole, a known agent of microtubular depolymerization, and Taxol, which induces tubulin polymerization and inhibits disassembly of microtubules, resulted in remarkable mitotic arrest but induced only negligible c-anaphase, tetraploidy, and diplochromosomes. Staurosporine, a kinase inhibitor that could effectively reduce arsenite-induced c-anaphase, could also decrease the development of diplochromosomes in the subsequent cell division cycle. These results imply that arsenite-induced c-anaphases mainly exited from mitosis without cell division and became tetraploid in the subsequent cell cycle. Antitubulin immunofluorescent staining confirmed no formation of bipolar spindles in nocodazole-arrested mitotic HFW cells, whereas in arsenite-arrested mitotic cells bipolar spindles were present but distorted in appearance and apparently dysfunctional. Mitotic arrest deficient 2 (Mad2) signal was, as expected, clearly visible at centromeres of nocodazole-arrested mitotic cells. However, the Mad2 signal at centrosomes became insignificant in either arsenite-arrested or nocodazole/arsenite-arrested mitotic cells. In addition, the association of Mad2 with the APC/C(cdc20) complex and the accumulation of Pds1, an anaphase inhibitor, were remarkably reduced in arsenite-arrested mitotic cells as compared with nocodazole-arrested mitotic cells. These results support the observation that nocodazole can inhibit spindle formation and, hence, activate spindle assembly checkpoint to arrest cells at metaphase. In contrast, the dysfunctional bipolar spindles in arsenite-arrested mitotic cells could not effectively activate spindle assembly checkpoint and, hence, resulted in formation of c-anaphase and diplochromosomes in the subsequent cell division.