A mitotic spindle requirement for DNA damage-induced apoptosis in Chinese hamster ovary cells

Promiscuously reactive electrophilic agents induce DNA and other cellular d amage. DNA repair-defective cells, when compared with genetically matched, repair-proficient parental cells, provide a means to distinguish cellular r esponses triggered by individual genetic lesions from other macromolecular damage. The Chinese hamster ovary (CHO) cell line EM9 is hypersensitive to the alkylating agent ethyl methanesulfonate (EMS) and-is unable efficiently to repair DNA single strand breaks in contrast to parental AA8 cells. EM9 was used to examine how CHO cells couple unrepaired DNA strand breaks:to lo ss of viability. Flow cytometry revealed that EMS-treated EM9 cells underwe nt prolonged cell cycle arrest in G(2), followed by entry into mitosis, mic ronucleation, and apoptosis, EM9 cells synchronized in G(1) prior to EMS tr eatment entered mitosis 24-36 h after release from synchrony, similar to 12 h after untreated control cells. Mitoses in EMS-treated cells were abnorma l, involving multipolar mitotic spindles and elongated and/or incompletely condensed chromosomes, The mitotic spindle poison nocodazole reduced DNA da mage-induced apoptosis by >60%, whereas the frequency of micronucleation wa s similar in the presence or absence of nocodazole. Flow cytometry revealed that nocodazole-treated cells sustained a second round of DNA replication without intervening mitosis, These results demonstrate that nuclear fragmen tation and inappropriate DNA replication are insufficient to trigger apopto sis following DNA strand breakage and demonstrate a requirement for mitotic spindle assembly for this process in CHO cells.