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A Mitotic Spindle Requirement for DNA Damage-induced Apoptosis in Chinese Hamster Ovary Cells

School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom [P. A. J., P. C., K. W. C.], and Zeneca Pharmaceuticals, Alderley Park, Alderley Edge, United Kingdom [K. H.]

Promiscuously reactive electrophilic agents induce DNA and other cellular damage. DNA repair-defective cells, when compared with genetically matched, repair-proficient parental cells, provide a means to distinguish cellular responses 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 loss of viability. Flow cytometry revealed that EMS-treated EM9 cells underwent prolonged cell cycle arrest in G₂, followed by entry into mitosis, micronucleation, and apoptosis. EM9 cells synchronized in G₁ prior to EMS treatment entered mitosis 24–36 h after release from synchrony, 12 h after untreated control cells. Mitoses in EMS-treated cells were abnormal, involving multipolar mitotic spindles and elongated and/or incompletely condensed chromosomes. The mitotic spindle poison nocodazole reduced DNA damage-induced apoptosis by >60%, whereas the frequency of micronucleation was 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 fragmentation and inappropriate DNA replication are insufficient to trigger apoptosis following DNA strand breakage and demonstrate a requirement for mitotic spindle assembly for this process in CHO cells.

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