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A mitotic spindle requirement for DNA damage-induced apoptosis in Chinese hamster ovary cells
P A Johnson1, P Clements, K Hudson
1School of Biological Sciences, University of Manchester, United Kingdom.
Abstract:
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, approximately 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.
Insights
DNA repair-defective Chinese hamster ovary (CHO) cells show that mitotic spindle assembly is required for apoptosis after DNA strand breaks, not nuclear fragmentation or replication errors.
Area of Science:
- Cell Biology
- Genetics
- Molecular Toxicology
Background:
- Electrophilic agents cause DNA damage, but cellular responses are complex.
- DNA repair-defective cells help isolate responses to specific genetic lesions.
- The EM9 Chinese hamster ovary (CHO) cell line is sensitive to ethyl methanesulfonate (EMS) and deficient in DNA single-strand break repair.
Purpose of the Study:
- To investigate how CHO cells link unrepaired DNA strand breaks to cell death.
- To elucidate the role of mitotic spindle assembly in apoptosis following DNA damage.
Main Methods:
- Utilized the DNA repair-defective EM9 CHO cell line and its parental AA8 cells.
- Treated cells with the alkylating agent ethyl methanesulfonate (EMS).
- Employed flow cytometry and nocodazole (a mitotic spindle poison) to analyze cell cycle progression, apoptosis, and micronucleation.
Main Results:
- EMS-treated EM9 cells exhibited prolonged G2 arrest, abnormal mitoses (multipolar spindles, abnormal chromosomes), micronucleation, and apoptosis.
- Nocodazole significantly reduced EMS-induced apoptosis but did not affect micronucleation frequency.
- Nocodazole treatment led to a second round of DNA replication without mitosis, indicating nuclear fragmentation and replication errors alone do not trigger apoptosis.
Conclusions:
- Mitotic spindle assembly is essential for triggering apoptosis in response to DNA strand breaks in CHO cells.
- Nuclear fragmentation and inappropriate DNA replication are insufficient to induce cell death following DNA damage.
- The study clarifies the specific role of the mitotic process in DNA damage-induced cell death pathways.