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Clonogenic Assays to Detect Cell Fate in Mitotic Catastrophe
José Manuel Bravo-San Pedro1,2,3, Oliver Kepp1,2, Allan Sauvat1,2
1Centre de Recherche des Cordeliers, Sorbonne Université, Inserm, Université de Paris, Equipe 11 Labellisée par la Ligue Contre le Cancer, Paris, France.
Abstract:
Mitotic catastrophe (MC) is a cell death modality induced by DNA damage that involves the activation of cell cycle checkpoints such as the "DNA structure checkpoint" and "spindle assembly checkpoint" (SAC) leading to aberrant mitosis. Depending on the signal, MC can drive the cell to death or to senescence. The suppression of MC favors aneuploidy. Several cancer therapies, included microtubular poisons and radiations, trigger MC. The clonogenic assay has been used to study the capacity of single cells to proliferate and to generate macroscopic colonies and to evaluate the efficacy of anticancer drugs. Nevertheless, this method cannot analyze MC events. Here, we report an improved technique based on the use of human colon cancer HCT116 stable expressing histone H2B-GFP and DsRed-centrin proteins, allowing to determine the capacity of cells to proliferate, and to determine changes in the nucleus and centrosomes.
Insights
Mitotic catastrophe (MC), a cell death pathway triggered by DNA damage, can now be analyzed using a new technique. This method tracks cell proliferation, nucleus, and centrosome changes, overcoming limitations of traditional clonogenic assays.
Area of Science:
- Cell Biology
- Cancer Research
- Genetics
Background:
- Mitotic catastrophe (MC) is a form of cell death resulting from DNA damage, involving cell cycle checkpoints and aberrant mitosis.
- MC can lead to cell death or senescence, and its suppression promotes aneuploidy.
- Cancer therapies like radiation and microtubule poisons can induce MC, but traditional clonogenic assays cannot analyze these events.
Purpose of the Study:
- To develop an improved technique for analyzing mitotic catastrophe (MC) events.
- To overcome the limitations of clonogenic assays in studying MC.
- To assess cell proliferation, nuclear, and centrosome changes during MC.
Main Methods:
- Utilized human colon cancer HCT116 cells stably expressing histone H2B-GFP and DsRed-centrin.
- Developed a novel method to monitor cell proliferation and dynamic changes in nucleus and centrosomes.
- Integrated live-cell imaging to observe MC-related cellular events.
Main Results:
- The new technique allows for the determination of cell proliferation capacity.
- The method enables the analysis of changes occurring in the nucleus and centrosomes.
- This approach provides a more comprehensive understanding of MC compared to clonogenic assays.
Conclusions:
- The developed technique offers a powerful tool for studying mitotic catastrophe.
- This method enhances the analysis of cell death modalities induced by DNA damage.
- The findings contribute to a better understanding of cancer therapy efficacy and cellular responses to DNA damage.
