Effects Of PI3K Inhibitors And Irradiation On Cell Fates

Mitotic cell death is a type of cell death that occurs specificly at mitotic stages and anti-mitotic drugs is the mainstay for tumor chemotherapy. Classic anti-mitotic drugs activate the spindle assembly checkpoint through disrupting microtubule dynamics or disturbing mitotic processes, causing mitotic arrest and mitotic cell death in order to combat cancer. However, cells stuck in mitosis may exit mitosis through mitotic slippage, generating polyploid cells, which restricts the clinical applications of anti-mitotic drugs.PI3K pathway is involved in regulating cell growth, survival, motility and metabolism. Aberrant activation of PI3K was closely correlated with tumor occrrence, progression and metastasis. PI3K has also been proposed to be a target for cancer therapy. However, studies on the relationship between PI3K pathway and mitotic cell death are lacking3-Methyladenien is the most widely used autophagy inhibitor. In this study, we found that3-methyladenine could cause caspase-dependent death in HeLa cells. However, depletion of autophagy essential protein beclinl in HeLa cells or deletion of autophagy essential gene atg5in mouse embryonic fibroblasts have no effects on3-methyladenine induced cell death, indicating that autophagy inhibition-independent functions of3-methladenine caused cell death. PI3K is the only reported target for3-methyladenine, and another PI3K inhibitor wortmannin caused similar effects to3-methyladenine. Live cell imaging performed on H2B-mCherry expressing HeLa cells demonstrated that inhibitors of PI3K caused increased lagging chromosomes at prometaphase, delayed anaphase onset and increased cell death at prometaphase. Those cells that died at prometaphase induced by PI3K inhibition stayed at mitosis for an average of only six hours, which was much shorter than that induced by classic anti-mitotic drugs. We further showed that inhibiting PI3K accelerated microtubule inhibitor nocodazole induced mitotic cell death, and concominately reduced mitotic slippage.Our data showed for the first time that inhibitors of PI3K could cause arrest and cell death at prometaphase, prvoding the theoretical basis for combining PI3K inhibitors and anti-mitotic drugs to improve the efficacy of tumor therapy. Micronuclei are closely related to DNA damage. The presence of micronuclei in mammalian cells is a common phenomenon post ionizing radiation. The level of micronucleation in tumor cells has been used to predict prognosis after radiotherapy in many cancers. However, the justification for using micronuclei as the biomarker for radiosensitivity is lacking.In order to understand how irradiation-induced micronuclei affect cell fate, we performed extensive long-term live cell imaging on X-irradiated nasopharyngeal carcinoma (NPC) cells with different radiosensitivities. To visualize the dynamics of micronuclei more clearly, chromosomes were stably labeled with red fluorescent protein (RFP) by targeting to human histone H2B.Initially, significantly more micronuclei were observed in radiosensitive cells than in radioresistant cells post irradiation. Additionally, cells with micronuclei were found to be more likely to die or undergo cell cycle arrest when compared with micronucleus-free cells after irradiation, and the more micronuclei the cells contained the more likely they would die or undergo arrest. Moreover, micronucleated cells showed predisposition to produce daughter cells with micronuclei through chromosome lagging. Fluorescence in situ hybridization using human pan-centromeric probes revealed that about70%of these micronuclei and lagging chromosomes did not contain centromeric signals. Finally, DNA damage was more severe and p38stress kinase activity was higher in micronucleated cells than in micronucleus-free cells as shown by phospho-H2AX and phospho-p38immunofluorescence staining.Altogether, our observations indicated that the presence of micronuclei coupled with activated DNA damage response could compromise the proliferation capacity of irradiated cells, providing the evidence and justification for using micronucleus index as a valuable biomarker of radiosensitivity.