Distinct effects of heat shock temperatures on mitotic progression by influencing the spindle assembly checkpoint

Heat shock is a physiological and environmental stress that leads to the denaturation and inactivation of cellular proteins and is used in hyperthermia cancer therapy. Previously, we revealed that mild heat shock (42 & DEG;C) delays the mitotic progression by activating the spindle assembly checkpoint (SAC). However, it is unclear whether SAC activation is maintained at higher temperatures than 42 & DEG;C. Here, we demonstrated that a high temperature of 44 & DEG;C just before mitotic entry led to a prolonged mitotic delay in the early phase, which was shortened by the SAC inhibitor, AZ3146, indicating SAC activation. Interestingly, mitotic slippage was observed at 44 & DEG;C after a prolonged delay but not at 42 & DEG;C heat shock. Furthermore, the multinuclear cells were generated by mitotic slippage in 44 & DEG;C-treated cells. Immunofluorescence analysis revealed that heat shock at 44 & DEG;C reduces the kinetochore localization of MAD2, which is essential for mitotic checkpoint activation, in nocodazole-arrested mitotic cells. These results indicate that 44 & DEG;C heat shock causes SAC inactivation even after full activation of SAC and suggest that decreased localization of MAD2 at the kinetochore is involved in heat shock-induced mitotic slippage, resulting in multinucleation. Since mitotic slippage causes drug resistance and chromosomal instability, we propose that there may be a risk of cancer malignancy when the cells are exposed to high temperatures.