Mitotic catastrophe and cell death induced by depletion of centrosomal proteins

M Kimura,H Nagaoka,T Yoshioka,Y Okano,M Saio,Y Banno

doi:10.1038/cddis.2013.108

M Kimura, H Nagaoka + Show 4 more

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https://doi.org/10.1038/cddis.2013.108

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Abstract

Mitotic catastrophe, which refers to cell death or its prologue triggered by aberrant mitosis, can be induced by a heterogeneous group of stimuli, including chromosome damage or perturbation of the mitotic apparatus. We investigated the mechanism of mitotic catastrophe and cell death induced by depletion of centrosomal proteins that perturbs microtubule organization. We transfected cells harboring wild-type or mutated p53 with siRNAs targeting Aurora A, ninein, TOG, TACC3, γ-tubulin, or pericentriolar material-1, and monitored the effects on cell death. Knockdown of Aurora A, ninein, TOG, and TACC3 led to cell death, regardless of p53 status. Knockdown of Aurora A, ninein, and TOG, led to aberrant spindle formation and subsequent cell death, which was accompanied by several features of apoptosis, including nuclear condensation and Annexin V binding in HeLa cells. During this process, cleavage of poly(ADP-ribose) polymerase-1, caspase-3, and caspase-9 was detected, but cleavage of caspase-8 was not. Cell death, monitored by time-lapse imaging, occurred during both interphase and M phase. In cells depleted of a centrosomal protein (Aurora A, ninein, or TOG), the rate of cell death was higher if the cells were cotransfected with siRNA against BubR1 or Mad2 than if they were transfected with siRNA against Bub1 or a control siRNA. These results suggest that metaphase arrest is necessary for the mitotic catastrophe and cell death caused by depletion of centrosomal proteins. Knockdown of centrosomal proteins led to increased phosphorylation of Chk2. Enhanced p-Chk2 localization was also observed at the centrosome in cells arrested in M phase, as well as in the nuclei of dying cells. Cotransfection of siRNAs against Chk2, in combination with depletion of a centrosomal protein, decreased the amount of cell death. Thus, Chk2 activity is indispensable for apoptosis after mitotic catastrophe induced by depletion of centrosomal proteins that perturbs microtubule organization.

Highlights

Reagents that affect spindle organization, including Aurora inhibitor and microtubule-targeting agents, trigger mitotic catastrophe by disrupting the organization of the mitotic spindle.[3,4] In the case of mitotic catastrophe induced by cell fusion or DNA damage, caspases-2, -3, and -9 are activated,[5,6] but mitotic catastrophe can cause caspase-independent apoptosis by activation of the spindle checkpoint in Bub1-deficient cells.[7]
TOG and TACC3 localized at the centrosome only during M phase. g-Tubulin localized at the centrosome during both M phase and interphase, but more strongly during M phase
Localization of g-tubulin at the centrosome was observed throughout the cell cycle, but a stronger signal was observed in M phase

Summary

Introduction

Reagents that affect spindle organization, including Aurora inhibitor and microtubule-targeting agents, trigger mitotic catastrophe by disrupting the organization of the mitotic spindle.[3,4] In the case of mitotic catastrophe induced by cell fusion or DNA damage, caspases-2, -3, and -9 are activated,[5,6] but mitotic catastrophe can cause caspase-independent apoptosis by activation of the spindle checkpoint in Bub1-deficient cells.[7]. A few studies have investigated the mechanisms underlying cell death in response to abnormalities in centrosomal proteins that perturbs microtubule organization. VX-680 increased the Bax/Bcl-2 protein ratio, a favorable proapoptotic predictor for survival.[8] TACC3 knockdown leads to mitotic arrest and cell death, which is accompanied by apoptotic features, such as Annexin V binding and caspase-3 activation.[18] the induction of mitotic catastrophe and subsequent cell death by inhibition of centrosomal proteins has not been investigated in detail. Knockdown of centrosomal proteins, including Aurora A, ninein, TOG, and TACC3, led to high rates of aberrant spindle formation and subsequent cell death. Spindle assembly checkpoint (SAC) proteins and Chk[2] were required for the induced mitotic catastrophe and subsequent cell death induced by depletion of Aurora A, ninein, or TOG

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