A delay prior to mitotic entry triggers caspase 8-dependent cell death in p53-deficient Hela and HCT-116 cells.
A delay prior to mitotic entry triggers caspase 8-dependent cell death in p53-deficient Hela and HCT-116 cells.
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DOI：
10.1080/15384101.2015.1007781
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Cassimeris L
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文献类型：
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作者：
Silva VC;Plooster M;Leung JC;Cassimeris L
Stathmin/Oncoprotein 18是一种微管不稳定蛋白，是p53缺陷细胞存活所必需的。缺乏stathmin的细胞进入有丝分裂较慢，但是否延迟有丝分裂进入触发细胞死亡或stathmin是否具有单独的促存活功能尚不清楚。为了测试这些可能性，我们废除了细胞周期延迟抑制Wee 1在同步，stathmin耗尽细胞，并发现细胞凋亡减少到控制水平。用4小时脉冲的CDK 1或Aurora A和PLK 1抑制剂处理的同步化细胞延迟有丝分裂进入，并且仅在p53缺陷细胞中触发凋亡。我们没有检测到下游的有丝分裂缺陷的延迟有丝分裂进入，表明细胞死亡是由一种机制激活的，不同于那些激活的延长有丝分裂停滞。细胞死亡由引发剂胱天蛋白酶8触发，基于其裂解为活性形式，并通过在胱天蛋白酶8耗尽或用胱天蛋白酶8抑制剂处理后挽救活力。相比之下，启动caspase 9，激活延长有丝分裂停滞，不激活，是不需要在我们的实验条件下的细胞凋亡。P53上调cFLIPL的表达，cFLIPL是一种阻断caspase 8活化的蛋白质。cFLIPL水平在缺乏p53的细胞中较低，并且这些水平在stathmin耗尽后降低到更大程度。FLAG标记的cFLIPL在p53缺陷细胞中的表达使它们免于G2期间stathmin耗尽或CDK 1抑制引发的凋亡。这些数据表明，在G2期细胞周期延迟激活caspase 8启动凋亡，特别是在p53缺陷细胞。
Stathmin/Oncoprotein 18, a microtubule destabilizing protein, is required for survival of p53-deficient cells. Stathmin-depleted cells are slower to enter mitosis, but whether delayed mitotic entry triggers cell death or whether stathmin has a separate pro-survival function was unknown. To test these possibilities, we abrogated the cell cycle delay by inhibiting Wee1 in synchronized, stathmin-depleted cells and found that apoptosis was reduced to control levels. Synchronized cells treated with a 4 hour pulse of inhibitors to CDK1 or both Aurora A and PLK1 delayed mitotic entry and apoptosis was triggered only in p53-deficient cells. We did not detect mitotic defects downstream of the delayed mitotic entry, indicating that cell death is activated by a mechanism distinct from those activated by prolonged mitotic arrest. Cell death is triggered by initiator caspase 8, based on its cleavage to the active form and by rescue of viability after caspase 8 depletion or treatment with a caspase 8 inhibitor. In contrast, initiator caspase 9, activated by prolonged mitotic arrest, is not activated and is not required for apoptosis under our experimental conditions. P53 upregulates expression of cFLIPL, a protein that blocks caspase 8 activation. cFLIPL levels are lower in cells lacking p53 and these levels are reduced to a greater extent after stathmin depletion. Expression of FLAG-tagged cFLIPL in p53-deficient cells rescues them from apoptosis triggered by stathmin depletion or CDK1 inhibition during G2. These data indicate that a cell cycle delay in G2 activates caspase 8 to initiate apoptosis specifically in p53-deficient cells.