Silva Victoria C, Plooster Melissa, Leung Jessica C, Cassimeris Lynne
a Department of Biological Sciences ; Lehigh University ; Bethlehem , PA USA.
Cell Cycle. 2015;14(7):1070-81. doi: 10.1080/15384101.2015.1007781.
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.
微管解聚蛋白Stathmin/癌蛋白18是p53缺陷细胞存活所必需的。Stathmin缺失的细胞进入有丝分裂的速度较慢，但延迟的有丝分裂进入是否会触发细胞死亡，或者Stathmin是否具有独立的促存活功能尚不清楚。为了检验这些可能性，我们通过在同步化的、Stathmin缺失的细胞中抑制Wee1来消除细胞周期延迟，发现细胞凋亡减少到了对照水平。用CDK1抑制剂或Aurora A和PLK1两者的抑制剂处理4小时脉冲的同步化细胞会延迟有丝分裂进入，并且仅在p53缺陷细胞中触发凋亡。我们在延迟的有丝分裂进入下游未检测到有丝分裂缺陷，这表明细胞死亡是由一种不同于长期有丝分裂停滞所激活机制的机制所激活的。细胞死亡是由起始半胱天冬酶8触发的，这基于其切割成活性形式以及在半胱天冬酶8缺失或用半胱天冬酶8抑制剂处理后活力的恢复。相比之下，在我们的实验条件下，由长期有丝分裂停滞激活的起始半胱天冬酶9未被激活，并且对于细胞凋亡不是必需的。p53上调cFLIPL的表达，cFLIPL是一种阻断半胱天冬酶8激活的蛋白质。在缺乏p53的细胞中cFLIPL水平较低，并且在Stathmin缺失后这些水平会更大程度地降低。在p53缺陷细胞中表达FLAG标签的cFLIPL可使它们免于在G2期由Stathmin缺失或CDK1抑制触发的凋亡。这些数据表明，G2期的细胞周期延迟激活半胱天冬酶8以特异性地在p53缺陷细胞中启动凋亡。
