Computerized video time lapse study of cell cycle delay and arrest, mitotic catastrophe, apoptosis and clonogenic survival in irradiated 14-3-3σ and CDKN1A (p21) knockout cell lines
Computerized video time lapse study of cell cycle delay and arrest, mitotic catastrophe, apoptosis and clonogenic survival in irradiated 14-3-3σ and CDKN1A (p21) knockout cell lines
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DOI：
10.1667/rr3221
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发表时间：
2004-09-01
影响因子：
3.4
通讯作者：
Dewey, WC
中科院分区：
文献类型：
--
作者：
Chu, K;Teele, N;Dewey, WC
使用计算机视频延时（CVTL）显微镜观察电离辐射（10-12戈伊）在野生型HCT 116结直肠癌细胞系及其三个等基因衍生系（其中p21（CDKN 1A）、14-3- 3 sigma或两个检查点基因（双敲除）已被敲除）的非克隆细胞中诱导的细胞事件。有丝分裂后融合或未能完成有丝分裂的细胞被归类为经历有丝分裂灾难的细胞。17%的野生型细胞和34-47%的敲除细胞经历有丝分裂灾难以进入具有4 N DNA含量的第1代，即，与第0代结束时停滞在G（2）的照射细胞相同的DNA含量。辐射引起细胞分裂或发生有丝分裂灾难之前的第0代短暂分裂延迟。与表达CDKN 1A和14-3-3sigma的野生型细胞的分裂延迟相比，敲除CDKN 1A减少了在G（1）中照射的细胞的延迟（从近似15小时减少到近似3 -5小时），而敲除14-3-3sigma减少了在S和G（2）晚期照射的细胞的延迟（从近似18小时减少到3-4小时）。然而，27%的野生型细胞和17%的14-3-3sigma（-/-）细胞在第0代中在96小时被阻滞，而CDKNIA（-/-）和双敲除细胞的阻滞率小于1%。因此，CDKN 1A的表达对于第0代的延长延迟或停滞是必需的。此外，CDKN 1A在第1代中发挥着至关重要的作用，极大地抑制了有丝分裂灾难产生的二倍体细胞和多倍体细胞向后代的进展。因此，在CDKN 1A缺陷细胞系中，在第3代和第4代期间发生了一系列有丝分裂灾难事件，产生了高度多倍体后代。最重要的是，有丝分裂灾难事件产生的多倍体后代并不比分裂细胞的后代死得早。死亡被确定为细胞运动的丧失，即代谢活性。因此，有丝分裂灾难本身并不是一种直接的死亡方式。相反，单核细胞和多倍体细胞的间期细胞凋亡是在四种细胞类型中观察到的主要死亡模式。敲除CDKN 1A或14-3-3sigma增加了96小时的细胞死亡量，从52%增加到接近70%，当两个基因都被敲除时，甚至增加到90%。因此，除了CDKN 1A和14-3-3sigma表达对瞬时细胞周期延迟的影响之外，CDKN 1A具有抗增殖和抗凋亡功能，而14-3-3sigma仅具有抗凋亡功能。最后，细胞死亡量的大幅变化总体上与克隆形成存活率的小幅变化不相关（剂量修饰比为1.05-1.13）;然而，敲除CDKN 1A导致停滞细胞减少和存活率增加，而敲除14-3-3sigma导致凋亡增加和存活率降低。(C)2004年，辐射研究协会。
Computerized video time lapse (CVTL) microscopy was used to observe cellular events induced by ionizing radiation (10-12 Gy) in nonclonogenic cells of the wild-type HCT116 colorectal carcinoma cell line and its three isogenic derivative lines in which p21 (CDKN1A), 14-3-3sigma or both checkpoint genes (double-knockout) had been knocked out. Cells that fused after mitosis or failed to complete mitosis were classified together as cells that underwent mitotic catastrophe. Seventeen percent of the wild-type cells and 34-47% of the knockout cells underwent mitotic catastrophe to enter generation 1 with a 4N content of DNA, i.e., the same DNA content as irradiated cells arrested in G(2) at the end of generation 0. Radiation caused a transient division delay in generation 0 before the cells divided or underwent mitotic catastrophe. Compared with the division delay for wild-type cells that express CDKN1A and 14-3-3sigma, knocking out CDKN1A reduced the delay the most for cells irradiated in G(1) (from similar to15 h to similar to3-5 h), while knocking out 14-3-3sigma reduced the delay the most for cells irradiated in late S and G(2) (from similar to18 h to 3-4 h). However, 27% of wild-type cells and 17% of 14-3-3sigma(-/-) cells were arrested at 96 h in generation 0 compared with less than 1% for CDKNIA(-/-) and double-knockout cells. Thus expression of CDKN1A is necessary for the prolonged delay or arrest in generation 0. Furthermore, CDKN1A plays a crucial role in generation 1, greatly inhibiting progression into subsequent generations of both diploid cells and polyploid cells produced by mitotic catastrophe. Thus, in CDKN1A-deficient cell lines, a series of mitotic catastrophe events occurred to produce highly polyploid progeny during generations 3 and 4. Most importantly, the polyploid progeny produced by mitotic catastrophe events did not die sooner than the progeny of dividing cells. Death was identified as loss of cell movement, i.e. metabolic activity. Thus mitotic catastrophe itself is not a direct mode of death. Instead, apoptosis during interphase of both uninucleated and polyploid cells was the primary mode of death observed in the four cell types. Knocking out either CDKN1A or 14-3-3sigma increased the amount of cell death at 96 h, from 52% to similar to70%, with an even greater increase to 90% when both genes were knocked out. Thus, in addition to effects of CDKN1A and 14-3-3sigma expression on transient cell cycle delay, CDKN1A has both an anti-proliferative and antiapoptosis function, while 14-3-3sigma has only an anti-apoptosis function. Finally, the large alterations in the amounts of cell death did not correlate overall with the small alterations in clonogenic survival (dose-modifying ratios of 1.05-1.13); however, knocking out CDKN1A resulted in a decrease in arrested cells and an increase in survival, while knocking out 14-3-3sigma resulted in an increase in apoptosis and a decrease in survival. (C) 2004 by Radiation Research Society.