Chromatin condensation during apoptosis is accompanied by degradation of lamin A+B, without enhanced activation of cdc2 kinase.
Chromatin condensation during apoptosis is accompanied by degradation of lamin A+B, without enhanced activation of cdc2 kinase.
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DOI：
10.1083/jcb.126.4.827
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发表时间：
1994-08
影响因子：
7.8
通讯作者：
Pavelka, M
中科院分区：
文献类型：
--
作者：
Oberhammer, F A;Hochegger, K;Froschl, G;Tiefenbacher, R;Pavelka, M
DNA片段化引起的染色质凝聚是鉴定凋亡细胞的重要指标之一。然而，在用有丝分裂细胞的细胞提取物处理的间期核中也观察到类似的变化。在这方面，已知在有丝分裂中，由于核纤层蛋白溶解，核纤层结构被破坏，并且在凋亡细胞中可能发生类似的过程。本研究中描述的实验使用了胚胎成纤维细胞系的融合培养物，该细胞系可以在低血清条件下被诱导进行细胞凋亡或有丝分裂。通过免疫印迹和间接免疫荧光分析核纤层蛋白A+B的溶解。这些研究表明，在有丝分裂细胞中，纤层的破坏伴随着纤层蛋白的溶解。在凋亡细胞中，少量的核纤层蛋白在凋亡开始前溶解，此后，染色质浓缩伴随着核纤层蛋白A+B降解为46 kD片段。通过用抗PSTAIR随后用抗磷酸酪氨酸探针印迹分析细胞裂解物显示，与有丝分裂相反，在凋亡细胞中不发生酪氨酸残基的去磷酸化。在所有的时间点后，细胞凋亡的发生有没有显着增加，在组蛋白H1激酶测定中确定的p34cdc2的激活。阿非迪霉素阻滞释放细胞后，细胞凋亡和有丝分裂的共同诱导表明，细胞凋亡可与S期平行诱导。染色质结构的突然破坏可能是染色质环从其在核基质的锚定处脱离的结果，因为通过场反转凝胶电泳（FIGE）可检测到50 kbp的条带和相应的多聚体。在凋亡细胞中，所有的DNA片段化，但只有14%的DNA小于50 kbp。通过原位加尾（ISTAIL）在凝聚的染色质周围检测到DNA链断裂。细胞凋亡过程中染色质凝聚似乎是由于核基质蛋白的快速蛋白水解，不涉及p34cdc2激酶。
Chromatin condensation paralleled by DNA fragmentation is one of the most important criteria which are used to identify apoptotic cells. However, comparable changes are also observed in interphase nuclei which have been treated with cell extracts from mitotic cells. In this respect it is known that in mitosis, the lamina structure is broken down as a result of lamin solubilization and it is possible that a similar process is happening in apoptotic cells. The experiments described in this study have used confluent cultures of an embryonic fibroblast cell line which can be induced to undergo either apoptosis at low serum conditions or mitosis. Solubilization of lamin A+B was analyzed by immunoblotting and indirect immunofluorescence. These studies showed that in mitotic cells lamina breakdown is accompanied by lamin solubilization. In apoptotic cells, a small amount of lamin is solubilized before the onset of apoptosis, thereafter, chromatin condensation is accompanied by degradation of lamin A+B to a 46-kD fragment. Analysis of cellular lysates by probing blots with anti- PSTAIR followed by anti-phosphotyrosine showed that in contrast to mitosis, dephosphorylation on tyrosine residues did not occur in apoptotic cells. At all timepoints after the onset of apoptosis there was no significant increase in the activation of p34cdc2 as determined in the histone H1 kinase assay. Coinduction of apoptosis and mitosis after release of cells from aphidicolin block showed that apoptosis could be induced in parallel with S-phase. The sudden breakdown of chromatin structure may be the result of detachment of the chromatin loops from their anchorage at the nuclear matrix, as bands of 50 kbp and corresponding multimers were detectable by field inversion gel electrophoresis (FIGE). In apoptotic cells all of the DNA was fragmented, but only 14% of the DNA was smaller than 50 kbp. DNA strand breaks were detected at the periphery of the condensed chromatin by in situ tailing (ISTAIL). Chromatin condensation during apoptosis appears to be due to a rapid proteolysis of nuclear matrix proteins which does not involve the p34cdc2 kinase.