Chen Q M, Liu J, Merrett J B
Department of Pharmacology, Skaggs Pharmaceutical Sciences Building, Room 130, University of Arizona, 1703 E. Mabel Street, Tucson, AZ 85721, USA.
Biochem J. 2000 Apr 15;347(Pt 2):543-51. doi: 10.1042/0264-6021:3470543.
Early-passage human diploid fibroblasts (HDFs) undergo senescence-like growth arrest in response to sublethal concentrations of H(2)O(2) [Chen and Ames (1994) Proc. Natl. Acad. Sci. USA. 95, 4130-4134]. We determine here whether H(2)O(2) can cause apoptosis in HDFs and the molecular changes that differ between apoptosis and senescence-like growth arrest. When exponentially growing early-passage IMR-90 cells were treated for 2 h with 50-200 microM (or 0.25-1 pmol/cell) H(2)O(2), a fraction of cells detached at 16-32 h after the treatment. The cells remaining attached were growth-arrested and developed features of senescence in 1 week. The detached cells showed caspase-3 activation and typical morphological changes associated with apoptosis. Caspase-3 activation was H(2)O(2) dose-dependent and preceded nuclear condensation or plasma membrane leakage. Apoptotic cells were mainly distributed in the S-phase of the cell cycle, while growth-arrested cells exhibited predominantly G1- and G2/M-phase distributions. H(2)O(2) pretreatment induced G1 arrest and prohibited induction of apoptosis by a subsequent H(2)O(2) challenge. The p53 protein showed an average 6.1-fold elevation in apoptotic cells and a 3.5-fold elevation in growth-arrested cells. Reduction of p53 levels with human papillomavirus E6 protein prohibited the activation of caspase-3 and decreased the proportion of apoptotic cells. Growth-arrested cells had elevated p21, while p21 was absent in apoptotic cells. Bcl-2 was elevated in both growth-arrested and apoptotic cells. Finally, although the overall level of bax did not change in growth-arrested or apoptotic cells, the solubility of bax protein increased in apoptotic cells. Our data suggest that in contrast with growth-arrested cells, apoptotic cells show an S-phase cell cycle distribution, a higher degree of p53 elevation, an absence of p21 protein and increased solubility of bax protein.
早期传代的人二倍体成纤维细胞（HDFs）在亚致死浓度的H₂O₂作用下会经历类似衰老的生长停滞[Chen和Ames（1994年），《美国国家科学院院刊》95，4130 - 4134]。我们在此确定H₂O₂是否能在HDFs中引发凋亡以及凋亡和类似衰老的生长停滞之间存在差异的分子变化。当对数生长期的早期传代IMR - 90细胞用50 - 200微摩尔/升（或0.25 - 1皮摩尔/细胞）的H₂O₂处理2小时后，一部分细胞在处理后16 - 32小时脱落。剩余贴壁的细胞生长停滞，并在1周内出现衰老特征。脱落的细胞显示出caspase - 3激活以及与凋亡相关的典型形态变化。caspase - 3激活呈H₂O₂剂量依赖性，且先于核浓缩或质膜渗漏出现。凋亡细胞主要分布在细胞周期的S期，而生长停滞的细胞主要表现为G1期和G2/M期分布。H₂O₂预处理诱导G1期停滞，并阻止后续H₂O₂刺激诱导凋亡。p53蛋白在凋亡细胞中平均升高6.1倍，在生长停滞的细胞中升高3.5倍。用人乳头瘤病毒E6蛋白降低p53水平可阻止caspase - 3激活，并降低凋亡细胞的比例。生长停滞的细胞中p21升高，而凋亡细胞中不存在p21。生长停滞和凋亡细胞中Bcl - 2均升高。最后，尽管生长停滞或凋亡细胞中bax的总体水平没有变化，但凋亡细胞中bax蛋白的溶解度增加。我们的数据表明，与生长停滞的细胞相比，凋亡细胞表现为S期细胞周期分布、更高程度的p53升高、缺乏p21蛋白以及bax蛋白溶解度增加。
