Kwak In Hae, Kim Hong Seok, Choi Ok Ran, Ryu Min Sook, Lim In Kyoung
Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, Suwon, Korea.
Cancer Res. 2004 Jan 15;64(2):572-80. doi: 10.1158/0008-5472.can-03-1856.
We evaluated the nuclear actin accumulation as a new marker of cellular senescence, using human diploid fibroblast (HDF), chondrocyte primary cultures, Mv1Lu epithelial cells, and Huh7 cancer cells. Nuclear accumulation of globular actin (G-actin) and dephosphorylated cofilin was highly significant in the senescent HDF cells, accompanied with inhibition of LIM kinase (LIMK) -1 activity. When nuclear export of the actin was induced by 12-O-tetradecanoylphorbol-13-acetate, DNA synthesis of the senescent cells increased significantly, accompanied with changes of morphologic and biochemical profiles, such as increased RB protein phosphorylation and decreased expressions of p21(WAF1), cytoplasmic p-extracellular signal-regulated kinase 1/2, and caveolins 1 and 2. Significance of these findings was strengthened additionally by the fact that nuclear actin export of young HDF cells was inhibited by the treatment with leptomycin B and mutant cofilin transfection, whose LIMK-1 phosphorylation site was lost, and the old cell phenotypes were duplicated with nuclear actin accumulation, suggesting that nuclear actin accumulation was accompanied with G1 arrest during cellular senescence. The aforementioned changes were observed not only in the replicative senescence but also in the senescence induced by treatment of HDF cells, Mv1Lu, primary culture of human chondrocytes, or Huh7 cells with H-ras virus infection, hydroxyurea, deferoxamine, or H(2)O(2). Nuclear actin accumulation was much more sensitive and an earlier event than the well-known, senescence-associated beta-galactosidase activity.
我们使用人二倍体成纤维细胞（HDF）、软骨细胞原代培养物、Mv1Lu上皮细胞和Huh7癌细胞，评估核肌动蛋白积累作为细胞衰老的一种新标志物。在衰老的HDF细胞中，球状肌动蛋白（G-肌动蛋白）和去磷酸化的丝切蛋白的核积累非常显著，同时伴有LIM激酶（LIMK）-1活性的抑制。当用12-O-十四烷酰佛波醇-13-乙酸酯诱导肌动蛋白的核输出时，衰老细胞的DNA合成显著增加，同时伴有形态学和生化特征的变化，如RB蛋白磷酸化增加以及p21（WAF1）、细胞质磷酸化细胞外信号调节激酶1/2和小窝蛋白1和2的表达降低。用莱普霉素B处理和转染失去LIMK-1磷酸化位点的突变型丝切蛋白抑制了年轻HDF细胞的核肌动蛋白输出，且核肌动蛋白积累使旧细胞表型重现，这一事实进一步强化了这些发现的意义，表明在细胞衰老过程中核肌动蛋白积累伴随着G1期阻滞。上述变化不仅在复制性衰老中观察到，在用H-ras病毒感染、羟基脲、去铁胺或H₂O₂处理HDF细胞、Mv1Lu、人软骨细胞原代培养物或Huh7细胞诱导的衰老中也观察到。核肌动蛋白积累比众所周知的衰老相关β-半乳糖苷酶活性更敏感且是更早出现的事件。
