Ullrich O, Ciftci O, Hass R
Department of Cell Biology and Neurobiology, Institute of Anatomy, Medical Faculty (Charité), Humboldt University Berlin, Berlin, Germany.
Free Radic Biol Med. 2000 Nov 15;29(10):995-1004. doi: 10.1016/s0891-5849(00)00399-3.
Cytotoxic action of a variety of antitumor drugs generate oxidatively modified proteins that are predominantly metabolized via the proteasome. In the present study, a differentiation-retrodifferentiation cell system was exposed to oxidative stress by hydrogen peroxide treatment. Thus, the activity of the nuclear proteasome in proliferating human U937 leukemic cells increased by 2.5-fold after hydrogen peroxide treatment. In contrast, growth-arrested differentiated U937 cells demonstrated 40% less constitutive proteasomal activity, which was not inducible after hydrogen peroxide exposure. After a retrodifferentiation process, however, in which differentiated U937 cells resume autonomous growth again, the proteasomal activity was indistinguishable from that in U937 control cells, both constitutively and after induction of oxidative stress. Moreover, cells of TUR, a differentiation-resistant U937 subclone, expressed an elevated constitutive proteasomal activity that increased by 2.5-fold after oxidative stress. Immunoblot analysis revealed that these differences in proteasomal activities did not correlate with proteasome protein expression but with protein levels of the nuclear enzyme poly-ADP-ribose-polymerase (PARP). Further studies using specific PARP inhibitors revealed that the noninducible proteasome activity in differentiated U937 cells was PARP independent, whereas the increased activity level in oxidatively stressed TUR cells was downregulated upon PARP inhibition. Immunoprecipitation experiments demonstrated a protein-protein interaction of the functional active PARP with the proteasome in correlation with the proteasome activity. Similar results were obtained by analyzing protein carbonyls after oxidative stress. Taken together, these data suggest that proliferating, rather than growth-arrested, cells metabolize oxidatively damaged nuclear proteins via the proteasome by expressing high levels of PARP.
多种抗肿瘤药物的细胞毒性作用会产生主要通过蛋白酶体代谢的氧化修饰蛋白。在本研究中，一个分化-逆向分化细胞系统通过过氧化氢处理暴露于氧化应激。因此，过氧化氢处理后，增殖的人U937白血病细胞核蛋白酶体活性增加了2.5倍。相比之下，生长停滞的分化U937细胞的组成型蛋白酶体活性降低了40%，过氧化氢暴露后该活性不可诱导。然而，在逆向分化过程中，分化的U937细胞再次恢复自主生长，其蛋白酶体活性在组成型和氧化应激诱导后与U937对照细胞无差异。此外，TUR（一种分化抗性U937亚克隆）细胞表达升高的组成型蛋白酶体活性，氧化应激后增加了2.5倍。免疫印迹分析表明，蛋白酶体活性的这些差异与蛋白酶体蛋白表达无关，而是与核酶聚ADP-核糖聚合酶（PARP）的蛋白水平有关。使用特异性PARP抑制剂的进一步研究表明，分化的U937细胞中不可诱导的蛋白酶体活性与PARP无关，而氧化应激的TUR细胞中增加的活性水平在PARP抑制后下调。免疫沉淀实验证明功能性活性PARP与蛋白酶体之间存在蛋白-蛋白相互作用，与蛋白酶体活性相关。通过分析氧化应激后的蛋白羰基也得到了类似结果。综上所述，这些数据表明，增殖而非生长停滞的细胞通过表达高水平的PARP通过蛋白酶体代谢氧化损伤的核蛋白。
