Tsai Shwu Y, Ardelt Barbara, Hsieh Tze-Chen, Darzynkiewicz Zbigniew, Shogen Kuslima, Wu Joseph M
Department of Biochemistry & Molecular Biology, New York Medical College, Valhalla, NY 10595, USA.
Int J Oncol. 2004 Dec;25(6):1745-52.
Onconase (Ranpirnase), a novel ribonuclease isolated from Rana pipiens oocytes, was reported to suppress cancer cell growth in vitro, reduce tumor size in animals, and augment cytotoxicity of several chemotherapeutic agents. Since onconase is currently in phase III clinical trials tested in treatment of mesothelioma, much emphasis has been placed on the mechanism of its anti-tumor activity. Previous studies have shown that onconase-responsive cells become arrested at the G1/S checkpoint of the cell cycle and also undergo apoptosis. A proposed mechanism for these effects is that the enzymatic activity of onconase targets cellular RNAs, in particular tRNA, with an accompanying inhibition of protein synthesis. In the present study, we have investigated the time- and dose-dependent effects of onconase on growth of Jurkat SN acute T-lymphocytic leukemia cells. Significant suppression of cell proliferation became evident after 72 and 96 h of treatment, and was most pronounced at the highest concentration (10 microg/ml; 8.3x10(-7) M) of onconase. This reduction of cell proliferation, however, was not accompanied by measurable changes in distribution of cells at different phases of the cell cycle, but was paralleled by the induction of apoptosis, as assayed by flow cytometry, and with a modest decrease in the expression of a cell cycle regulatory retinoblastoma protein (Rb). Further biochemical analysis revealed that growth suppression was closely coordinated with a down-regulation in the steady state and subcellular distribution of NF-kappaB, a transcription factor known to be functionally associated with cell survival. The reduction in expression of NF-kappaB by onconase appeared to coincide or even precede growth suppression, suggesting a causal relationship. To further test the hypothesis that cellular localization and expression of NF-kappaB may be critical to cellular response to onconase, we also studied the growth effects of onconase in Jurkat-BalphaM cells, which, unlike the parent SN T cells, contain a stably transfected dominant-negative IkappaB gene. Growth suppression by onconase in BalphaM cells was more pronounced and occurred earlier compared to SN cells, although still did not affect changes in cell cycle phase distribution. Contrary to expectation, however, diminution in NF-kappaB expression by onconase was even more pronounced in BalphaM cells, suggesting that this transcription factor, while presumably prevented from dissociation from its inhibitory protein IkappaB in these cells, is even more efficiently targeted for degradation by onconase. These results implicate NF-kappaB and its turnover as important determinants in the anti-proliferative/apoptotic effects of onconase in acute T-lymphocytic leukemia cells.
癌蛙酶（Ranpirnase）是一种从豹蛙卵母细胞中分离出的新型核糖核酸酶，据报道它能在体外抑制癌细胞生长，减小动物体内肿瘤大小，并增强几种化疗药物的细胞毒性。由于癌蛙酶目前正处于治疗间皮瘤的III期临床试验阶段，因此人们非常关注其抗肿瘤活性的机制。先前的研究表明，对癌蛙酶有反应的细胞会在细胞周期的G1/S检查点停滞，并发生凋亡。对于这些效应的一种推测机制是，癌蛙酶的酶活性作用于细胞RNA，特别是tRNA，同时抑制蛋白质合成。在本研究中，我们研究了癌蛙酶对Jurkat SN急性T淋巴细胞白血病细胞生长的时间和剂量依赖性效应。在处理72小时和96小时后，细胞增殖受到显著抑制，且在癌蛙酶最高浓度（10微克/毫升；8.3×10⁻⁷摩尔）时最为明显。然而，这种细胞增殖的减少并没有伴随着细胞周期不同阶段细胞分布的可测量变化，而是伴随着凋亡的诱导，这通过流式细胞术检测得到，同时细胞周期调节性视网膜母细胞瘤蛋白（Rb）的表达有适度下降。进一步的生化分析表明，生长抑制与转录因子NF-κB的稳态和亚细胞分布的下调密切相关，NF-κB已知在功能上与细胞存活相关。癌蛙酶导致的NF-κB表达降低似乎与生长抑制同时发生甚至先于生长抑制，提示存在因果关系。为了进一步验证NF-κB的细胞定位和表达可能对细胞对癌蛙酶的反应至关重要这一假说，我们还研究了癌蛙酶对Jurkat-BalphaM细胞生长的影响，与亲本SN T细胞不同，Jurkat-BalphaM细胞含有稳定转染的显性负性IκB基因。与SN细胞相比，癌蛙酶对BalphaM细胞的生长抑制更明显且发生得更早，尽管仍然不影响细胞周期阶段分布的变化。然而，与预期相反，癌蛙酶导致的BalphaM细胞中NF-κB表达的减少甚至更明显，这表明该转录因子虽然在这些细胞中可能被阻止与抑制蛋白IκB解离，但更有效地被癌蛙酶靶向降解。这些结果表明，NF-κB及其周转是癌蛙酶对急性T淋巴细胞白血病细胞抗增殖/凋亡作用的重要决定因素。
