Cytostatic and cytotoxic properties of amphinase - A novel cytotoxic ribonuclease from Rana pipiens oocytes
Cytostatic and cytotoxic properties of amphinase - A novel cytotoxic ribonuclease from Rana pipiens oocytes
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DOI：
10.4161/cc.6.24.5045
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发表时间：
2007-12-15
期刊：
影响因子：
4.3
通讯作者：
Darzynkiewicz, Zbigniew
中科院分区：
文献类型：
--
作者：
Ardelt, Barbara;Ardelt, Wojciech;Darzynkiewicz, Zbigniew
Onconase（Onc）是一种新型的两栖类细胞毒性核糖核酸酶，具有抗肿瘤活性，目前正在进行治疗恶性间皮瘤的确证性III期临床试验。最近有报道称尖音蛙卵母细胞中还含有另一种核糖核酸酶，称为两栖酶（Amphinase，Amph）。Amph显示出与癌蛋白酶38-40%的氨基酸序列同一性，呈现为四种变体，其氨基酸序列同一性在87-99%之间变化，并且具有类似于13，000的分子量。在本研究中，我们描述了Amph对几种肿瘤细胞系生长的影响。所有四种变体均表现出对人早幼粒细胞HL-60、Jurkat T细胞和U-937单核细胞白血病细胞的细胞生长抑制和细胞毒性活性。Amph的活性模式与Onc有一定的相似性。因此，细胞增殖在0.5-10.0 μ g/ml（38-770 nM）Amph浓度下受到抑制，细胞明显积聚在细胞周期的G（1）期。此外，细胞正在发生凋亡，表现为DNA片段化（存在“亚G1”细胞，TUNEL阳性）、半胱天冬酶和丝氨酸蛋白酶活化以及转氨酶活化。Amph的细胞抑制和细胞毒性作用需要其核糖核酸酶活性：在活性位点具有组氨酸烷基化的无酶活性的Amph-2无效。对于所有四种Amph变体，有效性和细胞周期特异性通常相似，并且在等摩尔浓度下比Onc更明显。所观察到的Amph对肿瘤细胞系的细胞生长抑制和细胞毒性活性表明，类似于Onc，这种细胞毒性核糖核酸酶可能具有抗肿瘤活性，并在临床肿瘤学中找到应用。
Onconase (Onc) is a novel amphibian cytotoxic ribonuclease with antitumor activity and is currently in a confirmatory phase III clinical trial for the treatment of malignant mesothelioma. It was recently reported that Rana pipiens oocytes contain still another ribonuclease, named Amphinase (Amph). Amph shows 38-40% amino acid sequence identity with onconase, presents as four variants varying between themselves from 87-99% in amino acid sequence identity and has a molecular mass similar to 13,000. In the present study we describe the effects of Amph on growth of several tumor cell lines. All four variants demonstrated cytostatic and cytotoxic activity against human promyelocytic HL-60-, Jurkat T-cell-and U-937 monocytic leukemia cells. The pattern of Amph activity to certain extent resembled that of Onc. Thus, cell proliferation was suppressed at 0.5-10.0 mu g/ml (38-770 nM) Amph concentration with distinct accumulation of cells in G(1) phase of the cell cycle. In addition, the cells were undergoing apoptosis, which manifested by DNA fragmentation (presence of "sub-G1" cells, TUNEL-positivity), caspases and serine proteases activation as well as activation of transglutaminase. The cytotostatic and cytotoxic effects of Amph required its ribonuclease activity: the enzymatically inactive Amph-2 having histidine at the active site alkylated was ineffective. The effectiveness and cell cycle specificity was generally similar for all four Amph variants and at the equimolar concentrations was somewhat more pronounced than that of Onc. The observed cytostatic and cytotoxic activity of Amph against tumor cell lines suggests that similar to Onc this cytotoxic ribonuclease may have antitumor activity and find an application in clinical oncology.