Hint2, a mitochondrial apoptotic sensitizer down-regulated in hepatocellular carcinoma
Hint2, a mitochondrial apoptotic sensitizer down-regulated in hepatocellular carcinoma
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DOI：
10.1053/j.gastro.2006.03.024
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发表时间：
2006-06-01
期刊：
影响因子：
29.4
通讯作者：
Dufour, Jean-Francois
中科院分区：
文献类型：
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作者：
Martin, Juliette;Magnino, Fabrice;Dufour, Jean-Francois
背景与目的：组氨酸三联体结合蛋白是一类生物学功能尚不明确的腺苷酸-赖氨酸水解酶。在这里，我们报告了人类Hint 2的表征。研究方法：通过实时定量聚合酶链反应和免疫印迹，免疫细胞化学细胞定位，并转染绿色荧光蛋白构建体，确定组织分布。在Hint 2存在下，测量蛋白激酶C和腺苷磷酰胺酶的酶活性。建立了Hint 2过表达或敲低的HepG 2细胞系。通过半胱天冬酶免疫印迹和流式细胞术评估细胞凋亡。在SCID小鼠中测量肿瘤生长。通过微阵列研究人肿瘤中的表达。结果：Hint 2主要表达于肝脏和胰腺。Hint 2定位于线粒体。Hint 2水解的连接到氨基的腺苷一磷酸（AMP-pNA; k（cat）：0.0223 s（-1）; K-m：128 μ mol/ L）。暴露于凋亡应激，较少的过表达Hint 2的HepG 2细胞保持存活（32.2 +/- 0.6% vs 57.7 +/- 4.6%），更多的细胞显示线粒体膜电位的变化（87.8 +/- 2.35 vs 49.7 +/- 1.6%），与对照细胞相比具有更多的切割的半胱天冬酶。在Hint 2基因敲低表达的HepG 2细胞中观察到相反的结果。在SCID小鼠中皮下注射过表达Hint 2的HepG 2细胞导致较小的肿瘤（0.32 +/- 0.13 g vs 0.85 +/- 0.35 g）。微阵列分析显示，HINT 2信使RNA在肝细胞癌中下调（-0.42 +/- 0.58 log（2）vs-0.11 +/- 0.28 log（2））。HINT 2信使RNA丰度低与生存率低相关。结论：Hint 2是一种在肝癌中下调的线粒体凋亡增敏剂。
Background & Aims: Hints, histidine triad nucleotide-binding proteins, are adenosine monophosphate-lysine hydrolases of uncertain biological function. Here we report the characterization of human Hint2. Methods: Tissue distribution was determined by real-time quantitative polymerase chain reaction and immunoblotting, cellular localization by immunocytochemistry, and transfection with green fluorescent protein constructs. Enzymatic activities for protein kinase C and adenosine phosphoramidase in the presence of Hint2 were measured. HepG2 cell lines with Hint2 overexpressed or knocked down were established. Apoptosis was assessed by immunoblotting for caspases and by flow cytometry. Tumor growth was measured in SCID mice. Expression in human tumors was investigated by microarrays. Results: Hint2 was predominantly expressed in liver and pancreas. Hint2 was localized in mitochondria. Hint2 hydrolyzed adenosine monophosphate linked to an amino group (AMP-pNA; k(cat):0.0223 s(-1); K-m:128 mu mol/ L). Exposed to apoptotic stress, fewer HepG2 cells overexpressing Hint2 remained viable (32.2 +/- 0.6% vs 57.7 +/- 4.6%), and more cells displayed changes of the mitochondrial membrane potential (87.8 +/- 2.35 vs 49.7 +/- 1.6%) with more cleaved caspases than control cells. The opposite was observed in HepG2 cells with knockdown expression of Hint2. Subcutaneous injection of HepG2 cells overexpressing Hint2 in SCID mice resulted in smaller tumors (0.32 +/- 0.13 g vs 0.85 +/- 0.35 g). Microarray analyses revealed that HINT2 messenger RNA is downregulated in hepatocellular carcinomas (-0.42 +/- 0.58 log(2) vs -0.11 +/- 0.28 log(2)). Low abundance of HINT2 messenger RNA was associated with poor survival. Conclusion: Hint2 defines a novel class of mitochondrial apoptotic sensitizers down-regulated in hepatocellular carcinoma.