Darpp-32:: a novel antiapoptotic gene in upper gastrointestinal carcinomas
Darpp-32:: a novel antiapoptotic gene in upper gastrointestinal carcinomas
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DOI：
10.1158/0008-5472.can-05-1433
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发表时间：
2005-08-01
期刊：
影响因子：
11.2
通讯作者：
El-Rifai, W
中科院分区：
文献类型：
--
作者：
Belkhiri, A;Zaika, A;El-Rifai, W
我们揭示了DARPP-32过表达的分子机制及其在上胃肠腺癌(UGC)中的生物学作用。建立了一个包含377个样本的肿瘤组织阵列，用于检测DARPP-32DNA扩增和蛋白过度表达，这两种情况分别发生在32%和60%的UGCs中。在68%的肿瘤中观察到DARPP-32及其截短亚型t-DARPP的高表达(P<；0.001)。当DARPP-32和t-DARPP在AGS和RKO胃肠细胞中过表达时，在喜树碱、丁酸钠和神经酰胺的作用下，细胞凋亡率(末端脱氧核苷酸转移酶介导的缺口末端标记和Annexin V分析)减少了4倍。然而，磷酸化位点突变的引入消除了这一效应。DARPP-32和t-DARPP的表达保护了线粒体的跨膜电位，并与Bcl2蛋白水平的升高有关。利用对DARPP-32和t-DARPP的小干扰RNA逆转了Bcl2蛋白的水平。用p53和p21报告质粒检测荧光素酶，以及用针对p53转录靶点的抗体如hdm2和p21进行免疫印迹的探查表明，DARPP-32和t-DARPP都不干扰p53的功能。总之，我们显示DARPP-32的mRNA和蛋白质过度表达比DNA扩增更频繁，这表明除了扩增外，转录或转录后机制可能发挥重要作用。DARPP-32和t-DARPP的表达与癌细胞具有潜在的抗凋亡优势有关，这是通过一种与P53无关的机制来实现的，该机制涉及保护线粒体潜力和增加1302水平。
We show the molecular mechanisms involved in Darpp-32 overexpression and its biological role in upper gastrointestinal adenocarcinomas (UGC). A tumor tissue array of 377 samples was developed and used to detect DARPP-32 DNA amplification and protein overexpression, which occurred in 32% and 60% of UGCs, respectively. Concomitant overexpression of mRNA for Darpp-32 and its truncated isoform t-Darpp was observed in 68% of tumors (P < 0.001). When Darpp-32 and t-Darpp were overexpressed in AGS and RKO gastrointestinal cells, up to a 4-fold reduction in the apoptosis rate was observed (terminal deoxynucleotidyl transferase-mediated nick-end labeling and Annexin V assays) in response to camptothecin, sodium butyrate, and ceramide. However, the introduction of mutations in phosphorylation sites abrogated this effect. Expression of Darpp-32 and t-Darpp preserved the mitochondrial transmembrane potential and was associated with increased levels of Bcl2 protein. A reversal of Bcl2 protein level was obtained using small interfering RNAs for Darpp-32 and t-Darpp. Luciferase assays using the p53 and p21 reporter plasmids and probing of immunoblots with antibodies specific for p53 transcriptional targets, such as Hdm2 and p21, indicated that neither Darpp-32 nor t-Darpp interfere with p53 function. Altogether, we show more frequent mRNA and protein overexpression of Darpp-32 than DNA amplification, suggesting that, in addition to amplification, transcriptional or posttranscriptional mechanisms may play an important role. The expression of Darpp-32 and t-Darpp is associated,with a potent antiapoptotic advantage for cancer cells through a p53-independent mechanism that involves preservation of mitochondrial potential and increased 1302 levels.