Overexpression of human aspartyl(asparaginyl)beta-hydroxylase in hepatocellular carcinoma and cholangiocarcinoma
Overexpression of human aspartyl(asparaginyl)beta-hydroxylase in hepatocellular carcinoma and cholangiocarcinoma
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DOI：
10.1172/jci118918
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发表时间：
1996-09-15
影响因子：
15.9
通讯作者：
Friedman, PA
中科院分区：
文献类型：
--
作者：
Lavaissiere, L;Jia, S;Friedman, PA
为了鉴定随着人肝细胞恶性转化而上调的基因，我们建立了一个针对肝细胞癌细胞系的单抗文库。抗体FB-50与一种抗原反应，该抗原在10例原发性肝细胞癌中有4例高表达，在我们研究的所有20例胆管细胞癌中均有高表达，并在各种转化细胞系中表达。这种抗原在乳腺癌和结肠癌的肿瘤上皮细胞中也有高表达，而在正常肝细胞和非肿瘤上皮细胞中的表达水平很低。在研究的正常成人组织中，仅在胎盘和肾上腺的增殖滋养层细胞中观察到高水平的表达。从HepG2人肝母细胞瘤细胞产生的GT11表达文库中分离出636个碱基的部分c DNA，并用逆转录-聚合酶链式反应(RT-PCR)获得一个完整的c DNA，鉴定该抗原为人形式的天冬氨酸(天门冬氨酸基)β-羟基酶。这种酶催化某些蛋白质的EGF样区中特定天冬氨酸基和天冬酰胺基残基的β碳的翻译后羟化。对几种人类肿瘤细胞系提取液与正常组织提取液的分析表明，羟基酶的增加类似于10倍，控制在转录水平，蛋白质以酶活性的形式表达。在类似的分析中，将五名患者的肝细胞癌与邻近的未受影响的肝脏进行比较，四名患者的肿瘤组织中的酶活性要高得多，这四名患者的免疫印迹显示恶性组织中的羟基酶蛋白增加。EGF重复序列位于Notch的胞外区或其同源物中，包含用于羟化的共同序列。缺失该结构域的突变体是功能增益型突变体，表明该结构域通过细胞质结构域调控信号转导。虽然β-羟化所赋予的功能尚不清楚，但我们的研究提出了在哺乳动物Notch同源物等蛋白质中调控β-羟化的可能性，这些蛋白质的细胞质结构域已被证明是致癌的。
To characterize genes that become upregulated with malignant transformation of human hepatocytes, a library of monoclonal antibodies was produced against the FOCUS hepatocellular carcinoma cell line. Antibody FB-50 reacted with an antigen that was highly expressed in 4 of 10 primary hepatocellular carcinomas, in all 20 cholangiocarcinomas we studied, and in a variety of transformed cell lines. This antigen was also highly expressed in neoplastic epithelial cells of breast and colon carcinomas in contrast to its low level of expression in normal hepatocytes and in nonneoplastic epithelial cells. Among the normal adult tissues studied, high levels were observed only in proliferating trophoblastic cells of the placenta and in adrenal glands. A 636-bp partial cDNA, isolated from a gamma GT11 expression library generated with HepG2 human hepatoblastoma cells, and a complete cDNA, generated by reverse transcriptase-PCR, identified the antigen as the human form of aspartyl(asparaginyl)beta-hydroxylase. This enzyme catalyzes posttranslational hydroxylation of beta carbons of specific aspartyl and asparaginyl residues in EGF-like domains of certain proteins. Analyses of extracts prepared from several human tumor cell lines compared to their normal tissue counterparts indicate that the increase in hydroxylase, similar to 10-fold, is controlled at the level of transcription and the protein is expressed in an enzymatically active form. In similar analyses, comparing hepatocellular carcinomas to adjacent uninvolved liver from five patients, enzymatic activity was much higher in the tumor tissue from the four patients whose immunoblots revealed increased hydroxylase protein in the malignant tissue. EGF repeats in the extracellular domain of Notch or its homologs contain the consensus sequence for hydroxylation. Deletion mutants lacking this domain are gain-of-function mutants, suggesting that the domain modulates signal transduction by the cytoplasmic domain. While the function imparted by beta hydroxylation is unknown, our studies raise the possibility that beta hydroxylation is regulated in proteins like the mammalian Notch homologs, whose cytoplasmic domains have been shown to be oncogenic.