ARHI: AN IMPRINTED TUMOR SUPPRESSOR GENE

差异显示PCR已被用于寻找在正常卵巢表面上皮细胞中表达而在卵巢癌细胞中不表达的基因。NO3 EY 2（由人类基因命名委员会命名为ARHI）在正常卵巢和乳腺上皮细胞中持续表达，但在卵巢癌或乳腺癌中不表达。该基因编码与ras和rap同源的26 Kd的小G蛋白。ARHI表达的缺失由多种机制引起。该基因以单等位基因方式表达，并具有母系印记。在41%的卵巢癌和乳腺癌中检测到染色体1 p31上该基因的杂合性缺失。在tumo 5 rs的洛缺失中，9例中有7例非印迹功能等位基因缺失。在正常卵巢上皮细胞中，ARHI的表达也可以通过启动子活性进行转录调控，但在大多数癌症中则不然。不同长度的启动子片段的活性已在正常和恶性卵巢上皮细胞中进行了评估。180 bp的截短构建体在卵巢癌中的活性显著低于正常细胞。该区域中的26 bp DNA元件与不能表达ARHI的卵巢癌细胞中的推定调节蛋白结合，但不与表达ARHI的正常卵巢表面上皮细胞中的核蛋白结合。通过转染重新表达ARHI基因抑制了已经失去ARHI表达的卵巢癌和乳腺癌细胞系的克隆生长。生长抑制与细胞周期蛋白D1启动子活性的下调和P21/WAF 1/CIP 1的诱导有关。EGF通过Fas/map诱导的信号传导被ARHI的表达减少和截短，而JNK激酶通路被激活。携带人ARHI转基因的小鼠体型较小，类似于细胞周期蛋白D1敲除小鼠。此外，ARHI转基因的强表达与乳腺发育缺陷、不育、多器官萎缩以及海马和小脑皮质变性有关。因此，ARHI是一个假定的生长抑制性肿瘤抑制基因，其缺失可能对卵巢癌的发展很重要。这项提案的总体目标是探索ARHI作为生长调节基因的作用。我们的具体目标有三个方面：1）评估恶性卵巢上皮细胞中ARHI表达缺失的临床意义和（B）机制; 2）确定ARHI表达对卵巢细胞增殖、侵袭、转移和存活的影响; 3）确定ARHI干扰信号转导、抑制细胞增殖或诱导细胞凋亡的机制

Differential display PCR ha been used to search for genes that are expressed in normal ovarian surface epithelial cells, but not in ovarian cancer cells. NO3EY2 (designated ARHI, by the Human Gene Nomenclature Committee) is expressed consistently by normal ovarian and breast epithelial cells, but not be ovarian or breast cancers. This gene encodes a small G protein of 26 Kd that is homologous to ras and rap. Loss of ARHI expression results from multiple mechanisms. The gene is expressed monoallelically and is maternally imprinted. Loss of heterozygosity of the gene was detected in 41% of ovarian and breast cancers on chromosome 1p31. In tumo5rs with LOH, the non-imprinted functional allele was deleted in 7 of 9 instances. Expression of ARHI can also be transcriptionally regulated with promoter activity in normally ovarian epithelial cells, but not in most cancers. The activities of promoter segments of different lengths have been evaluated in normal and malignant ovarian epithelial cells. A truncated construct of 180 bp was significantly less active in ovarian cancers that in normal cells. A 26 bp DNA element in this region binds to a putative regulatory protein in ovarian cancer cells that fail to express ARHI, but does not bind to nuclear proteins in normal ovarian surface epithelial cells that express ARHI. Reexpression of the ARHI gene by transfection inhibits the clonogenic growth of ovarian and breast cancer cell lines that have lost expression of ARHI. Growth inhibition is associated with down- regulation of cyclin D1 promoter activity and induction of P21/WAF1/CIP1. EGF-induced signaling through Fas/map is reduced and truncated by expression of ARHI whereas the JNK kinase pathway is activated. Mice bearing the human ARHI transgene are small in size, resembling cyclin D1 knockout mice. In addition, strong expression of the ARHI transgene is associated with a defect in mammary gland development, sterility, atrophy of multiple organs and degeneration of the hippocampus and cerebellar cortex. Thus, ARHI is a putative growth inhibitory tumor suppressor gene whose loss may be important for the development of a significant fraction of ovarian cancers. The overall goal of this proposals is to explore the role of ARHI as a growth regulatory gene. Our specific aims are three fold: 1) to evaluate (a) the clinical significance and (b) the mechanism of loss of ARHI expression in malignant ovarian epithelial cells; 2) to determine the effect ARHI expression on proliferation, invasion, metastasis and survival of ovarian cells; 3) to determine mechanisms by which ARHI interferes with signal transduction, inhibits cell proliferation or induces apoptosis