ARHI: AN IMPRINTED TUMOR SUPPRESSOR GENE

差分显示PCR用于寻找在正常卵巢表面上皮细胞中表达的基因，而在卵巢癌细胞中不表达。NO3EY2（由人类基因命名委员会指定为ARHI）在正常卵巢和乳腺上皮细胞中一致表达，但在卵巢癌或乳腺癌中不表达。该基因编码一个26kd的小G蛋白，与ras和rap同源。ARHI表达的丧失是由多种机制引起的。该基因以单等位基因表达，并在母体中留下印记。41%的卵巢癌和乳腺癌患者在染色体1p31上检测到该基因的杂合性缺失。在LOH肿瘤中，非印迹功能等位基因在9例中有7例被删除。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节，但在大多数癌症中则不然。不同长度的启动子片段在正常和恶性卵巢上皮细胞中的活性已被评估。截断的180bp结构在卵巢癌中的活性明显低于正常细胞。在不表达ARHI的卵巢癌细胞中，该区域有一个26 bp的DNA元件与一个推测的调节蛋白结合，但在表达ARHI的正常卵巢表面上皮细胞中，该元件不与核蛋白结合。通过转染重新表达ARHI基因可以抑制失去ARHI表达的卵巢癌和乳腺癌细胞系的克隆生长。生长抑制与cyclin D1启动子活性下调和P21/WAF1/CIP1的诱导有关。egf通过Fas/map诱导的信号被ARHI的表达减少和截断，而JNK激酶途径被激活。携带人类ARHI转基因的小鼠体型小，类似于cyclin 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是一种推定的生长抑制肿瘤抑制基因，其缺失可能对很大一部分卵巢癌的发展很重要。本提案的总体目标是探索ARHI作为生长调节基因的作用。我们的具体目的有三个方面：1)评估(a)临床意义和(b)恶性卵巢上皮细胞中ARHI表达缺失的机制；2)测定ARHI表达对卵巢细胞增殖、侵袭、转移及存活的影响；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