Isolation of a cervical cancer tumor suppressor gene

描述(由申请人提供)： 抽象的。我们以前已经证明了宫颈癌抑制基因定位在染色体11q13的300kb区间。我们现在已经确定位于该区间的半胱氨酸氨基转移酶E/M是宫颈癌抑制基因。逆转录聚合酶链式反应(RT-PCR)和Western blotting检测发现，6株宫颈癌细胞株和21例原发癌中17例未见cystatin E/M表达。免疫组织化学检测20例肿瘤标本中15例表达缺失或降低。该基因在部分样本正常区域的表达作为阳性对照。在癌前病变的宫颈病变(CINS)中也有表达，表明表达缺失是一种肿瘤特异性事件。此外，在正常组织和含有半胱氨酸蛋白酶E/M的宫颈鳞癌组织中，溶酶体蛋白水解酶组织蛋白酶L的表达降低，而在肿瘤组织中发现组织蛋白酶L的过度表达，表明组织蛋白L的表达与胱抑素E/M的表达呈负相关。对19例原发肿瘤和21例正常组织进行检测，发现其中1例肿瘤组织中存在外显子1的纯合缺失。在其他六种肿瘤中也观察到了点突变。2例肿瘤中存在组织蛋白酶L共同结合位点的突变。通过定点突变引入结合位点突变导致组织蛋白酶L与半胱氨酸氨基转移酶E/M的结合减少，尽管在细胞系中未观察到突变，但4株细胞株和18例肿瘤中12例存在启动子高甲基化。5‘-氮杂脱氧胞苷和/或曲古抑素A处理宫颈癌细胞系后，观察到cystatin E/M的重新表达，证实存在启动子高甲基化。此外，对正常的人表皮角质形成细胞(宫颈癌的细胞型来源)以及HPV16E6、E7或E6&E7转化的角质形成细胞的分析表明，在含有E7的角质形成细胞中，cystatin E/M的表达降低。混合培养(HeLa与E6或E7角质形成细胞)的研究表明，E6细胞对HeLa细胞的生长有抑制作用，表明E7角质形成细胞中的cystatin E/M降解。免疫沉淀研究表明，HPV16E7与胱抑素E/M蛋白结合。Cystatin E/M在宫颈癌细胞系中的异位表达导致细胞生长抑制，同时细胞内外组织蛋白酶L水平降低。组织蛋白酶L过表达导致细胞生长增加，而导入cystatin E/M导致细胞生长抑制。我们现在建议通过以下目标来确定cystatin E/M介导的肿瘤抑制机制：1)鉴定与cystatin E/M表达缺失相关的肿瘤特异性突变，2)证实cystatin E/M通过组织蛋白L失活而抑制细胞生长，3)确定HPV16E7蛋白降解cystatin E/M的机制，4)利用四环素诱导载体系统研究cystatin E/M的抑瘤功能，5)确定cystatin E/M表达与组织蛋白酶L与宫颈癌表型的关系。 公共卫生相关性： 对普通退伍军人保健的潜在影响：尽管妇女在退伍军人中占少数，但近年来有更多的妇女在武装部队服役。宫颈癌是导致全球女性癌症相关死亡的第二大常见癌症。这种癌症影响到处于特权和服务不足的女性。它也是发展中国家的一大癌症。这种疾病经常在有多个性伴侣、吸烟习惯和免疫系统功能障碍的女性中发现。

DESCRIPTION (provided by applicant): ABSTRACT. We have previously shown that a cervical cancer suppressor gene is localized to a 300 kb interval of chromosome 11q13. We have now identified cystatin E/M mapped to this interval as a cervical cancer suppressor gene. RT-PCR (reverse transcriptase PCR), and western blotting studies revealed absence of cystatin E/M expression in 6 cervical cancer cell lines and 17 of 21 primary tumors. Immunohistochemistry on 20 tumor samples revealed loss or reduced expression in 15 tumors. Expression of the gene in normal areas of some samples served as positive controls. Expression was also seen in pre-neoplastic cervical intraneoplasisa (CINs) lesions, indicating loss of expression as a tumor specific event. Further, normal and CINs containing cystatin E/M showed reduced expression of the lysosomal protease cathepsin L. However, overexpression of cathepsin L was observed in tumors pointing to an inverse relationship between the expression of cystatin E/M and cathepsin L. Examination of the three exons of cystatin E/M in 19 primary tumors and 21 normal tissues revealed homozygous deletion of exon 1 in one tumor. Point mutations were observed in six other tumors. Two tumors contained mutations at the consensus binding sites for cathepsin L. Introduction of the binding site mutations using site directed mutagenesis resulted in reduced binding of cystatin E/M to cathepsin L. Although mutations were not observed in the cell lines, four cell lines and 12 of 18 tumors contained promoter hypermethylation. Reexpression of cystatin E/M was observed after 5'aza 2-deoxycytidine and/or Trichostatin A treatment of cervical cancer cell lines confirming the presence of promoter hypermethylation. Further, analysis of normal human epidermal keratinocytes (cell type origin of cervical cancer), and HPV16 E6, E7 or E6 & E7 transformed keratinocytes showed decreased expression of cystatin E/M in E7 containing keratinocytes. Mixed culture (HeLa with E6 or E7 keratinocytes) studies showed HeLa cell growth inhibition by E6 cells indicating degradation of cystatin E/M in E7 keratinocytes. Immunoprecipitation studies indicated binding of HPV 16 E7 to the cystatin E/M protein. Ectopic expression of the cystatin E/M in cervical cancer cell lines resulted in growth inhibition accompanied by decreased intracellular and extracellular level of cathepsin L. Finally, overexpression of cathepsin L led to increased cell growth and introduction of cystatin E/M resulted in growth inhibition. We now propose to determine the mechanism of cystatin E/M mediated tumor suppression using the following objectives: 1) Identification of tumor specific mutations correlating to loss of cystatin E/M expression in primary cervical tumors, 2) confirmation of cell growth inhibition by cystatin E/M through the inactivation of cathepsin L, 3) determine the mechanism of cystatin E/M degradation by the HPV 16 E7 protein, 4) use of tetracycline inducible vector system to study the tumor suppressor function of cystatin E/M, and 5) determine the relationship between the expression of cystatin E/M and cathepsin L to cervical cancer phenotypes. PUBLIC HEALTH RELEVANCE: Potential Impact on General VA Health care: Although women comprise minority of the veteran population, more women are serving in the armed services in recent years. Cervical cancer is the second most common cancer responsible for cancer related death in women around the world. The cancer affects women who are under privileged and under served. It is also a major cancer of the developing world. The disease is frequently found in women having multiple sex partners, smoking habits, and immune system dysfunctions.