Characterization of Human 3p Recessive Oncogenes

人类 3p 隐性癌基因的表征

• 批准号: 6399702
• 负责人: JOHN D. MINNA
• 金额: $ 35.1万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-13 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6399702
• 关键词: DNA directed DNA polymerase; DNA footprinting; apoptosis; athymic mouse; autosomal recessive trait; cell growth regulation; enzyme activity; gene deletion mutation; gene expression; human genetic material tag; human tissue; lung neoplasms; molecular cloning; molecular pathology; neoplasm /cancer genetics; northern blottings; nucleic acid sequence; oncogenes; polymerase chain reaction; protein sequence; pulsed field gel electrophoresis; single strand conformation polymorphism; southern blotting; statistics /biometry; telomerase; telomere; tissue /cell culture

This project focuses on identification and functional characterization of human 3p recessive oncogenes (tumor suppressor genes, TSGs), with the initial emphasis being on discovery of TSGs in a 630 kb, completely sequenced, 3p21.3 region found to be homozygously deleted in lung and breast cancers. Our overall hypothesis is that biallelic inactivation or haploinsufficiency of one or more of these genes is of fundamental importance in the pathogenesis of many human cancers and that this inactivation occurs early in the multistep process of carcinogenesis. We have just completed the isolation and initial characterization of 25 genes in this area (23 of which are new) and found two, RASSF1A (Ras interacting protein with a DAG binding domain), and SEMA3B (secreted class III semaphorin) to have biallelic expression loss and potent tumor suppressing activity. We also found several others to have some characteristics of TSGs (loss of expression, mutation, suppression of the malignant phenotype) including CACNA2D2, 101F6, NPR21/G21, BLU, FUSI, HYAL1, and FUS2. The current proposal will: Confirm the tumor suppressing function of RASSF1A and SEMA3B and further implicate or dismiss the other candidate 3p21.3 TSGs in multiple human cancer cell lines with different genetic abnormalities by in vitro and in vivo xenograft assays (Aim 1); Confirm that tumor acquired loss of expression of RASSF1A and SEMA3B and some of the other 3p21.3 TSG candidates occurs in human cancers but not in normal tissues, and is explained by tumor acquired methylation of CpG islands in their promoter regions (Aim 2); Study the potential mechanisms for tumor suppression of RASSF1A and SEMA3B using human cancer cell lines and thus determine the pathways involved in RASSF1A and SEMA3B suppression of the tumor phenotype; in the process also determine if acquired and germline amino acid sequence alterations in these genes inactivate their function potentially predisposing to cancer development (Aim 3); Determine whether a Sema3b knockout mouse model challenged with carcinogens confirms SEMA3B as a TSG and also test whether SEMA3B haploinsufficiency is sufficient for aiding carcinogen induced lung cancer in the mouse. (Aim 4). The characterization of these gene(s) should ultimately have translational benefit for the development of new cancer diagnostics and therapeutics, including use in cancer early detection, prevention, and treatment.

该项目致力于人类3p隐性癌基因(TSGs)的鉴定和功能鉴定，最初的重点是在肺癌和乳腺癌中发现一个630kb的3p21.3区的TSGs，该区域完全测序。我们的总体假设是，这些基因中的一个或多个的双等位基因失活或单倍体不足在许多人类癌症的发病机制中具有基本的重要性，并且这种失活发生在癌症发生的多步骤过程的早期。我们刚刚完成了该区域25个基因的分离和初步鉴定(其中23个是新基因)，发现了两个具有双等位基因表达缺失和强大的肿瘤抑制活性的基因，RASSF1A(带有DAG结合域的RAS相互作用蛋白)和SEMA3B(分泌型III类信号素)。我们还发现了其他几个具有TSGs(表达缺失、突变、恶性表型抑制)的特征，包括CACNA2D2、101F6、NPR21/G21、BLU、FUSI、HYAL1和FUS2。目前的建议将：通过体外和体内异种移植实验，确认RASSF1A和SEMA3B的抑瘤功能，并进一步在具有不同遗传异常的多个人类癌细胞系中牵连或排除其他候选3p21.3 TSG(目标1)；确认RASSF1A和SEMA3B以及其他一些3p21.3候选TSG的表达缺失存在于人类癌症中，而不是在正常组织中，并通过肿瘤获得的启动子区域CpG岛的甲基化来解释(AIM 2)；利用人类癌细胞株研究RASSF1A和SEMA3B的潜在抑瘤机制，从而确定RASSF1A和SEMA3B抑制肿瘤表型的途径；在这一过程中，还将确定这些基因中的获得性和胚系氨基酸序列变化是否会使它们的功能失活，从而可能导致癌症的发生(目标3)；确定致癌物攻击的Sema3b基因敲除小鼠模型是否确认SEMA3B为TSG，并且还测试SEMA3B单倍体不足是否足以帮助致癌物诱导的小鼠肺癌。(目标4)。这些基因(S)的特征最终将对新的癌症诊断和治疗方法的开发产生翻译上的好处，包括用于癌症的早期发现、预防和治疗。