IDENTIFICATION OF 3P RECESSIVE ONCOGENES IN LUNG CANCER

肺癌中 3P 隐性癌基因的鉴定

• 批准号: 6269751
• 负责人: JOHN D. MINNA
• 金额: $ 30.05万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6269751
• 关键词: DNA directed DNA polymerase; apoptosis; athymic mouse; autosomal recessive trait; carcinogenesis; carcinogenesis inhibitor; clone cells; disease /disorder proneness /risk; genetic markers; human genetic material tag; human tissue; lung neoplasms; molecular cloning; neoplasm /cancer genetics; nucleic acid sequence; oncogenes; open reading frames; preneoplastic state; single strand conformation polymorphism; telomere; tumor suppressor genes

Recessive oncogenes (tumor suppressor genes) play a major role in the pathogenesis of human lung cancer. However, 3p is the most frequently involved chromosomal region in lung cancer suggesting the location of one or more new recessive oncogene(s). Cytogenetic and molecular evidence for allele loss of 3p occurs in >80% of small cell (SCLC) and >50% of non-small cell (NSCLC) lung cancers. In fact the evidence is suggestive of several 3p distinct recessive oncogenes located at 3p25, 3p21.3 (two sites), 3p14.2, and 3p12-13. Studies of preneoplasia indicate the loss of 3p21 alleles is one of the earliest alterations found in preneoplastic lesions (occurring at the stage of hyperplasia) suggesting one or more 3p recessive oncogenes functions as "gatekeepers" in the molecular pathogenesis of lung cancer. The specific aims of this project are: (specific aim #1) to isolate by positional cloning a new recessive oncogene residing at 3p21.3. This will be done using the unique reagents including a complete cosmid contig and cDNA clones for >25 different genes, that have been assembled by Dr. Minna and his collaborators covering the shortest region of overlap in a series of nested homozygous deletions found in SCLC genomic DNAs. The gene will be identified by searching for mutations in the open reading frame of these cDNAs using SSCP and DNA sequencing techniques. Specific aim #2 is to determine the functional characteristics of this locus by introducing, through microcell mediated chromosome transfer, a portion of human chromosome 3p into a human lung cancer line bearing a homozygous deletion for the 3p21.3 region and then testing for suppression of tumorigenicity in nude mice, soft agarose colony formation in cell culture, and induction of apoptosis (programmed cell death) by Dr. Killary. Specific aim #3 proposed to test for suppression of telomrase activity, development of telomere shortening, and loss of immortal cell growth in lung cancer, following introduction of candidate cosmids and cDNAs from the 3p21.3 homozygous deletion as well as portions of chromosome 3 using unique assays developed by Dr. Shay's lab. The translational goal is to apply this information to develop new methods for identification of genetic changes in preneoplastic lesions for very early lung cancer diagnosis; use as a surrogate molecular marker; search for genetic predisposition via germline mutations in the gene; and potentially to develop tumor specific therapy. The research being translated involves cytogenetic, allele loss, genetic changes in preneoplasia, homozygous deletion discovery, and positional cloning information. The steps required are identification of candidate 3p21.3 recessive oncogenes in the homozygous deletion, determining their open reading frame sequence and expression in lung cancer, screening for mutations that alter the primary sequence, demonstration of mutations in tumor cell lines, primary tumors, and preneoplastic lesions, testing for whether the presence of the mutation identifies very high risk individuals and the reversibility of the lesions with chemoprevention (surrogate markers), tests for germline inheritance of mutant genes and whether these lead to an inherited cancer predisposition syndrome; and, based on studies of the function of the gene, designing other diagnostic tests and potential therapies. Thus, this project interacts with Project #2 (Genetic Susceptibility to Lung Cancer), Project #3 (Molecular early Detection of Lung Cancer), and Project #4 (Chemoprevention of Lung Cancer).

隐性癌基因（肿瘤抑制基因）在 人肺癌的发病机理。 但是，3p是最常的 涉及肺癌中的染色体区域，表明 一个或多个新的隐性致癌基因。细胞遗传学和分子 3p等位基因损失的证据发生在> 80％的小细胞（SCLC）和 > 50％的非小细胞（NSCLC）肺癌。 实际上，证据是 提示位于3p25的几个3P不同的隐性发病 3P21.3（两个站点），3P14.2和3P12-13。肿瘤的研究 表明3p21等位基因的损失是最早的变化之一 在肿瘤性病变中发现（发生在增生阶段） 建议一个或多个3p隐性癌基因作为“守门人”的功能 在肺癌的分子发病机理中。 这个特定的目的 项目是：（特定目标＃1）通过定位克隆隔离 驻留在3P21.3的粘剂癌基因。 这将使用 独特的试剂，包括完整的宇宙重叠群和cDNA克隆 > 25种不同的基因，这些基因由Minna博士和他的 合作者涵盖了一系列重叠区域的最短区域 SCLC基因组DNA中发现的嵌套纯合缺失。 该基因会 可以通过在开放阅读框架中搜索突变来识别 这些cDNA使用SSCP和DNA测序技术。 特定目标＃2 是确定该基因座的功能特征 通过Microcell介导的染色体转移引入一部分 人类3p染色体的人类肺癌系 3p21.3区域的纯合删除，然后测试 在裸鼠，软琼脂糖菌落中抑制肿瘤性 细胞培养和凋亡的诱导（程序性细胞）的形成 死亡）Killary博士。 提议测试抑制的特定目标＃3 端粒活动，端粒缩短的发展和丧失 引入候选者后，肺癌的不朽细胞生长 来自3p21.3纯合删除的宇宙和cDNA以及 Shay博士开发的独特测定法3染色体的部分 实验室。 翻译目标是应用此信息来开发新的 鉴定产肿瘤病变遗传变化的方法 对于早期的肺癌诊断；用作替代分子 标记；通过种系突变搜索遗传易感性 基因;并有可能发展肿瘤特定疗法。 研究 被翻译涉及细胞遗传学，等位基因丧失，遗传变化 preneoplasia，纯合删除发现和位置克隆 信息。 所需的步骤是识别候选3P21.3 纯合缺失中的隐性致癌基因，确定其开放 阅读框架序列和肺癌的表达，筛查 改变主要序列的突变，突变的表现 在肿瘤细胞系，原发性肿瘤和肿瘤性病变中，测试 对于突变的存在是否识别出很高的风险 通过化学预防的个人和病变的可逆性 （替代标记），对突变基因种系遗传的测试和 这些是否导致遗传性癌症易感综合征；和， 基于对基因功能的研究，设计其他诊断 测试和潜在疗法。 因此，这个项目与 项目＃2（对肺癌的遗传敏感性），项目＃3 （分子早期检测肺癌）和项目＃4 （肺癌化学预防）。