IDENTIFICATION OF 3P RECESSIVE ONCOGENES IN LUNG CANCER

肺癌中 3P 隐性癌基因的鉴定

• 批准号: 6296132
• 负责人: JOHN D. MINNA
• 金额: $ 13.22万
• 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/6296132
• 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(抑癌基因)在肿瘤发生、发展中起主要作用。 人类肺癌的发病机制。然而，3P是最常见的 肺癌中涉及的染色体区域提示肺癌的位置 一个或多个新的隐性癌基因(S)。细胞遗传学和分子生物学 有证据表明3p的等位基因丢失发生在80%的小细胞(SCLC)和 50%的非小细胞(NSCLC)肺癌。事实上，证据是 提示位于3p25的几个3p不同的隐性癌基因， 3p21.3(两个位点)、3p14.2和3p12-13。关于创业精神的研究 表明3p21等位基因的丢失是最早的变化之一 发现于癌前病变(发生在增生期) 提示一个或多个3P隐性癌基因具有“把关人”的功能 在肺癌的分子发病机制中起重要作用。这样做的具体目的是 项目是：(具体目标#1)通过位置克隆分离新的 隐性癌基因位于3p21.3。这将使用 独特的试剂，包括完整的粘粒重叠群和cDNA克隆 &gt；25个不同的基因，由Minna博士和他的 合作者涵盖了一系列 在小细胞肺癌基因组DNA中发现嵌套纯合子缺失。基因将会 通过搜索开放阅读框中的突变来鉴定 这些cDNA使用SSCP和DNA测序技术。具体目标2 是通过以下方式来确定该基因座的功能特征 通过微细胞介导的染色体转移，引入一部分 将人类染色体3p导入人肺癌细胞系 3p21.3区域的纯合缺失，然后检测 软琼脂糖集落对裸鼠成瘤的抑制作用 在细胞培养中形成，并诱导细胞凋亡(程序化细胞 死亡)，由Killary博士。为测试抑制而提出的具体目标3 端粒酶活性、端粒缩短和端粒缺失 在引入候选基因后，肺癌中永生细胞的生长 来自3p21.3纯合缺失的Cosmids和cDNA以及 使用Shay博士开发的独特分析方法对3号染色体的部分进行分析 实验室。翻译的目标是应用这些信息来开发新的 癌前病变遗传变化的鉴定方法 用于肺癌的早期诊断；用作替代分子 标记；通过种系突变寻找遗传易感性 基因；并有可能开发肿瘤特异性治疗方法。这项研究 翻译涉及细胞遗传学、等位基因丢失、基因变化 创业发育、纯合子缺失发现和位置克隆 信息。所需的步骤是识别候选人3p21.3 隐性癌基因在纯合缺失中的表达，决定其开放程度 肺癌中阅读框序列和表达的筛查 改变初级序列的突变，突变的演示 在肿瘤细胞系、原发肿瘤和癌前病变中，检测 这种突变的存在是否表明存在非常高的风险 个体化与采用化学预防的皮损可逆性 (替代标记)，突变基因的种系遗传测试和 这些是否会导致遗传性癌症易感综合征；以及 基于对基因功能的研究，设计其他诊断方法 测试和潜在的治疗方法。因此，该项目与 项目2(肺癌的遗传易感性)，项目3 (肺癌的分子早期检测)和项目#4 (肺癌的化学预防)