ISOLATION OF TUMOR SUPPRESSOR GENE ON CHROMOSOME 3P

3P染色体上抑癌基因的分离

• 批准号: 2376865
• 负责人: SUSAN L NAYLOR
• 金额: $ 27.69万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 1998-03-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2376865
• 关键词: animal genetic material tag; artificial chromosomes; athymic mouse; breast neoplasms; chromosome deletion; genetic mapping; human tissue; lung neoplasms; molecular cloning; neoplasm /cancer genetics; neoplastic transformation; northern blottings; nucleic acid sequence; ovary neoplasms; polymerase chain reaction; protooncogene; pulsed field gel electrophoresis; restriction mapping; small cell lung cancer; southern blotting; tumor suppressor genes

Deletion of chromosome 3 has been implicated in a number of human cancers including small cell lung cancer (SCLC). Chromosomal deletion in solid tumors is one of the hallmarks of a tumor suppressor gene. Microcell transfer experiments from our laboratory support the contention that there is a tumor suppressor gene on the short arm of chromosome 3. The goals of this research are to clone the putative tumor suppressor gene in the region chromosome 3p21 and to determine its role in tumorigenesis and its function in normal cells. A 2Mb fragment from the p2l-p22 region of chromosome 3 which suppresses the growth of mouse A9 cells will be the starting material for cloning this gene. This human DNA fragment contains sequences which are homozygously deleted in SCLC. Both genomic and cDNA strategies will be pursued in the cloning of the gene. For the genomic approach, YAC, P1 and cosmid libraries will be screened with markers which are known to be in this fragment and are also deleted in SCLC. cDNAs corresponding to this region will be isolated by hybrid selection, exon trapping, and direct screening methods. Tumor cell lines either from revertants of the somatic cell hybrid or from SCLC will be analyzed for altered gene structure or expression for each candidate locus. Mutation analysis of tumor samples will indicate the frequency of aberration of candidate genes in SCLC. To prove the tumor suppressor gene- lies on a clone, genomic or cDNA clones will be transfected into mouse A9 cells. The stable transfectants will be analyzed for tumor growth. Once the tumor suppressor is identified, this gene will be examined in tumors which have shown deletions of chromosome 3 including breast and ovarian cancer. Characterization of this gene and gene product will include sequencing the cDNA and production of antibodies to define the expression pattern of this protein. These experiments will define a tumor suppressor gene on chromosome 3 and initiate new research on determining the role of this gene in many types of human cancer.

染色体3的缺失已与许多人类癌症有关 包括小细胞肺癌（SCLC）。固体中的染色体缺失 肿瘤是肿瘤抑制基因的标志之一。 Microcell 我们实验室的转移实验支持了这样的论点 是染色体3的短臂上的肿瘤抑制基因。 这项研究是为了克隆推定的肿瘤抑制基因 区域3p21染色体并确定其在肿瘤发生及其中的作用 在正常细胞中的功能。来自P2L-P22区域的2MB片段 抑制小鼠A9细胞生长的染色体3将是 克隆该基因的起始材料。这个人的DNA碎片包含 在SCLC中被纯合的序列。基因组和cDNA 将在基因的克隆中采取策略。对于基因组 方法，YAC，P1和Cosmid库将用标记筛选 已知在此片段中，也被删除在SCLC中。 cDNA 与该区域相对应的将通过混合选择外显子隔离 捕获和直接筛选方法。肿瘤细胞系来自 将分析体细胞杂种或从SCLC的恢复物的 每个候选基因座的基因结构或表达改变。突变 肿瘤样品的分析将表明异常的频率 SCLC中的候选基因。为了证明肿瘤抑制基因 克隆，基因组或cDNA克隆将被转染到小鼠A9细胞中。这 将分析稳定的转染物以分析肿瘤生长。一旦肿瘤 鉴定抑制剂，将在具有的肿瘤中检查该基因 显示了3染色体的缺失，包括乳腺癌和卵巢癌。 该基因和基因产物的表征将包括测序 cDNA和抗体的产生以定义了这一点的表达模式 蛋白质。这些实验将定义抑制肿瘤基因 染色体3并启动有关确定此作用的新研究 许多类型的人类癌中的基因。