ISOLATION OF TUMOR SUPPRESSOR GENE ON CHROMOSOME 3P

3号染色体的缺失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。微蜂窝 我们实验室的转移实验支持这样一种观点 是一种位于3号染色体短臂上的肿瘤抑制基因。 本研究的目的是克隆肿瘤抑制基因。 染色体3p21区域及其在肿瘤发生和发展中的作用 在正常细胞中发挥作用。来自p21-p22区域的2Mb片段 抑制小鼠A9细胞生长的3号染色体将是 克隆该基因的起始材料。这个人类DNA片段包含 在SCLC中纯合缺失的序列。基因组和cDNAs 在克隆该基因方面将采取各种策略。对于基因组而言 Approach、YAC、P1和COSMID文库将用下列标记进行筛选 已知存在于该片段中，并且在SCLC中也被删除。CDNA 与该区域对应的基因将通过杂交选择、外显子 诱捕法和直接筛查法。肿瘤细胞系或来自 体细胞杂交或小细胞肺癌的返回体将被分析 改变每个候选基因座的基因结构或表达。突变 对肿瘤样本的分析将表明 小细胞肺癌的候选基因。为了证明肿瘤抑制基因-依赖于 克隆、基因组或cDNA克隆将被导入小鼠A9细胞。这个 将对稳定的转染体进行肿瘤生长分析。一旦肿瘤 抑制子被确定，这个基因将在有 显示包括乳腺癌和卵巢癌在内的3号染色体缺失。 这种基因和基因产物的特征将包括对 基因和抗体的制备以确定该基因的表达模式 蛋白。这些实验将定义一种肿瘤抑制基因 3号染色体，并启动新的研究，以确定这一作用 基因存在于多种类型的人类癌症中。

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.