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）。固体中染色体缺失 肿瘤是肿瘤抑制基因的标志之一。微小区 我们实验室的转移实验支持这样的论点， 是3号染色体短臂上的肿瘤抑制基因。 的目标 本研究旨在克隆人乳腺癌组织中的抑癌基因， 区域染色体3 p21，并确定其在肿瘤发生中的作用， 在正常细胞中发挥作用。从大肠杆菌p21-p22区的一个2 Mb片段， 抑制小鼠A9细胞生长的3号染色体将是 克隆这个基因的起始材料这个人类DNA片段含有 在SCLC中同源缺失的序列。基因组和cDNA 将在基因克隆方面采取各种战略。对于基因组 方法，YAC、P1和粘粒文库将用标记物筛选， 已知存在于该片段中，并且在SCLC中也被删除。的cdna 将通过杂交选择分离对应于该区域的外显子， 诱捕和直接筛选方法。肿瘤细胞系来自 将分析体细胞杂种或来自SCLC的回复突变体， 改变每个候选基因座的基因结构或表达。突变 对肿瘤样品的分析将表明 SCLC中的候选基因。为了证明肿瘤抑制基因-在于一个 克隆、基因组或cDNA克隆将转染到小鼠A9细胞中。的 分析稳定转染子的肿瘤生长。一旦肿瘤 抑制基因被鉴定，该基因将在具有以下特征的肿瘤中进行检查： 显示3号染色体缺失，包括乳腺癌和卵巢癌。 该基因和基因产物的表征将包括对 cDNA和抗体的产生来确定这种表达模式。 蛋白这些实验将确定一个肿瘤抑制基因， 3号染色体，并启动新的研究，以确定这一作用， 基因在许多类型的人类癌症中。

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.