FUNCTIONAL ANALYSIS--GENE FROM METASTATIC BREAST CANCER

功能分析--转移性乳腺癌基因

• 批准号: 2103568
• 负责人: SAYEEDA B ZAIN
• 金额: $ 19.78万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-15 至 1997-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2103568
• 关键词: SDS polyacrylamide gel electrophoresis; breast neoplasms; calcium binding protein; cell differentiation; cell motility; complementary DNA; embryo /fetus tissue /cell culture; embryo implantation; endopeptidases; gene expression; human pregnant subject; immunocytochemistry; in situ hybridization; laboratory mouse; metastasis; neoplasm /cancer genetics; neoplasm /cancer invasiveness; neoplastic growth; phenotype; polymerase chain reaction; protein structure function; structural genes; transfection

Mts1 is a unique gene isolated from metastatic mammary carcinomas belong to the S100 family of calcium binding proteins. It is selectively expressed during embryogenesis and metastasis. Both the murine as well as human cDNAs have been cloned and characterized in our laboratory. It is encoded in humans on chromosome 1 and on chromosome 3 in the murine system. The gene product is an approximately 10 KD protein moiety, with very high homology to the Ca++ binding domain of the other members of S100 family of proteins, which are highly conserved. In view of the relevance of this gene product to the process of invasion, motility, embryonic transplantation, and metastasis, we propose to study the biological significance of this gene using a variety of molecular and cell biological techniques. Mouse and human cDNAs will be overexpressed, both in bacterial as well as eukaryotic expression systems, and the protein obtained will be used to generate highly specific mono and polyclonal antibodies. To determine whether there is a causal relationship between mts1 expression and metastatic phenotype, additional copies of the mouse mts1 will be introduced into the mouse benign breast tumor cell line CSML0 by DNA transfection techniques. Similarly, constructs containing Mts1 cDNA in opposite orientation will be introduced into the highly metastatic CSML100 cell line. In addition, these transfectants will be cotransfected with beta-gal gene to be used as a visual, sensitive marker to detect mice metastases. These permanently transfected cell lines along with their parent control lines will be utilized in testing multiple aspects of the tumorigenic and metastatic phenotype, i.e. growth rates, invasiveness, and motility, levels of proteolytic enzymes, etc. Using immunohistochemical and in situ hybridization techniques, mts1 expression patterns will be generated on murine embryonic tissue (days 5-10) when trophoblasts are the most invasive) to examine the correlation between invasiveness and mts1 expression. Serial sections of the tw73 implantation defective mouse embryonic and extraembryonic tissues (days 5-10) will also be tested similarly. Human trophoblast cultures (villous cultures of the first trimester human placenta) will be used to examine mts1 gene involvement in cellular differentiation, motility and invasiveness during embryonic implantation. Similar studies will be conducted on tumor tissues at various stages of tumor progression to evaluate mts1 protein's role in aspects of tumor metastasis.

Mts1是从转移性乳腺癌中分离出来的独特基因 与钙结合蛋白S100家族有关。它是有选择的 在胚胎发生和转移过程中表达。无论是小鼠还是 本实验室已成功克隆并鉴定了人的cDNA。它是 人类编码在1号染色体上，小鼠在3号染色体上 系统。该基因产物约为10kD的蛋白质部分，具有 与S100其他成员的钙结合结构域高度同源 蛋白质家族，高度保守。鉴于这种关联性 该基因产物的侵袭、运动、胚胎发育过程 移植和转移，我们建议研究生物学 利用多种分子和细胞生物学方法研究该基因的意义 技巧。小鼠和人类的cDNA将在细菌中过度表达 以及真核表达系统，获得的蛋白质将是 用于产生高度特异的单抗和多克隆抗体。至 确定mts1表达之间是否存在因果关系 和转移性表型，小鼠mts1的额外拷贝将是 DNA导入小鼠乳腺良性肿瘤细胞系CSML0 转基因技术。类似地，含有Mts1基因的构建体在 相反的取向将被引入高转移的CSML100 细胞系。此外，这些转染体将与 β-半乳糖基因将作为检测小鼠的直观、灵敏的标志 转移瘤。这些永久转染的细胞系和它们的 父控制线将用于测试 致瘤和转移表型，即生长率、侵袭性和 运动性、蛋白水解酶水平等免疫组织化学方法 和原位杂交技术，mts1的表达模式将是 产生于小鼠胚胎组织(5-10天)，此时滋养层细胞是 最具侵袭性)，以检查侵袭性和MTS1之间的相关性 表情。TW73植入缺陷小鼠的连续切片 胚胎和胚胎外组织(5-10天)也将进行测试 同样的。人滋养层细胞培养(第一代绒毛培养 人胎盘)将用于检测mts1基因在 胚胎发育过程中的细胞分化、运动和侵袭 植入。将对肿瘤组织进行类似的研究， 评估mts1蛋白在肿瘤进展的不同阶段中的作用 肿瘤转移的各个方面。