FUNCTIONAL ANALYSIS--GENE FROM METASTATIC BREAST CANCER

功能分析--转移性乳腺癌基因

• 批准号: 2103569
• 负责人: SAYEEDA B ZAIN
• 金额: $ 19.36万
• 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/2103569
• 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的一个独特基因， S100家族的钙结合蛋白。其选择性地 在胚胎发生和转移过程中表达。 无论是鼠类还是 我们的实验室已经克隆并鉴定了人cDNA。是 人类1号染色体和小鼠3号染色体编码 系统基因产物是约10 KD的蛋白质部分， 与S100的其他成员的Ca ++结合结构域具有非常高的同源性 蛋白质家族，高度保守。鉴于相关性， 这种基因产物的过程中的入侵，运动，胚胎 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 技术.小鼠和人的cDNA将在细菌中过表达， 以及真核表达系统，并且所获得的蛋白质将 用于产生高度特异性的单克隆和多克隆抗体。到 确定mts1表达之间是否存在因果关系 和转移表型，小鼠MTS 1的额外拷贝将被 通过DNA导入小鼠乳腺良性肿瘤细胞系CSML0 转染技术。类似地，含有Mts1 cDNA的构建体在 相反的方向将被引入高转移性CSML100 细胞系此外，这些转染子将与 β-gal基因可作为一种直观、灵敏的标记物，用于检测小鼠 转移这些永久转染的细胞系沿着其 将利用母管制线测试的多个方面 肿瘤发生和转移表型，即生长速率、侵袭性，以及 运动性、蛋白水解酶水平等，使用免疫组织化学 和原位杂交技术，MTS1表达模式将被 在鼠胚胎组织（第5 - 10天）上产生， 最具侵袭性）以检查侵袭性和MTS 1之间的相关性 表情TW 73植入缺陷小鼠的连续切片 还将检测胚胎和胚外组织（第5 - 10天 同样的。人滋养层培养物（第一次的绒毛培养物） 三个月的人胎盘）将被用于检查MTS1基因参与 胚胎期的细胞分化、运动和侵袭 置入将在肿瘤组织上进行类似的研究， 肿瘤进展的不同阶段，以评估MTS1蛋白在 肿瘤转移方面。