GENETIC AND BIOCHEMICAL ANALYSIS OF CELL BEHAVIOR

细胞行为的遗传和生化分析

• 批准号: 3963034
• 负责人: M M GOTTESMAN
• 金额: --
• 依托单位: DIVISION OF CANCER BIOLOGY AND DIAGNOSIS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3963034
• 关键词: Rous sarcoma virus; acyltransferase; cell growth regulation; chloramphenicol; cyclic AMP; cytogenetics; drug resistance; fibroblasts; gel electrophoresis; gene expression; genetic manipulation; interferons; linkage mapping; mitotic spindle apparatus; molecular biology; molecular cloning; morphology; mutant; natural gene amplification; neoplasm /cancer genetics; protein kinase; tissue /cell culture; transposon /insertion element; tumor promoters; viral carcinogenesis

We are utilizing the Chinese hamster ovary (CHO) fibroblast to study the genetics and biochemistry of some aspects of the behavior of cultured cells. Our work has emphasized morphology and its relationship to growth control, and response to cyclic AMP. We have isolated a variety of different mutants with altered microtubules which express mutated Alpha or Beta-tubulin subunits and used these mutants to study spindle formation and the mechanism of anti-microtubule drug action. These mutants are defective in spindle formation because the mutant tubulins are present in spindles and interfere with their normal function. One Beta-tubulin mutation has been transferred by DNA mediated gene transfer to wild-type cells where it is amplified, overexpressed and renders the cells dependent on Colcemid for growth at 37 degrees. We have also established two cell systems for examining the ways in which AMP can positively and negatively regulate cell growth. CHO cell growth is inhibited by cAMP; mutants selected for resistance to growth inhibition have defective cAMP dependent protein kinases. We have used DNA from cells carrying dominant cAMP-resistant defects to transfer the cAMP-resistance phenotype to sensitive cells and are cloning these genes from cosmid libraries prepared from our mutant cell lines. CHO cells malignantly transformed by RSV are also cAMP-resistant. Formation of tumors by CHO-RSV cells and human melanoma calls is dependent on prior treatment with cholera toxin which raises cAMP levels within the cells. This increased tumorigenicity is an example of positive regulation of cell growth by cAMP.

我们正在利用中国仓鼠卵巢（CHO）成纤维细胞来研究 培养物行为某些方面的遗传学和生物化学 细胞。 我们的工作强调形态及其与生长的关系 控制和对环 AMP 的响应。 我们隔离了多种 不同的突变体具有改变的微管，表达突变的α或 β-微管蛋白亚基并使用这些突变体来研究纺锤体的形成和 抗微管药物的作用机制。 这些突变体有缺陷 纺锤体形成中，因为突变的微管蛋白存在于纺锤体中 并干扰其正常功能。 一种 β-微管蛋白突变 通过DNA介导的基因转移被转移到野生型细胞中 被扩增、过表达并使细胞依赖秋水酰胺 增长37度。 我们还建立了两个细胞系统 检查 AMP 正向和负向调节细胞的方式 生长。 cAMP 抑制 CHO 细胞生长；选择的突变体 对生长抑制的抵抗力有缺陷的 cAMP 依赖性蛋白 激酶。 我们使用了来自携带显性 cAMP 抗性的细胞的 DNA 将 cAMP 抗性表型转移到敏感细胞的缺陷 正在从我们的突变细胞制备的粘粒文库中克隆这些基因 线。 由 RSV 恶性转化的 CHO 细胞也具有 cAMP 抗性。 CHO-RSV 细胞和人类黑色素瘤细胞形成肿瘤具有依赖性 先前接受霍乱毒素治疗，可提高体内 cAMP 水平 细胞。 这种致瘤性的增加是正向调节的一个例子 cAMP 影响细胞生长。