IMMORTALIZATION OF SV40-TRANSFORMED HUMAN CELLS

SV40 转化人类细胞的永生化

• 批准号: 3115377
• 负责人: HARVEY L OZER
• 金额: $ 28.19万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-12-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3115377
• 关键词: DNA replication; RNA; cell cycle; cell differentiation; cell line; chromosome aberrations; clone cells; complementary DNA; diploidy; fibroblasts; gene expression; genetic library; genetic manipulation; genetic mapping; human tissue; in situ hybridization; karyotype; laboratory mouse; molecular cloning; mutagens; natural gene amplification; northern blottings; nucleic acid sequence; polymerase chain reaction; protooncogene; simian virus 40; tissue /cell culture; tumor suppressor genes; virus antigen

DESCRIPTION (Adapted from the applicant's abstract): Human diploid cells derived from normal tissue undergo a characteristic pattern of growth in cell culture, ending with a non-replicative phase. Models of cellular aging have been developed which suggest analogy between this pattern and behavior of the organism in vivo; hence, the term senescence. Human diploid fibroblasts (HF) which have been transformed in vitro by the DNA virus SV40 have been found to overcome this phenomenon (i.e., immortalize) at low frequency. The investigators have isolated a series of SV40- transformed HF using origin-defective viral genomes containing either a wild-type T antigen (SV/HF) or that of tsA58 (SVtsA/HF). In each system, they have subsequently isolated immortalized derivatives for comparative studies. The data are most consistent with a 2-step model for immortalization. SV40 large T antigen(LT) function is required in both stages, as demonstrated by temperature-dependent (ts) cell proliferation in concordance with ts LT function in preimmortal and immortal SVtsA/HF-A cells. At step 1, LT functions as a mitogenic agent, in large part through its interaction with cellular proteins as pRb and p53. Step 2, which is specific to immortalization, involves changes in functions not directly dependent on LT. A rearrangement on the long arm of chromosome 6 (6q21) consistent with inactivation of a suppressor gene has been found to be specifically associated with step 2. The applicant has identified changes in gene expression through differential hybridization with cDNA libraries of preimmortal and immortal SV40- transformants and propose to identify the genes involved. The investigator will focus initially on those genes whose expression is reduced in several matched immortal transformants as compared to their parental preimmortal transformants, most consistent with the recessive nature of the latter in cell hybrids. Strategies are also presented to identify the responsible gene on 6q. In both cases, the genes identified will be assessed for growth suppression of immortal cell lines, a phenomenon relevant to both senescence and carcinogenesis. The investigators have also found that the cellular proto-oncogene c-myb is induced in an immortal HF cell line transformed by an intact but not a truncated LT. They propose to assess the viral and cellular functions responsible because of the importance of c-myb to cell proliferation and as a prototype of genes overexpressed in immortal SV40-transformants.

描述（改编自申请人的摘要）：人类二倍体细胞 源自正常组织，经历特有的生长模式 细胞培养，以非复制阶段结束。 蜂窝模型 老化的发展表明这种模式与 生物体内的行为；因此，出现了“衰老”这个词。 人类 DNA 体外转化的二倍体成纤维细胞 (HF) 病毒 SV40 被发现可以克服这种现象（即永生化） 低频时。 研究人员分离出一系列 SV40- 使用包含以下任一来源的起源缺陷病毒基因组转化 HF： 野生型 T 抗原 (SV/HF) 或 tsA58 抗原 (SVtsA/HF)。 在每个系统中， 他们随后分离出永生衍生物用于比较 研究。数据与 2 步模型最为一致 永生化。 两者都需要 SV40 大 T 抗原 (LT) 功能 阶段，如温度依赖性 (ts) 细胞增殖所示 与永生前和永生 SVtsA/HF-A 中的 ts LT 功能一致 细胞。 在第 1 步，LT 在很大程度上起到促有丝分裂剂的作用 通过其与 pRb 和 p53 等细胞蛋白的相互作用。步骤2， 这是永生所特有的，涉及功能的变化，而不是 直接依赖于LT。 染色体长臂的重排 6 (6q21) 与抑制基因失活一致已被发现 与步骤 2 特别相关。 申请人通过以下方式确定了基因表达的变化 与永生前和永生的 cDNA 文库进行差异杂交 SV40-转化体并建议鉴定所涉及的基因。这 研究人员将首先关注那些表达量较低的基因 与它们相比，在几个匹配的永生转化体中减少 亲本前永生转化体，与隐性遗传最一致 后者在细胞杂交中的性质。 还提出了策略 确定 6q 上的相关基因。在这两种情况下，鉴定的基因 将评估永生细胞系的生长抑制， 与衰老和癌变有关的现象。 研究人员还发现细胞原癌基因c-myb 在由完整细胞而非非细胞转化的永生 HF 细胞系中诱导 截断的 LT。 他们建议评估病毒和细胞功能 由于 c-myb 对细胞增殖的重要性和 作为永生 SV40 转化体中过度表达的基因的原型。