IMMORTALIZATION OF SV40-TRANSFORMED HUMAN CELLS

SV40 转化人类细胞的永生化

• 批准号: 3115386
• 负责人: HARVEY L OZER
• 金额: $ 28.06万
• 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/3115386
• 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 从正常组织派生的人在体内经历了特有的生长模式 细胞培养，以非复制阶段结束。元胞模型 衰老的研究表明，这种模式与 生物体在体内的行为；因此，术语衰老。人类 DNA体外转化的二倍体成纤维细胞 病毒SV40已被发现可以克服这种现象(即永生) 在低频率下。调查人员已经分离出一系列SV40- 使用含有A或A的来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， 他们随后分离出不朽的衍生品，以便进行比较 学习。数据与以下两步模型最为一致 永垂不朽。两者都需要SV40大T抗原(LT)功能 温度依赖性(Ts)细胞增殖所证明的阶段 长生不老和长生不老SVtsA/HF-A的TS-LT功能一致 细胞。在步骤1中，LT在很大程度上起着促有丝分裂的作用 通过它与细胞蛋白如pRb和p53的相互作用。第二步， 这是特定于不朽的，涉及到功能的改变 直接依赖于LT。染色体长臂上的重排 6(6q21)与抑制基因失活一致 具体与步骤2相关联。 申请人通过以下途径确定了基因表达的变化 永生与永生基因文库的差异杂交 SV40-转化子，并建议鉴定涉及的基因。这个 研究人员首先将重点放在那些表达 在几个匹配的不朽转化子中减少，与他们的 亲本永生前转化体，最符合隐性 后者在细胞杂交中的性质。还提出了策略以 确定6q上的致病基因。在这两种情况下，基因都被识别出来 将被评估为对永生细胞系的生长抑制， 与衰老和致癌有关的现象。 研究人员还发现，细胞原癌基因c-myb 在一种永生的HF细胞系中被诱导，该细胞系由完整的而不是 截断LT。他们建议评估病毒和细胞功能 负责是因为c-myb对细胞增殖和 作为在不朽的SV40转化子中过度表达的基因的原型。