REC-DNA ANALYSIS OF HUMAN HEMATOPOIETIC DIFFERENTIATION

人类造血分化的 REC-DNA 分析

• 批准号: 3170165
• 负责人: WINSTON SALSER
• 金额: $ 25.82万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-05-01 至 1993-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3170165
• 关键词: actins; alleles; athymic mouse; cell bank /registry; cytoskeleton; electroporation; epithelium; gene expression; gene mutation; genetic manipulation; genome; hematopoietic stem cells; human tissue; leukemia; messenger RNA; molecular cloning; molecular oncology; myeloid stem cell; natural gene amplification; neoplasm /cancer genetics; neoplastic cell; neoplastic transformation; nucleic acid probes; nucleic acid sequence; oncogenes; oncoproteins; transfection; tubulin; vimentin

We have recently found that sequences of alpha-tubulin and gamma- actin cDNA clones derived from the human leukemic cell line HL-60 contain evidence of multiple somatic mutations. These changes, occurrings in highly conserved regions, suggest a causual relationship to the neoplastic growth of HL-60 cells. The alpha- tubulin changes include drastic alterations of a GTP-binding site homology. Out of the very small number of cancer-cell cytoskeletal proteins examined by ourselves and others, three have shown multiple mutations. Taken together, these and other results have prompted us to propose the existence of a new class of oncogenes which involves the cytokeletal system. Cytoskeletal oncogenes would appear to have roles different from the nuclear-acting (e.g. myc, fos) or the growth-factor/receptor/protein-kinase (e.g., vis, erb-B, ras) classes of oncogenes. To further define the role of cystokeletal mutations occurring in neoplastic cells we will first isolate genomic clones of the mutant gamma-actin and alpha-tubulin genes from HL-60 cells. We will then directly test the oncogene potential of these genes in model systems for multistage tumorigenesis. Because of the assistance of expert collaborators we will be able to test the oncogene potential of these mutant genes in myeloid, lymphoid and epithelial tumorigenesis in addition to the more conventional fibroblast systems. These systems will permit us to detect collaboration between mutant cytoskeletal genes and others such as ras and myc. We may detect such collaborations as increase in anchorage independence, decreases in latent period or effects on the invasiveness of resulting tumors. We will also use new rapid screening procedures to screen a large number of tumor cell lines or fresh tumors for evidence of cytoskeletal mutations in other neoplasms. RNase A detecting candidate mutants. The new polymerase Chain Reaction technique provides a rapid way to clone any such candidates in order to precisely determine the nature of the mutations involved. These experiments will test the suggestion that a high percentage of cancer cells may contain cytoskeletal mutations and investigate the way in which cytoskeletal mutations may collaborate with other oncogenes in cancer initiation or progression.

我们最近发现α-微管蛋白和γ-微管蛋白的序列 人白血病细胞系HL-60肌动蛋白cDNA克隆 都有多重体细胞突变的证据 这些变化， 出现在高度保守的区域，这表明 与HL-60细胞肿瘤生长的关系。 头狼- 微管蛋白的变化包括GTP结合位点的剧烈改变 同源性 在极少数的癌细胞骨架中 我们和其他人研究了三种蛋白质， 多重突变 综合起来，这些和其他结果 促使我们提出了一类新的致癌基因的存在 这涉及到一个电子邮件系统。 细胞骨架癌基因 似乎具有与核作用不同的作用（例如， myc，fos）或生长因子/受体/蛋白激酶（例如，维斯， erb-B，ras）类癌基因。 为了进一步明确 肿瘤细胞中发生的膀胱癌突变，我们将首先 分离突变体γ-肌动蛋白和α-微管蛋白的基因组克隆 HL-60细胞的基因。 然后我们将直接测试致癌基因 这些基因在多阶段模型系统中的潜力 肿瘤发生 由于专家合作者的协助， 我们将能够测试这些突变体的致癌基因潜力， 此外， 更传统的成纤维细胞系统。 这些系统将 允许我们检测突变细胞骨架基因之间的协作 以及其它如ras和myc。 我们可能会发现这种合作 随着锚定独立性的增加，潜伏期缩短 或对所产生的肿瘤的侵袭性的影响。 我们还将 使用新的快速筛查程序筛查大量 肿瘤细胞系或新鲜肿瘤的细胞骨架证据 其他肿瘤中的突变。 RNase A检测候选突变体。 新的聚合酶链反应技术提供了一种快速的方法 克隆任何这样的候选人，以准确地确定 所涉及的突变的性质。 这些实验将测试 这表明高百分比的癌细胞可能含有 细胞骨架突变，并研究 细胞骨架突变可能与其他癌基因协作， 癌症开始或进展。