DERIVATION OF REVERTANTS OF RAS-TRANSFORMED HUMAN CELLS

RAS 转化人类细胞回复体的衍生

• 批准号: 3459313
• 负责人: LEONARD E BENADE
• 金额: $ 10.35万
• 依托单位: AMERICAN NATIONAL RED CROSS
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-05-01 至 1993-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3459313
• 关键词: athymic mouse; cell bank /registry; cell line; cell transformation; gene interaction; genetic mapping; helper virus; human genetic material tag; human tissue; hybrid cells; molecular cloning; mouse sarcoma virus; murine leukemia virus; mutant; natural gene amplification; neoplasm /cancer genetics; neoplastic cell; nitrosoguanidines; nucleic acid sequence; oncogenes; oncogenic virus; ouabain; regulatory gene; transfection; viral rescue; western blottings

Revertants of ras-transformed cells will be derived from the KHOS cell line. These cells, transformed by a single copy of the Kirsten murine sarcoma virus (KiMuSV) genome, were derived from HOS cells, which were originally cloned from a human osteosarcoma. Although it is a continuous, immortalized human line, HOS exhibits none of the recognized properties of transformed cells. It is readily transformed, however, by various oncogenic viruses and by chemical means. A second Ki-ras gene has been introduced into the KHOS cells to reduce the frequency of revertants due to alterations of the transforming gene itself. Following nitrosoguanidine mutation, flat revertants will be selected using ouabain treatment. KHOS is much more highly susceptible to ouabain than non- transformed, parental HOS cells. Revertant cells thus obtained will be assayed-for anchorage-independent growth in soft agar and tumorigenicity in nude mice. Those lines failing to exhibit these transformed characteristics will then be examined for active transforming genes by rescuing transforming virus from KHOS revertants using amphotropic murine leukemia virus (MuLV) as a helper and assaying focus-forming activity on NIH 3T3 cells. The presence of the ras gene product p21 will be confirmed by Western (protein) blotting. Those revertant lines which can be demonstrated to have retained active transforming genes will be tested for re-transformation by ras-containing Harvey and Kirsten viruses. We will also perform cell fusions with other transformed lines to look for dominance of transformation suppression in the resulting hybrids. In making hybrids with mouse cell lines we will attempt to map the loci responsible for suppression to specific human chromosomes. Finally, we will try to isolate the sequence(s) involved in suppression by transfecting KiMuSV-transformed NIH 3T3 cells, once again using ouabain to select for revertants.

ras 转化细胞的回复体将源自 KHOS 细胞系。 这些细胞被单个拷贝转化 Kirsten 鼠肉瘤病毒 (KiMuSV) 基因组，源自 HOS 细胞，最初是从人类骨肉瘤中克隆出来的。 尽管HOS是一个连续的、不朽的人类世系，但它却展示了 没有任何公认的转化细胞特性。 这是 然而，很容易被各种致癌病毒和 化学手段。 第二个 Ki-ras 基因已被引入 KHOS 细胞可减少由于改变而产生回复突变的频率 转化基因本身。 继亚硝基胍之后 突变，将使用哇巴因治疗选择扁平回复体。 KHOS 比非 Ouabain 更容易受到哇巴因的影响。 转化的亲本 HOS 细胞。 由此获得的回复细胞 将在软琼脂和 裸鼠的致瘤性。 那些未能展示这些的线路 然后将检查转化后的特征是否活跃 通过从 KHOS 中拯救转化病毒来转化基因 使用双嗜性鼠白血病病毒（MuLV）作为回复突变体 帮助并分析 NIH 3T3 细胞的焦点形成活性。 这 ras 基因产物 p21 的存在将由 Western 证实 （蛋白质）印迹。 那些可恢复的线 证明保留了活性转化基因 经含有 ras 的 Harvey 和 Kirsten 进行再转化测试 病毒。 我们还将与其他转化的细胞进行融合 线寻找转型抑制的主导地位 由此产生的杂种。 在与小鼠细胞系进行杂交时，我们将 尝试将负责抑制的位点映射到特定的 人类染色体。 最后，我们将尝试分离序列 通过转染 KiMuSV 转化的 NIH 3T3 参与抑制 细胞，再次使用哇巴因来选择回复体。