DERIVATION OF REVERTANTS OF RAS-TRANSFORMED HUMAN CELLS

ras转化细胞的回复突变体将来自KHOS 细胞系 这些细胞，由一个单一的拷贝， Kirsten鼠肉瘤病毒（KiMuSV）基因组，来自HOS 这些细胞最初是从人类骨肉瘤中克隆出来的。 虽然这是一个连续的，不朽的人类线， 没有转化细胞的公认特性。 是 然而，容易被各种致癌病毒和 化学手段。 第二个Ki-ras基因已经被引入到 KHOS细胞，以减少由于改变而产生的回复突变体的频率 转化基因本身。 继亚硝基胍 突变时，将使用哇巴因处理选择平坦回复突变体。 KHOS对哇巴因的敏感性比非哇巴因高得多。 转化的亲本HOS细胞。 由此获得的回复突变体细胞 将在软琼脂中测定锚定非依赖性生长， 裸鼠致瘤性。 那些没有表现出这些 然后将检查转换后的特征， 从KHOS中拯救转化病毒转化基因 使用嗜中性鼠白血病病毒（MuLV）作为 辅助细胞和测定对NIH 3 T3细胞的病灶形成活性。 的 ras基因产物p21的存在将通过蛋白质印迹法证实。 （蛋白质）印迹。 这些回复突变株 证明保留了活性转化基因， 用含有ras的哈维和克尔斯滕进行了再转化测试 病毒。 我们还将进行细胞融合与其他转化 线，以寻找在 产生的混合物。 在用小鼠细胞系制造杂交体时， 试图将负责抑制的位点定位到特定的 人类染色体 最后，我们将尝试分离序列 通过抑制KiMuSV转化的NIH 3 T3参与抑制 细胞，再次使用哇巴因选择回复突变体。

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.