DERIVATION OF REVERTANTS OF RAS-TRANSFORMED HUMAN CELLS

ras转化细胞的复归物将从KHOS中得到

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.