Serum-free selection of onc genes

We have described the development of a selective system useful for the identification and characterization of onc genes capable of conferring growth factor independence. The use of defined, serum-free media allows us to select for transformed cells which have lost specific growth factor requirements. We have used this system to show that several cloned onc genes generate different transformed phenotypes with respect to growth factor requirements. BPV-1 is active in relieving contact inhibition, yet these transformed NIH/3T3 cells retain their stringent requirement for FGF. In contrast, sis and H-ras were equally proficient at relieving contact inhibition and the requirement for FGF. Sis induced equal numbers of colonies regardless of the presence or absence of insulin, however, H-ras-mediated colony formation decreased four-fold when insulin was removed. This suggests that H-ras is less efficient in relieving the insulin requirement than is sis. To determine if colony formation by H-ras is a function of dosage, we are conducting experiments to measure the level of expression of p21 in transformants selected with and without insulin in the media. We have also presented data to show that loss of contact inhibition and loss of growth factor requirements are dissociable phenotypes under separate control in some cells. Thus, it should be possible to use this selective system to identify transforming genes in tumor DNA. Since some of these genes may be undetectable by the standard focus forming assay, selection in MSF medium may prove to be a useful tool for identifying and elucidating the action of activated cellular onc genes.