Functional identification and molecular cloning of a rat gene mediating growth inhibition and programmed cell death in normal and transformed rat cell lines.

We wished to identify DNA sequences conferring suppression of proliferation and transformed phenotypes. Thus, we have transfected DNA from normal rat cells, covalently linked to neo DNA coding for neomycin resistance into a tumorigenic, HRAS transformed rat cell line. Phenotypic revertants were selected after the first cycle of transfection by enrichment procedures that served to eliminate transformed cells. The revertant clones continued to express the HRAS oncogene, but exhibited a lower tumorigenicity, loss of anchorage-independent proliferation, flat morphology, and retardation of growth in monolayer culture. The reverted phenotype could be transferred in a second cycle of transfection into the HRAS transformed rat cells. Neo DNA ligated to genomic donor DNA was used as a tagging sequence to molecularly clone the transferred DNA sequence in a recombinant phage. Fragments of the cloned DNA detect a 2.5 kb transcript in parental cells and revertants. Thus, the recombinant phage harbors a putative growth inhibitory gene, designated trg, that is expressed at a higher level in rat embryo fibroblasts and in the REF52 cell line. Introduction of recombinant phage DNA into established 208F and Rat-2 cells and into HRAS-, v-fgr-, v-fms- and v-raf-transformed rat cell lines resulted in inhibition of growth and induction of programmed cell death.