CPH, A NOVEL ONCOGENE WHICH COOPERATES WITH H-RAS IN THE TRANSFORMATION OF NIH3T3 FIBROBLASTS

We have performed the molecular cloning of the non-ras transforming se quences previously detected in neoplastic Syrian hamster embryo fibrob lasts initiated in vitro with 3-methylcholanthrene (MCA) (Notario et a t, 1990). These sequences were isolated using cosmid-rescue techniques from a third-cycle NIH3T3 transformant obtained by sequential transfe ctions of genomic DNA from MCA-initiated hamster fetal cells. Rescued (C-5) clones encompassed about 42.5 kbp of Syrian hamster genomic DNA containing hamster-specific repetitive elements (HRS). An internal 19 kbp BamHI fragment (B-1) was the only C-5 fragment which recognized sp ecific transcripts in poly(A)(+) RNA from hamster embryo cells, The sa me mRNA species were present in both normal and MCA-initiated neoplast ic cells: a major transcript of about 2.5 kb, and other less abundant ones, ranging from approximately 2.0 kb to 5.0 kb. These mRNA species were detected consistently by each of several B-1 DNA subfragments loc ated at positions spanning almost the entire B-1 length. The nucleotid e sequence of some transcript-positive (S5P2 and S-6) genomic B-1 frag ments was determined. No significant homology exists between the nucle otide sequences of these B-1 subfragments and established DNA database s. Therefore, the C-5 cosmid clone contains novel genomic sequences. T ransfection of C-5 DNA into mouse NIH3T3 cells resulted in the appeara nce of transformed foci (about five foci per mu g of DNA) within 25 da ys post-transfection, thus demonstrating the transforming activity of the C-5 clone, which was consequently renamed as the cph oncogene. Co- transfection of the cph oncogene with the human H-ras oncogene (T24), demonstrated a synergistic action between the two oncogenes in the tra nsformation of murine fibroblasts.