2 NOVEL VARIANTS OF THE V-SRC ONCOGENE ISOLATED FROM LOW AND HIGH METASTATIC RSV-TRANSFORMED HAMSTER-CELLS

Four different transformed cell lines were isolated as a result of ind ependent infection of primary hamster fibroblasts by Rous sarcoma viru s (RSV SR-D stocks). These lines differ by the level of their spontane ous metastatic activity: HET-SR-1, HET-SR-8, and HET-SR-10 cell lines induced 70-200 metastatic nodules in the lung and/or lymph nodes of in oculated animals (high metastatic lines, HM). Metastatic activity was not identified after injection of HET-SR cells (low metastatic line, L M). All cell lines contained one copy of integrated and expressed inta ct RSV provirus. The difference in the amount of v-src protein in cell lines was not correlated with their metastatic potential in vivo. Com plete v-srcHM and v-srcLM genes were cloned from corresponding gene li braries and sequenced. In the unique region of both v-src isoforms a G C-rich insert of 60 nucleotides (20 a.a.) was found. The presence of t his insert explains the unusual apparent molecular weight of protein e ncoded by v-srcHM and v-srcLM: 62 kDa. Both genes had 10 identical ami no acid changes when compared to the known RSV SR-D v-src sequence. v- srcHM and v-srcLM differ by several amino acid changes. Most of them a re localized in the unique domain and the extreme carboxy-terminal reg ion of the oncoprotein. Both v-src variants and chimeric v-src with mu tually substituted parts were subcloned in a retroviral vector and int roduced into avian neuroretina cells. Significant differences in the m orphology of transformed neuroretina cells were associated with the mu tations in the carboxy-terminal region of the v-src oncogene. Low meta static HET-SR cells transfected with v-srcHM and the chimeric gene v-s rc-LH remarkably increased their metastatic potential. In contrast, th is effect was not observed when the same cells were transfected with v -srcLM and the chimeric v-srcHL gene. Specific changes in the distribu tion of fibronectin matrix typical for high metastatic cells were foun d in the lines transfected with v-srcHM. (C) 1996 Academic Press, Inc.