It is proposed to develop plasmids which express antisense RNA and to apply these plasmids to model systems and human tumor cell lines in order to determine the function and intervene in the expression of selected oncogenes. The c-fos and c-sis proto-oncogenes have been is chosen for analysis. Autocrine stimulation by the c-sis product, PDGF-II, has been implicated as a cause of transformation in several human tumor types including osteosarcoma, fibrosarcoma and glioblastoma. Further, PDGF is a potent fibroblast mitogen and stimulator of the c-fos gene which appears to act as a necessary intermediate in the recruitment of quiescent fibroblasts into the cell cycle. An activated c-fos gene has been shown to transform primary cells and the viral transduced forms cause murine osteosarcomas and avian nephroblastomas. It is proposed that the c-fos gene may be activated in and contribute to the properties of the phenotype of v-sis transformed cells. In model studies using a continuously expressing antisense c-fos RNA plasmid in v-sis transformed cells it has been observed that the cellular fos gene is indeed activated and that antisense-mediated subtraction of c-fos mRNA leads to restoration of contact inhibition. A systematic study is now proposed to extend these studies in order to confirm the mechanism of antisense c-fos RNA and to extend the application to cells transformed by the v-fos gene (RS-2 cells). Further we propose to develop a range of improved plasmids and to extend the approach to human tumor lines known to exhibit autocrine stimulation by PDGF-like molecules. Conditional plasmids directing the expression of sense and antisense sis RNA have already been obtained and a prototype of the proposed extended range of antisense c-fos RNA expressing plasmids has been prepared. Thus all the essential methods and materials to complete the proposals are available. In summary, experiments are designed to test whether the c-sis and/or c-fos genes function in certain human tumors and whether the antisense technique is a valid means of analysis and intervention.
