Carcinogenesis involves multiple, independent somatic mutations in proto-oncogenes and tumor suppressor genes. Such mutations can lead to deregulation of signal transduction cascades that control cell growth and differentiation. Although the individual functions of certain oncogenes and tumor suppressor genes are known in some detail, little is known about the components or regulation of the signalling pathways they control. A molecular genetic approach, i.e.; the analysis of revertants of oncogene-transformed cells, has been chosen to gain insight into aberrations in such pathways that result in neoplastic growth. The proposed studies focus on two independent revertant cell lines that were derived from rat fibroblasts that overproduce protein kinase C and are transformed by a ras oncogene. In contrast to their transformed parent line, these revertant cell lines do not form colonies in soft agar. Additionally, they suppress the transformed phenotype in somatic cell hybrids and are resistant to retransformation by several different oncogenes. Both revertant cell lines also exhibit defects in expression of metallothionein (MT) genes in response to diverse stimuli. These data suggest that dominantly- acting mutant genes in each revertant may drive the synthesis or activity of a repressor of a specific battery of genes, including MTs and yet to be identified genes that play a critical role in transformation.
The specific aims of this proposal are: 1.) To isolate the dominant mutant gene(s) that are responsible for the revertant phenotype in each of the two revertant cell lines. This will be done by insertional mutagenesis of these cell lines with a specialized retrovirus, followed by selection for retransformants and cloning of the insertionally-mutated gene. 2.) To analyze the mechanism of negative regulation of MT gene expression in the revertant lines, through the use of reporter gene constructs under the control of various MT gene promoter elements, followed by electrophoretic mobility shift assays and analysis of the proteins that interact with the promotor element(s) of interest. 3.) To isolate genes in addition to MTs whose expression is inhibited in the revertants by differential screening of cDNA libraries constructed from control and revertant cell lines. Such genes represent potential mediators of the transformed phenotype.
