Structure/function analysis of thec-myb locus in murine myeloid leukemias harboring an integrated MoMLV proviral genome

This research project involves the study of the myb locus in a set of tumors and their descendant cell lines from BALB/c mice subsequent to infection with acute transforming retroviruses. Previous studies of a subtype of tumors which evolved subsequent to the infection event revealed a virus insertional mutagenesis of the myb locus. These tumors, the Abelson Virus Induced Plasmacytoid Lymphosarcomas (ABPL tumors) have an integrated Moloney Murine Leukemia Virus genome between c-myb exons 3 and 4. Despite the invariant position of the virus integration event, three ABPL tumor cell lines studied in our laboratory produce three different sized myb proteins. The project began by determining the molecular mechanism associated with the observed heterogeneity. DNA and RNA from ABPL tumor cells were studied to determine the structure of their myb loci, and of their mRNA. Although no genetic lesions secondary to the virus insertion event were detected, all three ABPL tumors were found to produce three different myb mRNA species. Examination of ABPL tumor mRNA revealed that the heterogeneity of protein isoforms was caused by differential splicing patterns both within the integrated proviral genome, and within the myb sequences. Production of full length cDNAs corresponding to ABPL mRNAs were generated which, when translated in vitro, shared identical antigenic profiles with their in vivo produced counterparts. We then examined the biochemical and biological activities of these various proteins through expression in vitro of their cDNAs. We used a transient transfection strategy to determine the capacity of the various ABPL tumor mybs to transactivate transcription from a myb responsive promoter. We also used these cDNA clones to determine whether or not transgenic expression of an ABPL tumor myb gene could induce a change in the growth characteristics of non transformed cells. We were able to show that an ABPL tumor myb gene could induce a myeloid leukemia in a primary chicken splenocyte culture. Further, we were able to show that the well characterized growth factor requirements of 32D c13 cells could be altered through transgenic expression of ABPL tumor myb cDNAs