Neoplastic transformation begins at the level of DNA and ends at the levels of aberrant cellular proliferation. In between these two points is a complex set of events, and compounding the complexity is the realization that different mechanisms are most likely responsible for different cancers. The direction and fate of an initiated cell are dependent on exposure to specific progression/promotion factors. The phenotypic markers of tumor progression/promotion and overt neoplasia are numerous. One such marker is gap junctional communication (GJC) which is believed to be involved in the regulation of cell proliferation and which is generally diminished or abolished at some stage of progression towards neoplasia. Connexin (cx) 32 and cx43 are two gap junctional proteins which have putative tumor suppressor function, and our preliminary data strongly supports such a contention for cx43. Thus, transfection of transformed TRMP cells with cx43 reverted their transformed phenotype, including inability to grow in soft agar, lengthened G1 and S phase times, flatter morphology and greatly increased sensitivity to density dependent factors, serum deprivation and inhibition of proliferation by TGFBs. The mechanism by which expression of a single gene, cx43, induces this pleiotropic effect is not known; however, we have established several important links. The cx43-induced increased in G1 and S phase times are linked to a decreased expression of cyclin D1, D2, A, cyclin dependent kinases (CDK) 5 and 6. The increased sensitivity to the TGFBs is related to a significant increase in TGFB11 receptors (TGFB11R) in cx43 transfected cells. Thus, the tumor suppressive functions of cx43 and TGFB/TGFBR s may be linked and function through the cyclins and CDKs. The long-range goal of this proposal is to understand at the molecular level the mechanism by which cx43 reverses the transformation of neoplastic cells, and to this end we propose and will accomplish three specific aims: 1) Is a TGFB/TGFBR autocrine system involved in cx43-induced tumor suppression and is TGFB11R an early target of the cx43 pathway to phenotypic reversion; 2) Establish an inducible system under which cx43 is regulated and characterize with respect to cx43 TGFB/TGFBR and cyclin/CDK gene expression and protein function; 3) Clone the cDNA of the first order genes activated/inactivated after functional expression of cx43. a) Clone the cDNA of the first order genes after functional expression of cx43; b) determine the kinetics and functional significance of gene expressions identified in a) relative to the activation/inactivation of cyclin/CDK, TGF-B11R gene expressions, sensitivity to TGF-B and reversion of the transformed phenotype.
