Integrins are heterodimeric cell surface proteins that mediated cell-cell and cell-extracellular matrix interactions. The integrin alpha5Beta1 and its extracellular matrix ligand, fibronectin, play a key role in development and oncogenesis by effecting cell adhesion, migration, and growth. It is known that expression of the alpha5 subunit of the alpha5beta1 integrin, the subunit unique to this integrin, is down- regulated during development and oncogenesis. Thus understanding the regulation of alpha5 gene expression will provide insight into the mechanisms controlling these processes. As an initial step to understand the regulation of the expression of the alpha5 gene we isolated and characterized its promoter region. Using B cell maturation as a model we discovered that DNA methylation of the alpha5 gene promoter is an important mechanism regulating its expression; methylation of the promoter inhibits its transcription. In addition, we identified a novel methylated DNA-binding protein that binds to a specific sequence of the alpha5 gene only when a single CpG dinucleotide is methylated. We have also examined the factors regulating the expression of the alpha5 gene in small cell lung cancer and found that the oncogene N-myc, often over- expressed in this tumor, inhibits alpha5 gene transcription. N-myc over- expression inhibits specific DNA-protein interactions thought to be important in controlling alpha5 gene expression. Treatment of a small cell lung cancer cell line with a potential chemotherapeutic agent, bromodeoxyuridine, resulted in a loss of the amplified region of DNA encoding N-myc and decreased N-myc expression with a subsequent increase in alpha5 and fibronectin expression. The alpha5 expressing cells demonstrated a dramatic decrease in their tumorigenicity compared to the nontreated cells. This proposal focuses on studying alpha5 gene expression and its cell- extracellular matrix interaction to elucidate the role of DNA methylation and oncogenes in development and oncogenesis. To examine the role of DNA methylation in the regulation of alpha5 gene expression during development we have constructed a transgenic mouse that expresses human growth hormone under control of the alpha5 promoter. This will allow us to compare the methylation status of the alpha5 promoter to growth hormone production. Creation of additional transgenic mice that contain specific mutations or deletions in the alpha5 promoter will allow for the precise localization of sequences important in controlling alpha5 expression. We will continue to investigate the relationship between DNA methylation and alpha5 gene expression in cell culture. In addition, using small cell lung cancer as a model, we plan to pursue the molecular mechanisms by which N-myc repress a gene expression and how this relates to tumor growth as well as defining the role of alpha5 in tumorigenesis.
