During the previous years of this project, the applicant has found that TGF-alpha transfection of early malignant colon carcinoma cells results in the acquisition of growth factor independence (GFI) with increased tumorigenicity in athymic nude mice. Similarly, anti-sense TGF-alpha expression in late malignant colon cancer cells decreases tumorigenicity and induces the re-acquisition of growth factor dependence (GFD). GFI appears to be related to aberrant expression of TGF-alpha in non-dividing growth states where it is normally downregulated. This aberrant TGF-alpha expression in late malignant cells depends upon dysregulation of transcriptional control. The cis-element of the TGF-alpha promotor mediating dysregulation has been identified and characterized. In addition, the applicant has demonstrated that aberrant expression of TGF-alpha allowed for continued EGF receptor (EGFR) activation in non-dividing states. Thus, the applicant now wants to characterize the transcription factors necessary for autostimulatory regulation of the TGF-alpha promotor dysregulative cells. Gel shift analyses suggest that downregulation of trans-acting factors that bind with the cis-element for TGF-alpha autoregulation occurs in quiescent GFD cells, while GFI cells show constitutive expression of these factors in growth arrested states. In GFD cells, insulin-like growth factor I (IGF-I) stimulates EGFR and entry into S phase. Therefore, the applicant proposes to study how IGF-I-R acts as a cofactor for entry into DNA synthesis. Thus, the specific aims of this application are, first, to characterize the TGF-alpha autoregulatory transcription factors. The second specific aim is to determine the mechanistic basis for the downregulation of TGF-alpha autocrine activity in GFD cells and its dysregulation in GFI cells. The final specific aim is to determine the effects of autocrine TGF-alpha dysregulation on the control of cell cycle transit.
