It is well known that the overexpression of growth factors is often observed in human tumor cells and is thought to be responsible for the aberrant growth of these cells. A large body of current information suggests that transforming growth factor (TGF) alpha is one of the growth factors which plays an important role in many human tumors. Although the role of TGF alpha in human carcinogenesis is well established, the biochemical mechanism of the growth factor overexpression has not been examined. The present study will take a systematic approach to answering this question. The goal of this AREA grant is to obtain valuable information on the biochemical mechanism of TGF alpha overexpression in human tumor cells which will aid in the design of future treatment regiments and also be the basis for a competitive RO1. Normal human flbroblasts which don't express mRNA for TGF alpha, and fibrosarcoma cells which have a high expression of the growth factor mRNA will be used in this study. Initial experiments will involve transfecting both cell types with vector constructs which contain the TGF alpha promoter linked to the luciferase reporter gene. These constructs have been extensively characterized and will be kindly supplied by Dr. Michael Brattain from the Medical College of Ohio (see accompanying letter of support and reference 46). These experiments will determine if the normal TGF alpha promoter can drive luciferase expresion in the fibrosarcoma cells suggesting that a transcription factor may be activated in these cells. Alternatively, the normal TGF alpha promoter in these constructs may not be able to drive luciferase expression in the tumor cells which would suggest that the difference between the normal and fibrosarcoma cells lies within the promoter region. Additional experiments will then be conducted based upon the results of these initial transfection studies and may include deletion analysis to identify specific regulatory regions, gel-mobility shift assays and DNase I protection experiments to identify differential binding of transcription factors in the two cell types and sequences involved in the DNA/protein interaction, sequence analysis of the promoter from both normal and fibrosarcoma cells to determine if a mutation occurs in the tumor cells, examination of the methylation state of the promoters from each cell type, and Southern analysis to look for TGF alpha gene amplification in the fibrosarcoma cells.
