This research will test a specific hypothesis which attempts to explain how peptide growth factors regulate cell-cycle specific gene expression and how alterations in these mechanisms occur in chemically transformed cells. Based largely on recent findings in this laboratory, it is postulated that genes which are specifically induced by epidermal growth factor (EGF) are regulated in part by a trans-acting repressor of gene transcription and that repressor activity can be lost as a result of chemically induced neoplastic transformation. This model suggests that both mitogen stimulation and the loss of repressor activity are required for maximum phenotypic effect. Thus, this model has implications for multistage carcinogenesis. In order to test predictions which arise from this model, somatic cell hybrids between a nontransformed and chemically transformed cell line will be derived by fusing clonal transfectants containing two different dominant selectable markers. These two cell lines differ markedly in their patterns of regulation of at least two specific genes in respnose to EGF and inhibitors of protein synthesis. Hybrid cells will allow a determination of the complementation properties of specific trans-acting gene regulatory factors present in these two cell types. In addition hybrid genes will be constructed in which coding sequences for the E. coli enzyme chloramphenicol acetyltransferase (CAT) are fused to fragments of EGF-regulated cellular genes whose transcription can be stimulated by inhibitors of protein synthesis. These hybrid constructs will be introduced into nontransformed and chemically transformed cells in order to further study trans-acting factors involved in regulation. Finally, soluble extracts from both nontransformed and chemically transformed cells will be developed which retain the capacity to accurately transcribe IGF-regulated genes in vitro. These will be used in complementation experiments in order to further define the spectrum of transacting transcription factors present in both cell types.
