In our attempt to investigate the mechanism of carcinogenesis, we are using a human fibroblast system which was transformed by a chemical carcinogen into 4 clonal cell lines, 3 of which were immortalized but not tumorigenic, and one which was fully tumorigenic. Changes in expression of several proteins were noted in the cell lines such as a mutation in the beta-actin gene, tropomyosin isoform modulation, and the induction of plastin and metaschematin expression. We have previously shown that high expression of the mutant beta-actin gene promotes stable tumorigenic conversion of immortalized HuT-12 fibroblasts, and elicits limited neoplasia-related changes in diploid human fibroblasts. In a separate but parallel investigation, we have cloned and characterized the human gene encoding l-plastin, a 68 kd phosphoprotein induced In neoplastic human fibroblasts. The plastin gene has been put under the control of the beta-actin promoter, and is being transfected into human and rodent fibroblasts to ascertain its role in cell transformation. In this research we propose to isolate the human gene for metaschematin, a 27-28 kd polypeptide that is up-regulated 25 to 30 fold accompanying neoplastic transformation of human fibroblasts. We will determine the amino acid sequence for this protein from the nucleotide sequence of its cDNA and gene. We will look for structural changes in the gene isolated from the transformed cell to try to explain the mechanism of its elevation in expression in the transformed cell. We will examine the promoter and mechanism of its regulation by nucleotide sequencing around the promoter, and by linking a reporter gene such as E.coli beta-galactosidase or CAT to the promoter and subsequent mapping of regulatory sequences by deletion analysis. We will look for factors which activate metaschematin synthesis in transformed fibroblasts using gel shift experiments. We will express the metaschematin gene under control of the beta-actin promoter and examine the effect of its expression on cell growth, cytoarchitecture, and tumorigenicity. Finally, we will study the synergistic effect of metaschematin, plastin, and mutant beta-actin expression on the induction of the neoplastic phenotype in diploid human fibroblasts.
