Immunoprecipitation Assay of Alpha-fetoprotein Synthesis by Cultured Mouse Hepatoma Cells Treated with Estrogens and Glucocorticords

This investigation was to study the biosynthesis of ³H-labeled alpha-fetoprotein (AFP) by cultured mouse hepatoma (HEPA-2) cells. Both the function and regulation of this oncodevelopmental gene are unknown. However, evidence indicates that mechanisms controlling the expression of AFP involve aspects of both normal embryonic development and neoplastic transformation. The secretion of AFP was analyzed during different phases of the growth cycle to provide information on AFP production using standard culture conditions. The highest rate of secretion occurred during the stationary phase, followed by the late logarithmic and early logarithmic phases of growth, respectively. The production of AFP was then determined following the addition of glucocorticords and estrogens in an attempt to understand hormonal factors that may be involved. Studies utilizing estradiol-17β indicated that the secretion of AFP did not appear to be sensitive to this steroid even though sucrose density gradient analysis of HEPA-2 cytosol, for estrogenic receptors, revealed competitive binding moieties in the 8S and 4S regions of the gradient. In contrast, the secretion of the total complement of proteins, including AFP, was significantly stimulated by the glucocorticords, dexamethasone and corticosterone. Analysis of HEPA-2 cytosol for glucocorticord receptors revealed binding components in the 7S and 3–4S regions of the gradient. The ³H-dexamethasone binding appeared to be stereospecific since nonlabeled dexamethasone, but not nonlabeled estradiol-17β, effectively displaced the bound radioactivity. The glucocorticoid-binding component in HEPA-2 therefore displayed characteristics reported for glucocorticord receptors in normal liver and other hepatomas.