Variation in the Binding of ¹²⁵I-labeled Interferon-β_ser to Cellular Receptors during Growth of Human Renal and Bladder Carcinoma Cells in Vitro

Abstract

Studies of various established human bladder and renal carcinoma cell lines cultured in vitro demonstrated the presence of specific, saturable, high affinity binding sites for ¹²⁵I-labeled human interferon β_ser (IFN-β_ser). This recombinant produced interferon labeled with approximately one atom of ¹²⁵I/molecule of IFN expressed minimal or no loss of antiviral activity. A single class of binding sites (1000–2000/cell) with an affinity constant of 10¹⁰–10¹¹ L/M was measured at 4°C for cells exhibiting widely different sensitivity to the antiproliferative effect of IFN-β_ser. Major fluctuations in the binding of ¹²⁵I-labeled IFN-β_ser to cellular receptors were observed during in vitro proliferation of four of five cell lines examined. A significant decrease (P < 0.001) in specific binding was observed 48 h after cultures were established. Cell cycle analysis suggested that within the first 24 h and in the very late log and stationary phase of growth of ACHN (human renal carcinoma) cells, variations in the binding of ¹²⁵I-labeled IFN-β_ser were partially attributable to binding fluctuations during the mitotic cycle. The 2- to 3-fold decline 24 h following plating of ACHN cells corresponded to a 70% decrease in the number of cells in G₀-G₁. T24 (human transitional cell carcinoma) and ACHN cells, synchronized by serum starvation, demonstrated increased binding of ¹²⁵I-labeled IFN-β_ser 4–16 h following serum replenishment. This increase in receptor binding occurred prior to the onset of DNA and protein synthesis and was followed by a decline immediately prior to cell division. Binding site analysis indicated that the increased binding prior to DNA synthesis was due to a 5- to 6-fold increase in receptor affinity for the radiolabeled ligand. After an initial 40% decline in receptors per cell following serum stimulation, receptor concentration remained essentially unchanged. Induction of 2′,5′-oligoadenylate synthetase in ACHN cells and antiproliferative activity in RT112, RT4, T24 (human transitional cell carcinoma), and ACHN cells by IFN-β_ser decreased significantly 48 h following plating. These changes in the biological activity of this interferon corresponded to growth related fluctuations in the IFN-β_ser binding.