In Vitro Cellular Responses to Cytokines and Erythropoietin

Abstract

Cytokines and polypeptide hormones act through high-affinity binding to cognate transmembrane receptor molecules, expressed on target cells. The impact of such ligand molecules is conveyed to the cell nucleus by specific signal transduction mechanisms and is ultimately manifested as changes in gene expression, largely accomplished by transcription-regulatory factors. Depending on target cell maturation and receptor signalling pathways, cell-cycle progression or growth inhibition may follow from ligand/receptor interactions. We have employed cellular growth as an endpoint for potency determination of several human bioactive substances, such as interferons (IFNs), IL-2, G-CSF, GM-CSF and erythropoietin (Epo), using murine or human cell lines as indicators. The conversion of the tetrazolium salt MTT by mitochondrial reductase to blue formazan served as an endpoint in such estimations. In addition to a cellular growth suppression IFN assay, a reporter gene-modified human glioblastoma line was devised to provide an implement for high-throughput potency assessment of interferons. The bioassay systems were all designed according to the parallel line assay model and were subjected to extensive validation procedures. Both intra- and inter-assay variations were consistently within the range of immunometric counterparts; hence precision and reproducibility do not need to be compromised when using biological determination methods. Furthermore, the advantage of monitoring downstream signal transduction effects of ligand binding, particularly over immunometry, is evident since it reflects a pharmacodynamic cellular response. The assays were operating in the pM range and their sensitivity could hence compete with immunometric counterparts. When applicable, the aforementioned approaches were combined with physicochemical characterization of the respective ligands, which further enhanced the physiological relevance of the cellular readout. Accordingly, such two-part assays should provide alternatives to traditional in vivo activity determinations of biological substances.