IN-VITRO CELLULAR-RESPONSES TO CYTOKINES AND ERYTHROPOIETIN

Cytokines and polypeptide hormones act through high-affinity binding t o cognate transmembrane receptor molecules, expressed on target cells. The impact of such ligand molecules is conveyed to the cell nucleus b y 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 s ignalling pathways, cell-cycle progression or growth inhibition may fo llow from ligand/receptor interactions. We have employed cellular grow th as an endpoint for potency determination of several human bioactive substances, such as interferons (IFNs), IL-2, G-CSF, GM-CSF and eryth ropoietin (Epo), using murine or human cell lines as indicators. The c onversion of the tetrazolium salt MTT by mitochondrial reductase to bl ue formazan served as an endpoint in such estimations. In addition to a cellular growth suppression IFN assay, a reporter gene-modified huma n glioblastoma line was devised to provide an implement for high-throu ghput potency assessment of interferons. The bioassay systems were all designed according to the parallel line assay model and were subjecte d to extensive validation procedures. Both intra- and inter-assay vari ations were consistently within the range of immunometric counterparts ; hence precision and reproducibility do not need to be compromised wh en 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 pharma codynamic cellular response. The assays were operating in the pM range and their sensitivity could hence compete with immunometric counterpa rts. When applicable, the aforementioned approaches were combined with physicochemical characterization of the respective ligands, which fur ther enhanced the physiological relevance of the cellular readout. Acc ordingly, such two-part assays should provide alternatives to traditio nal in vivo activity determinations of biological substances. (C) 1998 Published by Elsevier Science Ltd. All rights reserved.