Abstract
Mutant cell lines B3 and B10, which are unresponsive to both interferon (IFN)-alpha and IFN-gamma, and line B9, which does not respond to IFN-gamma stimulation, are described. The mutants were submitted to fluorescence-activated cell sorting (FACS) from a cellular pool, which was obtained from the parental cell line 2C4 after several rounds of mutagenesis. The unresponsiveness to IFN stimulation was observed both in terms of expression of cell surface markers (CD2, class I and II HLAs) and mRNA expression of IFN-stimulated genes (2'-5' oligoadenylate synthetase (OAS), 9-27, and guanylate binding protein (GBP)). Genetic crossing of B3, B9 and B10 with U3 (STAT1-), gamma 2a (JAK2-) and U4 (JAK1-) mutants, respectively, did not restore IFN responsiveness to the hybrid cell lines. However, when these cell lines were crossed with the same mutants, but using the pairwise crosses B3 x U4, B9 x U3 and B10 x U3, the cell hybrids recovered full IFN responsiveness. The present genetic experiments permitted us to assign the mutant cell lines B3, B9 and B10 to the U3, gamma 2 and U4 complementation groups, respectively. These conclusions were supported by the analysis of IFN-stimulated genes in the mutants.
Highlights
Our knowledge of cytokine signalling has experienced enormous progress in the past few years due to the combined utilization of somatic cell genetics, biochemical analysis and transgenic animals [1,2,3,4]
9-27 GBP Actin nals triggered by cytokines, especially IFNs, upon ligand-receptor binding, owing to the converging approaches resulting from the utilization of somatic cell genetics, biochemical analysis and target gene disruption (2830)
The JAK/STAT signalling pathway was first associated to the IFNs and extended to the other cytokines, as the molecules that elicit the signals from the cell surface to the nucleus upon IFN-receptor binding, and that promote IFN-gene stimulation, which in turn results in the diverse biological activities exerted by these cytokines [31,32,33]
Summary
Our knowledge of cytokine signalling has experienced enormous progress in the past few years due to the combined utilization of somatic cell genetics, biochemical analysis and transgenic animals [1,2,3,4]. The U mutants mentioned earlier proved to be fundamental in the elucidation of the signal transduction pathway triggered by JAK/ STAT proteins upon cytokine stimulation [22,23,24,25] These mutants were characterized based on selection of the growing cells in HAT (hypoxanthine, aminopterin, and thymidine) plus IFN-α, constitutive mutants could be obtained under HAT selection without IFN (22,23; Bonjardim CA, unpublished observations). I describe the JAK/STAT-deficient cell lines B3, B9 and B10, which were cloned by an alternative selection procedure based on the expression of cell surface markers, followed by fluorescence-activated cell sorter (FACS) analysis These mutant cell lines were further characterized biochemically and were assigned to the U3 (STAT1-) [23], γ2 (JAK2-) [26] and U4 (JAK1-) [23] complementation groups, respectively
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Schedule a callMore From: Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas
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