Lanes I and J represent control samples for GR expression in GC sensitive CEM cells and leukaemic blast cells, respectively. prednisolone (562\fold and 1220\fold, respectively). Restoration of GC sensitivity in cells exposed to SSZ was provoked via GC induced apoptosis, coinciding with inhibition of NFB activation. Moreover, western blot analysis revealed a markedly increased expression of glucocorticoid receptor (GR) in cells exposed to SSZ. Since GR mRNA levels were only marginally increased, these results suggest Rusalatide acetate that an altered post\transcriptional mechanism was operable which conferred a stable GR protein on SSZ exposed cells. Conclusion These results suggest that chronic targeting of the NFB signalling pathway by SSZ may be exploited as SOX9 a novel strategy to stabilise GR expression and thereby sensitise primary resistant cells to GCs. The anti\inflammatory and antiproliferative properties of glucocorticoids (GCs) including prednisolone and dexamethasone have led to their widespread use in the treatment of (chronic) inflammatory diseases such as rheumatoid arthritis (RA) as well as several human cancers (eg, acute lymphoblastic leukaemia).1,2,3 The mechanistic basis for the anti\inflammatory and anticancer effects of GCs involves an interaction with cytosolic glucocorticoid receptor (GR).4,5 Upon nuclear translocation, the GC\GR complex can bind to GC responsive elements in the promoter region of several genes which control the expression of both cell death/apoptosis Rusalatide acetate proteins and proinflammatory cytokines such as tumour necrosis factor (TNF).2,6,7 Moreover, GR can physically interact and antagonise transcription factors, including Activator Protein\1 and nuclear factor kappa B (NFB), which facilitate transcription of proinflammatory and antiapoptotic genes.4,5,8 At least three isoforms of GR have been reportedGR, GR and GR9,10,11,12of which only the \isoform is capable of high affinity GC binding. The \isoform lacks the high affinity GC binding capacity and is known as a dominant negative regulator of GR. The functional and biological significance of GR is not yet clear. 13 The efficacy of GCs can be limited by primary or acquired resistance.9,14,15,16,17,18,19 Several modes of resistance to GC induced apoptosis have been described,2,9,17,18,20 including (1) enhanced drug efflux via the multidrug resistance transporter P\glycoprotein, (2) enhanced metabolism by 11\hydroxysteroid\dehydrogenase, (3) downregulation of GR expression, (4) an increased ratio of GR over GR expression, (5) post\transcriptional modifications of GR resulting in reduced GC binding affinity, or (6) impaired GC induced apoptosis. Several of these mechanisms have been found responsible for inherent clinical resistance to GCs.14,15,21 Elucidation of the molecular basis underlying GC sensitivity and resistance is therefore of key importance in improving the efficacy of GCs for the treatment of both inflammatory and malignant diseases. In clinical rheumatology the addition of prednisolone to a drug combination of methotrexate (MTX) and sulfasalazine (SSZ), also known as the COBRA combination, appeared to be markedly more effective than SSZ+MTX alone.22,23,24 These observations suggested that SSZ, which inhibits the activation of the transcription factor NFB,25,26,27 and MTX are capable of conditioning cells for enhanced prednisolone activity. Recent studies from our laboratory showed that chronic exposure of the human (T lymphocytic) cell line CCRF\CEM to SSZ markedly enhanced its primary sensitivity to dexamethasone (by 10C20\fold).28,29 This observation prompted us to investigate whether chronic exposure to SSZ would also provoke Rusalatide acetate restoration of GC sensitivity in myeloid cells with inherent resistance to GCs. Methods Cell culture Human THP1 and U937 (monocytic/macrophage) and CCRF\CEM (T lymphocytic) cell lines (ATCC, Manassas, Virginia, USA) were cultured in RPMI\1640 medium supplemented with 10% fetal calf serum, 2?mM l\glutamine and 100?g/ml penicillin+streptomycin. Cell cultures were seeded at an initial density of 3105 cells/ml and refreshed biweekly. Exposure of parental/wild type (WT) U937 Rusalatide acetate and THP1 cells to SSZ was performed essentially as described in detail by De Bruin et al.30 Briefly, THP1 and U937 cells were initially incubated with a concentration of SSZ (0.4?mM and 0.3?mM, respectively) that conveyed a 50% growth inhibitory effect. Following 2C3?weeks of adaptation to these SSZ levels, SSZ concentrations were gradually increased to 0.6?mM for both cell lines over a period of another 2.5?months. At this stage, cells had unchanged doubling times and unchanged phenotypic properties compared with parental cells.30 Cells kept at 0.6?mM SSZ (further designated as THP1/SSZ and U937/SSZ) were used for further characterisation of GC sensitivity. Other procedures Detailed technical protocols for cell growth inhibition assays, western blot analysis, RT\PCR analysis, assays for apoptosis, NFB activity assays, chemicals and statistical assays are given in the online supplement available at http://ard.bmj.com/supplemental. Results Sensitisation of myeloid cells to GCs by chronic exposure to SSZ.
This descriptor here includes a great effect and a high relationship coefficient (?93%) with pIC50 and dominating impact on both eqs 3 and 4 with a higher bad contribution (?12
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