BMC Cancer 2001, 1:19 http://www.biomedcentral.com/1471-2407/1/19 Figure 3 IL-6 expression of clones A4 (red) and D8 (green) of NRP-154 cells. Clones of NRP-154 cells were derived by limit dilution at 1 cell/well; calculated cloning efficiency was 17.8%. Clones were fixed, permeabilized, and stained with biotinylated anti-IL-6 (R&D Systems) plus streptavidin-phyco- erythrin (Pharmingen), as described in Materials & Methods. Fluorescence in FL2 was analyzed on a FACScan. The IL-6 expression of the 16 clones was either high, like A4 (shown in red), or low, like D8 (shown in green). A4 cells are 8 times as fluorescent as D8 cells. The IL-6 expression of 2 of the 16 clones is shown for the sake of clarity. The fluorescence intensity of clone A4 was 10× that of clone D8 in this exper- iment (p < 0.001 by KS statistics). A histogram from 1 deter- mination is shown here. ments, we used dexamethasone-free medium, and also treated the serum twice with activated charcoal, to re- move endogenous steroids (stripped serum), then added defined amounts of dexamethasone for carying out the experiments. We observed that dexamethasone treat- ment of NRP-152 cells inhibited IL-6 synthesis without affecting NRP-152 growth, as expected. In contrast, dex- amethasone treatment of NRP-154 cells did not inhibit IL-6 synthesis; instead dexamethasone treatment appar- ently enhanced IL-6 production (panel B). This effect was observed for high and low IL-6-expressing clones of NRP-154 (Figure 5, panels C and D), and therefore was independent of IL-6 production by the cells. However, clones expressing low amounts of IL-6 expressed less en- hancement of IL-6 production that clones expressing high amounts of IL-6 (Figure 5, panels C and D). Inhibition of JAK2 induced apoptosis in NRP-152 but not NRP-154 clones In continuing our studies on the role of IL-6 in STAT3 phosphorylation, we used the JAK2 phosphorylation in- hibitor AG490, to see if inhibiting JAK2 phosphorylation B A Figure 4 IL-6 receptor is expressed on untreated NRP-152 & NRP-154 cells. Confluent cells were removed with citrate- saline buffer and stained with 2 µ g/106 cells goat anti-IL-6 receptor (R&D Systems). Fluorescent anti-goat Ig was then added. Fluorescence in FL2 was analyzed using a FACScan. A, NRP-152 cells. The blue line is the fluorescence control; the red line is the fluorescence due to the IL-6 receptor on the cells. The anti-IL-6 receptor-stained cells were 30 times as fluorescent as the control stained cells (p < 0.001 by KS sta- tistics). B, NRP-154 cells. The green line is the fluorescence control; the purple curve is the IL-6 receptor on the cells. The anti-IL-6 receptor-stained cells (second red peak) were 10 times brighter than the control-stained cells (green peak; p < 0.001 by KS statistics). The first red peak was comprised of cells that did not stain positive for the IL-6 receptor. would inhibit survival of NRP-152 or NRP-154 cells, as had been described for LNCaP cells previously [29]. We examined clones of NRP-154, selected by amount of IL-6 expression each had: 2 high IL-6-expressing clones and 2 low-IL-6-expressing clones were used for these experi- ments. Treatment with the JAK2 inhibitor for 48 hrs in- duced apoptosis, measured by cellular binding of FITC- annexin V, in NRP-152 but not NRP-154 clones (Table 1). We detected no effect of the vehicle (DMSO) at the high- est concentration used. Our data imply that JAK2-medi- ated phosphorylation of STAT3 was essential for the