BMC Cancer 2001, 1:19 http://www.biomedcentral.com/1471-2407/1/19 Figure 1 NRP-154 cells, but not NRP-152 cells, have constitu- tively-activated STAT3. NRP-152 and NRP-154 cells were grown in their respective complete media. For IC flow cytometry, confluent cells were harvested, fixed, permeabi- lized, and stained for P-STAT3, as described in Materials & Methods. Cells were analyzed on a Becton-Dickinson FACS- can for fluorescence in FL2. The thin purple line on the histo- gram shows the results for NRP-152 cells + testosterone; the thick blue line shows the results for NRP-154 cells minus testosterone. The fluorescent staining of NRP-152 cells plus testosterone was the same as for the fluorochrome control on both cell lines, and the same for NRP-152 cells not treated with testosterone (data not shown). The NRP-154 cells were approximately 10 times more fluorescent than the NRP-152 cells (p < 0.001 by Kolmogorov-Smirnov {KS} sta- tistical analysis). A representative histogram from 4 inde- pendent experiments is shown here. peak of cells negative for IL-6 (Figure 2). This may have been due to heterogeneity of the NRP-154 cells, since the parental lines had not been subcloned by us. Therefore, we derived 16 clones of NRP-154 cells by limit-dilution cloning, and examined these for level of IL-6 expression. We observed that 9 clones were highly-positive for IL-6, while the rest expressed little IL-6, thereby accounting for the 2 peaks we observed in the parental NRP-154 line (Figure 3). IL-6 receptor was expressed on the surfaces of NRP-152 and NRP-154 cells When we examined NRP-152 and uncloned NRP-154 cells for expression of the IL-6 receptor, we observed that all NRP-152 cells expressed the IL-6 receptor, while the uncloned NRP-154 cells had a population that apparent- ly did not express the receptor (Figure 4). In studies cur- rently underway, we are analyzing the 16 NRP-154 clones we derived for differences in IL-6 receptor expression, A (A) NRP-152 (BB) NRP-154 Figure 2 Constitutive IL-6 expression by NRP-152 & NRP-154 cells. Confluent cells were harvested, fixed, and permeabi- lized as described in Materials & Methods. They were stained with goat anti-rat IL-6, then with fluorescent anti-goat Ig. Cells were analyzed on a FACScan for fluorescence in FL2. A, NRP-152 cells. B, NRP-154 cells. In both histograms, green = fluorescent anti-goat Ig only; purple = goat anti-rat IL-6 plus fluorescent anti-goat Ig. KS analysis revealed that the fluores- cence intensity of the NRP-154 cells in the second peak in panel B were 3 to 5 times the fluorescence intensity of the control-stained cells (green peak in panel B; p < 0.01). A his- togram from 1 experiment is shown here. and to see if there is any correlation with levels of IL-6 expression. Effect of dexamethasone treatment on NRP-152 and NRP- 154 cells To examine the role of autocrine IL-6 on NRP-152 and NRP-154 cells, we looked at the effect of dexamethasone treatment on IL-6 expression on both cell lines, and on high and low IL-6-expressing clones of NRP-154 cells. Dexamethasone is known to inhibit IL-6 synthesis by acting at the steroid-response elements of the IL-6 pro- moter . NRP-152 and NRP-154 cells were routinely grown in the presence of dexamethasone, so the effect on proliferation was not quantified. In the following experi-