BMC Cancer 2001, 1:19 http://www.biomedcentral.com/1471-2407/1/19 NRP-152 cells. These results would explain why we saw an effect of AG490 on NRP-152 cells, in the absence of phospho-STAT3 in these cells (Table 1). Table 1: Inhibition of JAK2 resulted in apoptosis of NRP-152 cells Cell NRP-154 (uncloned) 154 clone A3 (high IL-6) 154 clone B1 (high IL-6) 154 clone G3 (low IL-6) 154 clone G7 (low IL-6) NRP-152 Rx AG490 AG490 AG490 AG490 AG490 AG490 µ M 0 30 100 0 100 0 100 0 100 0 100 0 30 100 % Apoptotic +/- SD 8 + 4 8.5 + 5 7.5 + 5 7.2 + 2 9.8 + 3 8.5 + 5 9.3 + 4 3.7 + 3 8.3 + 5 13.2 + 5 11.9 + 7 7.5 + 4 12 + 0 45 + 10* Legend: NRP-152 and NRP-154 cells were placed in 60 mm plates for 48 hr with compound at the concentrations indicated. Zero concen- tration of the compound is the vehicle (DMSO) control. At the end of the incubation period, cells were harvested, washed, and stained with FITC-annexin V, to demonstrate apoptotic cells. Quantification of fluorescence was performed on a Becton-Dickinson FACScan flow cytometer using CellQuest software. * p < 0.005 by Student t-test, compared to vehicle-treated cells. We looked at the effect of anti-rat IL-6 Ab on both NRP- 152 or NRP-154 cell growth rate, but were unable to de- tect an effect, using 3H-thymidine incorporation to measure proliferation (data not shown). We think this is due to failure to reach high enough Ab (commercially- available anti-rat IL-6) concentrations necessary to neu- tralize the IL-6 produced by the NRP-154 cells (highest concentration achieved was only 20 µ g/ml). We are lim- ited in performing Ab studies by the lack of commercial- ly-available rat-specific reagents. However, we are using alternative strategies in more experiments currently un- derway to determine the role of IL-6 in STAT3 activation to answer this important question in NRP-154 cells. We found that dexamethasone treatment of NRP-152 cells inhibited IL-6 synthesis without affecting cell growth. In contrast, dexamethasone treatment of NRP- 154 cells did not inhibit IL-6 synthesis; instead dexame- thasone treatment eliminated the IL-6 negative peak and enhanced IL-6 production albeit to a smaller extent in low IL-6-expressing clones (Figure 5, panel D). Enhance- ment of IL-6 production by dexamethasone treatment has been previously observed in Kaposi's sarcoma cells [32]. In the case of NRP-154 cells, the steroid-responsive element for dexamethasone on the IL-6 promoter may have been mutated to a form that does not bind steroid receptors. Mutations in the IL-6 promoter region may play a role in the tumorigenic effects of constitutive IL-6 expression in prostatic carcinoma cells [33]. Such poly- morphisms have been described for the IL-6 receptor in Kaposi's sarcoma, and are believed to play a role in IL-6- mediated progression of this type of cancer [32]. We observed that inhibition of STAT3 activation by treatment with AG490, which inhibits JAK2 activation, resulted in apoptosis of NRP-152 but not NRP-154 cells (Table 1). A possible explanation of the data would be the use of JAK1 for phosphorylation of STAT3 in NRP-154 cells, which would not be inhibited by AG490. IL-6 re- ceptor binding activates JAK1 as well as JAK2, which in turn phosphorylates STAT3 [14]. This in fact has been shown to be the case for v-src-transformed fibroblasts [34]. Another hypothesis is that STAT3 activation in NRP-154 cells is not dependent upon a signaling cascade, but is constitutive, possibly due to a mutation not unlike that contained within the cSTAT3 plasmid generated by Bromberg, et al. [35]. We are investigating in detail the signaling pathway in NRP-152 and NRP-154 cells to an- swer these important questions. The importance of STAT3 activation via IL-6 in prostatic cancer development has been suggested by previous in- vestigators. For example, IL-6 acting via its receptor has been shown to activate STAT3 in LNCaP cells. IL-6 given exogenously, since LNCaP cells do not produce IL-6, re- sulted in increased growth of the cells concomitant with activation of STAT3 [36]. LNCaP cells transfected with a plasmid conferring constitutive IL-6 expression demon- strated increased growth, relative to untransfected or sham-transfected cells [36]. However, other investiga- tors have observed that IL-6 treatment of LNCaP cells re- ulted in terminal differentiation and inhibition of growth, associated with STAT3 activation [17,37,38]. The molecular basis for the apparent contradiction is un- known at this time. Continued use of the NRP-152 and NRP-154 cell lines in parallel experiments should be use- ful in elucidating the discrepancies among various labo- ratories. It is possible that exogenous or autocrine IL-6 is not re- quired for constitutive STAT3 activation in NRP-154 cells. For example, viral IL-6 might be incorporated into the genomes of prostatic carcinomas, as has been de- scribed for Kaposi sarcoma [5,32,33,39–41]. The route of introduction of the viral IL-6 is believed to be through previous herpesvirus infection [39,41]. Another possibil- ity is that the insertion of the oncogene BRCA1 results in the constitutive activation of STAT3 in NRP-154 cells, as