BMC Cancer 2001, 1:19 http://www.biomedcentral.com/1471-2407/1/19 cer biology. For example, early investigators observed that transfection of untransformed B cells with a plasmid for constitutive expression of IL-6 conferred the tumori- genic phenotype on the cells [2]. IL-6 is a key factor in myeloma progression and survival [3,4], and also in Ka- posi's sarcoma, a solid tumor [5]. In myeloma, the stand- ard therapy for treatment includes prednisone, which acts by inhibiting IL-6 synthesis. Experimental anti-IL-6 therapies for myeloma and B-lymphoproliferative disor- ders have been shown to be of some use in limited clini- cal trials [6–11], therefore this is an intensely-studied target for myeloma therapy. As mentioned above, IL-6 is a cytokine that functions as a necessary growth factor in several cancer types, most studied in multiple myeloma [12]. It is an essential factor in the development and maintenance of B cell neoplasms [13], and likely plays an important role in many other types of cancer. IL-6 signals through a set of signaling proteins of the JAK and STAT kinase families [14]. The JAK and STAT kinases are activated by phosphorylation initiated by the homodimerization of the IL-6/IL-6 re- ceptor complex on the cell surface. The major IL-6 sign- aling intermediates are JAK2 and STAT3 [15]. Homodimerization of the IL-6/receptor complex induc- es the autophosphorylation of JAK2. The now-activated JAK2 phosphorylates STAT3, which forms homodimers, can cross the nuclear membrane and function as a tran- scription factor, inducing various genes including genes involved in the cellular transformation process [15]. An association between autocrine IL-6 and PCA has been known for some time [16,17]. The change in prostate cell phenotype from paracrine IL-6-stimulated to autocrine IL-6-stimulated is believed to be a contributing factor in the progression from benign hyperplasia to neoplasia [17]. IL-6 is also implicated in the development of cancer cell resistance to chemotherapy in PCA patients [18,19]. In other studies, a chimeric protein consisting of an anti- IL-6 Ab fused to Pseudomonas exotoxin was found to in- hibit proliferation of prostate carcinoma cell lines [20]. Exogenous IL-6 activated androgen responsive gene ex- pression in the absence of androgens in human LNCaP cells [21]. More work is needed to clarify the role of IL-6 in prostate neoplasia. While there is some evidence suggesting IL-6-mediated neoplasia in PCA development [17,22], a system suitable for following the transformation of prostate cells during PCA development remains lacking. We chose to use the NRP-152 and NRP-154 cell lines, derived by Danielpour, et al. [23], to examine the question of IL-6-mediated ne- oplastic progression via STAT3 activation. The 2 lines were derived from the same part of the rat prostate, fol- lowing treatment in vivo with N-methyl-N-nitrosourea. The NRP-152 cells are immortalized but not trans- formed, require several growth factors for in vitro surviv- al, and do not give rise to tumors in vivo. The NRP-154 cells are transformed, grow in the absence of exogenous growth factors, and are tumorigenic [23–27]. These lines come from epithelial cells. While prostatic epithelium is resistant to neoplastic transformation, it is not resistant to the development of hyperplasia. Studying the neoplas- tic transformation events in a cell type inherently resist- ant to this type of change can yield much valuable information about the transformation process in pros- tate cells. Materials & Methods Cell lines and growth media The tumorigenic (NRP-154) and non-tumorigenic (NRP- 152) rat prostate epithelial cell lines were the gift of Dr. David Danielpour, Ireland Cancer Center, University Hospital of Cleveland, Case Western Reserve University, Cleveland, OH [23]. NRP-152 cells were propagated in DMEM/ Ham's F12 medium (1:1; GIBCO) supplemented with 10% fetal bovine serum (GIBCO), 2 mM glutamine (GIBCO), epidermal growth factor (20 ng/ml), insulin (5 µ g/ml), dexamethasone (0.1 µ M) and cholera toxin (10 µ g/ml; all reagents listed, Sigma), pH 7.3. NRP-154 cells were grown in DMED/F12 plus serum and dexametha- sone only. Both lines were grown in a humidified 37°C CO2 incubator until the monolayers reached about 90% confluence. For treatment with steroids, the cells were cultured in complete medium, in which the serum was replaced by charcoal-stripped serum overnight. Cells were treated with 20 nM testosterone for 6 hr. The cells were harvested with trypsin/EDTA solution, washed, and subjected to further analyses. Intracellular flow cytometry for analysis of IL-6 and phos- pho-STAT3 NRP-152 and NRP-154 cells were grown as described above. For analysis of IL-6 production, the cells were fixed in Cytofix (Pharmingen) for 30 min on ice, then washed and permeabilized with Cytoperm (Pharmingen) for 15 min on ice. After washing with Perm/Wash buffer (Pharmingen), cells were incubated in 5–10 mg/ml goat Ig for 1 hr on ice. Cells were washed three times in Perm/ Wash buffer, then incubated with 1 µ g biotinylated anti- rat IL-6 (Pharmingen)/106 cells in 100 l Perm/Wash buffer for 1 hr on ice. After washing with Perm/Wash buffer three times (first wash being a 1 hr period in which the cells remain in Perm/Wash buffer for 1 hr on ice), cells were incubated with phycoerythrin-labeled strepta- vidin (Pharmingen) for 1 hr on ice, and washed three times as described for the Ab incubation step, then brought to 1 ml with PBS [28].