Yeh et al. BMC Cancer 2011, 11:139 http://www.biomedcentral.com/1471-2407/11/139 Dimerization is generally required for activating RTKs [11]. In addition to heterodimeric complex formation of the same subfamily [6,11-14], heterologous RTK interac- tion is also involved in the pathogenesis of human can- cers, e.g. between EGFR and RON (a member of the c- Met family) [15,16]. The biological significance of inhibi- tion of both RTK signaling pathways of cancer cells was demonstrated in the context of cell proliferation, migra- tion, anti-apoptosis and transformation in vitro. [15]. Therefore, identification of cross-talk partners of c-Met involved in the tumorigenesis may provide important biomarkers for co-targeting therapy. In our prior RTK profiling experiment, c-Met was frequently co-expressed with Axl, platelet-derived growth factor receptor a (PDGFR-a), DDR2 and/or IGF1R in the same uroepithe- lial cells [17], suggesting the existence of yet unspecified cross-talk partners of c-Met. Axl overexpression is detected in various human can- cers, and is associated with invasiveness and/or metasta- sis of carcinoma of the breast [18], stomach [19], kidney [20], lung [21], and prostate [22]. High expression of PDGFR-a is also detected in a variety of tumors, such as prostatic intraepithelial neoplasia, and carcinoma of the ovary, kidney, breast and liver [23-26]. Furthermore, PDGFR-a expression provides additional predictive value related to breast cancer progression [23], and patient’s survival in the kidney cancer [27] or lung can- cer [28]. The implications of these two receptor-related signaling events in the bladder carcinogenesis, however, remain unclear. This study was aimed to identify the novel interaction partners of c-Met, investigate their regulation, effect on biological activity, and the potential significance in association with patient outcome. Methods Cell Lines, transfection, and stable cell line establishment NIH/3T3 mouse fibroblast cell line and bladder cancer cell line T24 were obtained commercially. The four bladder cancer cell lines UB09: stage B2; UB40: stage A, papillary; UB47: stage B1; TSGH8301: stage A were established from patients of transitional cell carcinoma of the urinary tract [6,29]. UB47 was cultured in RPMI medium 1640 supplemented with 15% fetal bovine serum (FBS). Other cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS. The plasmids pTRE-Met and pTet-Lac-Hyg were transfected into NIH/3T3 and T24 cells by Lipofecta- mine™ 2000 reagent according to manufacturer’s proto- col (Invitrogen, Carlsbad, CA, USA) [30]. Two stable cell lines: NIH-Met5 and T24-Met3 were established. Microarray array RNA was isolated using TRIzol reagent (GIBCO BRL, Gaithersburg, MD, USA), followed by mRNA purification using Oligotex™ mRNA kit (Qiagen, Valencia, CA, USA). RNA samples were reverse transcribed into cDNA fluores- cently labeled either with Cy3 or with Cy5. The labeled cDNA was hybridized with a microarray cDNA chip con- taining 192 RTK genes [31]. Data were imported and nor- malized using MeV: MultiExperiment Viewer (Dana- Farber Cancer Institute, http://www.tm4.org/mev.html) [32]. Clustering affinity search technique (CAST) was used for gene expression cluster analysis. There are 23 clusters after CAST analysis [33], the gene expression profiles of 8 genes showing the best correlation with c-Met gene were clustered as one group (table 1). Antibodies Anti-phospho-tyrosine antibody was purchased from BD Transduction Laboratories (Lexington, KY, USA), and antibodies to Axl, c-Met, p-Met (phosphorylation of c- Met;Tyr 1234), PDGFR-a, and p-PDGFR-a (phosphory- lation of PDGFR-a;Tyr 754) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The Ras antibody was obtained from Calbiochem (Merck, Darm- stadt, Germany), Sp1 from Upstate Biotechnology Inc. (Golden, CO, USA), p-Axl (phosphorylation of Axl;Tyr 702) from Cell Signaling Technology Inc. (Beverly, MA, USA) and b-actin was purchased from Sigma-Aldrich (St. Louis, MO, USA). The Src antibody was obtained from Millipore (Billerica, MA, USA) and p-Src (phos- phorylation of Src;Tyr 418) purchased from Invitrogen (Carlsbad, CA, USA). Western blot analysis The western blot analysis was performed as previously described [6]. Briefly, the total lysates were prepared using RIPA solution. Total protein (50 μg) was analyzed by polyacrylamide gel electrophoresis and transferred to the PVDF membrane. The membrane was probed with targeted protein antibodies and the immune complex was detected with an enhanced chemiluminescence Table 1 The mRNA expression levels of eight RTK genes clustered together with c-Met in RTK c-DNA microarray analysis NIH-Met5 day NIH-Met5 day NIH-Met5 day 1 4 7 RTK name Axl ERBB2 ERBB3 Met MST1R PDGFRa PDGFRb TIE1 TIE2 NIH/ 3T3 -0.43 -0.24 -0.34 0.02 -0.35 -0.40 -0.20 -0.24 -0.01 1.48 1.34 1.47 1.18 1.45 1.48 1.13 1.47 1.38 Page 2 of 12 -0.71 -1.07 -0.79 -1.26 -0.84 -0.70 -1.25 -0.76 -0.97 -0.34 -0.02 -0.35 0.06 -0.26 -0.38 0.32 -0.47 -0.40