Prostate carcinomas are surrounded by a layer of stromal fibroblastic cells

Prostate carcinomas are surrounded by a layer of stromal fibroblastic cells that are characterized by increased expression of CD90. stromal cell induction. CD90+ stromal cell secreted factors induced an increased expression of CD90 CDP323 and differential induction of genes involved in extracellular matrix remodeling and the RECK pathway in NCCIT. These results suggest that, compared to normal tissue stromal cells, signaling from cancer-associated stromal cells has a markedly different effect on stem cells as represented by NCCIT. Given that stromal cells are important in directing organ-specific differentiation, stromal cells in tumors appear to be defective in this function, CDP323 which may contribute to abnormal differentiation found in diseases such as cancer. Electronic supplementary material The online version of this article (doi:10.1007/s12307-010-0061-4) contains supplementary material, which is available to authorized users. or m3d?m0h. Functional and ontology enrichment analysis was performed using the DAVID web-based tool [22]. Freely available prediction software for determination of signal peptides and likely cell membrane-spanning sequences was also used. Signal peptides were predicted using SignalP 3.0 [23], CDP323 and transmembrane (TM) regions were predicted using TMHMM 2.0 [24] for protein topology and the number of TM helices. Information from both SignalP and TMHMM were combined to identify proteins that contained predicted cleavable signal peptides and no CDP323 predicted TM segments as reported previously [25]. Results Gene expression changes induced in NCCIT by secreted factors from CP stromal cells were determined by Affymetrix DNA microarray analysis. Following 3-days co-culture with CP stromal cells, the induced expression in NCCIT cells (CP-NCCIT) of smooth muscle genes ACTA2, CALD1, CNN1, prostate stromal genes PENK, CNTN1, ChGn, BMP2 [5, 7], androgen receptor (AR) and a stromal gene GFRA1 was significantly less than that previously shown by NP stromal cells (NP-NCCIT) [13]. The CP-NCCIT transcriptome dataset contained minimal signal levels for CALD1, and CNN1 compared to that of NP-NCCIT (Fig.?1a and b). Other queried prostate genes such as tenascin C (TNC) [5] were also lacking. The similar induction of stromal gene stanniocalcin (STC1) and increased expression of CD90/THY1 in CP-NCCIT showed that gene expression changes did occur in NCCIT co-cultured with CP stromal cells. Induction of CD90 was notably higher in CP-NCCIT than in NP-NCCIT (NCCIT cells are also positive for CD90, a stem cell marker). This reflected the increased CD90 expression in CP stromal cells. Fig.?1 Expression profiles of stromal and stem cell genes in treated NCCIT. a Increased expression of prostate stromal cell-specific genes relative to untreated NCCIT was detected in co-cultures of NP stromal + NCCIT cells (labeled NP-NCCIT). Expression of these … A comparison of stem cell gene expression in treated NCCIT showed higher signal levels of NANOG, POU5F1, TDGF1, and SOX2 in CP-NCCIT than in NP-NCCIT (Fig.?1c and d). Induction of NCCIT by NP stromal cells was found to lead to almost complete down-regulation of these stem cell genes [13], whereas CP stromal cells had apparently little effect. Of note was the detection of ABCG2 (a prostate progenitor cell marker) expression in NP-NCCIT but not CP-NCCIT (Fig.?1d). NCCIT is negative for ABCG2 expression. To identify genes encoding secreted Mouse monoclonal to beta Actin.beta Actin is one of six different actin isoforms that have been identified. The actin molecules found in cells of various species and tissues tend to be very similar in their immunological and physical properties. Therefore, Antibodies againstbeta Actin are useful as loading controls for Western Blotting. However it should be noted that levels ofbeta Actin may not be stable in certain cells. For example, expression ofbeta Actin in adipose tissue is very low and therefore it should not be used as loading control for these tissues proteins that might function in cell-cell signaling, the most differentially expressed genes in NP-NCCIT vs. CP-NCCIT (or NP stromal vs. CP stromal) were analyzed using the DAVID annotation tool. CP stromal induction led to up-regulation of several such genes including LEFTY2 and TAC1, while NP CDP323 stromal induction led to up-regulation of ADAMTS1, IGFBP5, WNT5A and others (Table S1). Overall, there were many more such genes induced in NP-NCCIT than CP-NCCIT. This showed a smaller pool of candidate signaling molecules produced by CP stromal cells. Differentially expressed genes were also analyzed for significant enrichment with respect to functional categories using DAVID. The top KEGG pathways identified were cytokine-cytokine receptor interaction, chemokine signaling pathway, extracellular matrix (ECM)-receptor interaction, cell adhesion and focal adhesion. Enrichment of these functional categories was prominent in the CP-NCCIT vs. NP-NCCIT datasets. Of particular interest were the genes that contribute to the functioning and maintenance of ECM. Matrix metalloproteinases (MMPs) are involved in the degradation of ECM proteins and have been associated with tumor cell invasion. The membrane-anchored reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is a potent inhibitor of MMP activity. RECK down-regulation has been identified in many cancers, and it has been reported that high RECK expression levels were associated with favorable prognosis in prostate cancer [26, 27]. In comparing the CP-NCCIT and NP-NCCIT expression profiles, several genes associated with RECK were differentially expressed (Fig.?2a). For example, induction of MMP9, a potential prostate cancer urine biomarker [28], was greater in CP-NCCIT than in NP-NCCIT (Fig.?2b)..

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