We provide novel functional data that posttranscriptional silencing of gene RPL19 using RNAi not only abrogates the malignant phenotype of PC-3M prostate cancer cells but is selective with respect to ...transcription and translation of other genes. Reducing RPL19 transcription modulates a subset of genes, evidenced by gene expression array analysis and Western blotting, but does not compromise cell proliferation or apoptosis in-vitro. However, growth of xenografted tumors containing the knocked-down RPL19 in-vivo is significantly reduced. Analysis of the modulated genes reveals induction of the non-malignant phenotype principally to involve perturbation of networks of transcription factors and cellular adhesion genes. The data provide evidence that extra-ribosomal regulatory functions of RPL19, beyond protein synthesis, are critical regulators of cellular phenotype. Targeting key members of affected networks identified by gene expression analysis raises the possibility of therapeutically stabilizing a benign phenotype generated by modulating the expression of an individual gene and thereafter constraining a malignant phenotype while leaving non-malignant tissues unaffected.
Microquantity differential display analysis of gene expression profiles between benign (PNT2) and malignant (PC3M) human prostate cell lines identified the gene encoding ribosomal protein L19 (RPL19) ...to be overexpressed in the malignant cells. Northern blot hybridization analysis done on a wide range of human cell lines and tissues confirmed the level of RPL19 mRNA to be 5-fold higher in malignant cell lines and 8-fold higher in malignant tissues, when compared with their benign counterparts. Analysis of RPL19 mRNA expression by in situ hybridization revealed a significant increase of RPL19 expression in a substantial number of prostate cancers. All of the eight normal prostatic tissues were unstained (100%). Of 32 benign prostatic hyperplasia (BPH) tissues, 15 (46.9%) were unstained, 9 (28.1%) stained weakly, and 8 (25%) stained moderately. Among 87 carcinomas, only 7 (8.1%) were unstained, whereas 22 (25.2%) stained weakly, 21 (24.1%) stained moderately, and 37 (42.61%) stained strongly. The intensity of staining of the malignant specimens was significantly higher than that of normal and BPH specimens (chi(2): n = 127, P < 0.001). Gleason scores of the carcinomas correlated with RPL19 expression (chi(2): n = 87, P < 0.001). Kaplan-Meier survival analysis confirmed increased RPL19 expression to be highly predictive of shorter patient survival (P < 0.05), revealing RPL19 to be a sensitive predictor of prostate cancer progression. Expression of this protein could be a valuable marker in prostate cancer diagnosis and patient management.
The gene FABP5 encodes cutaneous fatty acid binding protein (C-FABP) that is up-regulated in prostate cancer where it acts as a putative oncogene. To test the hypothesis that siRNA to FABP5 delivered ...to the external environment of a prostate cancer would reduce the level of C-FABP in vivo, experiments were established whereby siRNA to FABP5 suspended in atelocollagen was injected around tumour masses produced by PC-3M cells in Balb/c nude mice and compared with the effect of non-specific scrambled siRNA in atelocollagen. At autopsy, the average size of tumours from the groups treated with 10 and 15 microM siRNA in atelocollagen was significantly (p=0.02) reduced by more than 3-fold, when compared to the controls. In contrast, when compared to the tumours produced by the group treated with scrambled siRNA, treatment with 10 microM FABP5 siRNA in buffer and 1 or 5 microM siRNA in atelocollagen did not produce significant differences. Although the dosage of 15 microM siRNA produced a greater reduction in tumour sizes when compared with 10 microM, this difference was not significant (p=0.9). Immunohistochemistry and Western blotting revealed that the levels of C-FABP expression in tumours from mice treated with 10 and 15 microM dosages were lower than those from the other groups. These data demonstrate that FABP5 siRNA delivered by atelocollagen to the external environment surrounding a tumour mass can effectively inhibit prostate cancer cell growth in nude mice when administered in a dose-dependent manner at concentrations of >10 microM.
We show protein kinase C-zeta (PKC-ζ) to be a novel predictive biomarker for survival from prostate cancer (P < 0.001). We also confirm that transcription of the PRKC-ζ gene is crucial to the ...malignant phenotype of human prostate cancer. Following siRNA silencing of PRKC-ζ in PC3-M prostate cancer cells, stable transfectant cell line si-PRKC-ζ-PC3-M(T1-6) is phenotypically nonmalignant in vitro and in vivo. Genome-wide expression analysis identified 373 genes to be differentially expressed in the knockdown cells and 4 key gene networks to be significantly perturbed during phenotype modulation. Functional interconnection between some of the modulated genes is revealed, although these may be within different regulatory pathways, emphasizing the complexity of their mutual interdependence. Genes with altered expression following PRKC-ζ knockdown include HSPB1, RAD51, and ID1 that we have previously described to be critical in prostatic malignancy. Because expression of PRKC-ζ is functionally involved in promoting the malignant phenotype, we propose PKC-ζ as a novel and biologically relevant target for therapeutic intervention in prostate cancer.
Three canine cell lines, K1, K6 and DH82, derived from canine malignant neoplasms, were characterised. They were examined for expression of surface antigens, cytokines, neuropeptide receptors, matrix ...metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). The growth characteristics of the cell lines were established and bioassays used to detect production of TNF-α, IL-1 and IL-6. In the DH82 cell line, production of TNF-α and IL-6 was readily detected. Neither K1 or K6 cell lines produced any measurable amounts of TNF-α, IL-1 or IL-6. At a molecular level, using reverse transcription-polymerase chain reaction (RT-PCR) to detect specific mRNA, the DH82 cell line expressed TNF-α, IL-1 and IL-6, whereas the K1 and K6 cell lines expressed TNF-α. Canine IL-5, IL-8 and IL-10 mRNA were detected in the DH82 cell line but only IL-5 and IL-8 mRNA were detected in the K1 and K6 cell lines. Gelatin zymography was used for the detection of MMP-2 and MMP-9 and all three cell lines produced MMP-2 but only the DH82 cell line produced MMP-9. Reverse zymography was used to detect TIMP-1 and TIMP-2 and all three cell lines produced both proteins. The presence of these MMPs and TIMPs was confirmed at a molecular level using RT-PCR. Canine MMP-14 mRNA was detected in all three cell lines. For this investigation several genes for canine inflammatory molecules were cloned and sequenced for molecular detection; these included IL-1, IL-6, IL-8, TNF-α, MMP-9, MMP-14, TIMP-1, TIMP-2 and β-actin. Of all the cell surface antigens tested, only CD14 was expressed on the DH82 cell line although CD5 and CD45 was partially expressed. The K1 and K6 cell lines were negative for all of the CD markers tested. K1 and K6 were negative for Neurokinin 1 receptor (NK1-R) but positive for Calcitonin gene related peptide receptor type 1 (CGRP-1R) and Calcitonin gene related peptide receptor component protein (CGRP-RCP). The DH82 cell line expressed neither NK1-R or CGRP-1R; however, it did express CGRP-RCP. Generally the DH82 cell line exhibited considerable similarity to canine monocytes, but all three cell lines will be useful as standards and for the purification of various immunological and inflammatory mediators in the dog.
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