• Cd133: a marker of transit amplification rather than stem cell phenotype in the prostate?

      Grey, Benjamin R; Oates, Jeremy E; Brown, Michael D; Clarke, Noel W; Genito-Urinary Cancer Research Group, University of Manchester Paterson Institute for Cancer Research, Manchester, UK. bengrey@doctors.org.uk (2009-04)
    • Characterization of the Hoechst 33342 side population from normal and malignant human renal epithelial cells.

      Addla, Sanjai K; Brown, Michael D; Hart, Claire A; Ramani, Vijay A C; Clarke, Noel W; Genito-Urinary Cancer Research Group, School of Cancer and Imaging Sciences, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, UK. (2008-09)
      The fundamental changes which predispose for renal cell carcinoma (RCC) are poorly characterized. It is hypothesized that "cancer stem cells" may be influential in carcinogenesis, and the epithelial side population (SP) is enriched for stemlike cells in other epithelial cancers. In this study, we have isolated and characterized the SP and non-SP (NSP) populations from normal (NK) and malignant (RCC) human kidney tissue. NK specimens were taken from patients undergoing non-renal cancer surgery and paired malignant and macroscopically normal tissue samples were taken from patients undergoing surgery for RCC. The Hoechst 33342 dye efflux technique was used to isolate epithelial SP and NSP from normal and malignant human renal tissue. Cellular subpopulations were phenotyped for lineage, cell cycle, and putative stem cell markers, and functionally characterized using in vitro colony-forming and proliferation assays. The SP constituted 3.8 +/- 0.4 and 5.9 +/- 0.9% of epithelial cells in NK and RCC, respectively, of which 14.1 +/- 3.5 and 13.2 +/- 3.6% were shown to be in G(0). SP cells demonstrated greater proliferative potential in colony-forming efficiency, long-term culture, and spheroids assays and were shown to be maintained upon tissue culture passage. We have shown that the renal SP is enriched for quiescent cells, with a high proliferative capacity and stemlike properties. The population is, however, heterogeneous, confirming that the terms "SP cell" and "stem cell" cannot be used interchangeably.
    • CpG methylation profiling in VHL related and VHL unrelated renal cell carcinoma.

      McRonald, Fiona E; Morris, Mark R; Gentle, Dean; Winchester, Laura; Baban, Dilair; Ragoussis, Jiannis; Clarke, Noel W; Brown, Michael D; Kishida, Takeshi; Yao, Masahiro; et al. (2009)
      BACKGROUND: Renal cell carcinoma (RCC) is histopathologically heterogeneous with clear cell and papillary the most common subtypes. The most frequent molecular abnormality in clear cell RCC is VHL inactivation but promoter methylation of tumour suppressor genes is common in both subtypes of RCC. To investigate whether RCC CpG methylation status was influenced by histopathology and VHL status we performed high-throughput epigenetic profiling using the Illumina Goldengate Methylation Array in 62 RCC (29 RCC from von Hippel-Lindau (VHL) disease patients, 20 sporadic clear cell RCC with wild type VHL and 13 sporadic papillary RCC). RESULTS: 43 genes were methylated in >20% of primary RCC (range 20-45%) and most (37/43) of these had not been reported previously to be methylated in RCC. The distribution of the number of methylated CpGs in individual tumours differed from the expected Poisson distribution (p < 0.00001; log-likelihood G test) suggesting that a subset of RCC displayed a CpG Island Methylator Phenotype. Comparison of RCC subtypes revealed that, on average, tumour specific CpG methylation was most prevalent in papillary RCC and least in VHL RCC. Many of the genes preferentially methylated in pRCC were linked to TGFbeta or ERK/Akt signalling. CONCLUSION: These findings demonstrate differing patterns of tumour-specific CpG methylation in VHL and non VHL clear cell RCC and papillary RCC, and identify multiple novel potential CpG methylation biomarkers for RCC.
    • Discrimination of prostate cancer cells and non-malignant cells using secondary ion mass spectrometry.

      Baker, Matthew J; Brown, Michael D; Gazi, Ehsan; Clarke, Noel W; Vickerman, John C; Lockyer, Nicholas P; Manchester Interdisciplinary Biocentre, Centre for Instrumentation and Analytical Science, School of Chemical Engineering and Analytical Science, The University of Manchester, UK. M.Baker-2@postgrad.manchester.ac.uk (2008-02)
      This communication utilises Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) combined with multivariate analysis to obtain spectra from the surfaces of three closely related cell lines allowing their discrimination based upon mass spectral ions.
    • A FTIR microspectroscopic study of the uptake and metabolism of isotopically labelled fatty acids by metastatic prostate cancer.

      Gazi, Ehsan; Harvey, Tim J; Brown, Michael D; Lockyer, Nicholas P; Gardner, Peter; Clarke, Noel W; Genito Urinary Cancer research Group, School of Cancer and Imaging Sciences, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK (2009)
    • FTIR-based spectroscopic analysis in the identification of clinically aggressive prostate cancer.

      Baker, Matthew J; Gazi, Ehsan; Brown, Michael D; Shanks, Jonathan H; Gardner, Peter; Clarke, Noel W; Manchester Interdisciplinary Biocentre, Centre for Instrumentation and Analytical Science, School of Chemical Engineering and Analytical Science, The University of Manchester, Manchester, M1 7DN, UK. M.J.Baker@manchester.ac.uk (2008-12-02)
      Fourier transform infrared (FTIR) spectroscopy is a vibrational spectroscopic technique that uses infrared radiation to vibrate molecular bonds within the sample that absorbs it. As different samples contain different molecular bonds or different configurations of molecular bonds, FTIR allows us to obtain chemical information on molecules within the sample. Fourier transform infrared microspectroscopy in conjunction with a principal component-discriminant function analysis (PC-DFA) algorithm was applied to the grading of prostate cancer (CaP) tissue specimens. The PC-DFA algorithm is used alongside the established diagnostic measures of Gleason grading and the tumour/node/metastasis system. Principal component-discriminant function analysis improved the sensitivity and specificity of a three-band Gleason score criterion diagnosis previously reported by attaining an overall sensitivity of 92.3% and specificity of 99.4%. For the first time, we present the use of a two-band criterion showing an association of FTIR-based spectral characteristics with clinically aggressive behaviour in CaP manifest as local and/or distal spread. This paper shows the potential for the use of spectroscopic analysis for the evaluation of the biopotential of CaP in an accurate and reproducible manner.
    • Functional epigenomics approach to identify methylated candidate tumour suppressor genes in renal cell carcinoma.

      Morris, M R; Gentle, D; Abdulrahman, M; Clarke, Noel W; Brown, Michael D; Kishida, Takeshi; Yao, M; Teh, B T; Latif, Farida; Maher, Eamonn R; et al. (2008-01-29)
      Promoter region hypermethylation and transcriptional silencing is a frequent cause of tumour suppressor gene (TSG) inactivation in many human cancers. Previously, to identify candidate epigenetically inactivated TSGs in renal cell carcinoma (RCC), we monitored changes in gene expression in four RCC cell lines after treatment with the demethylating agent 5-azacytidine. This enabled us to identify HAI-2/SPINT2 as a novel epigenetically inactivated candidate RCC TSG. To identify further candidate TSGs, we undertook bioinformatic and molecular genetic evaluation of a further 60 genes differentially expressed after demethylation. In addition to HAI-2/SPINT2, four genes (PLAU, CDH1, IGFB3 and MT1G) had previously been shown to undergo promoter methylation in RCC. After bioinformatic prioritisation, expression and/or methylation analysis of RCC cell lines+/-primary tumours was performed for 34 genes. KRT19 and CXCL16 were methylated in RCC cell lines and primary RCC; however, 22 genes were differentially expressed after demethylation but did not show primary tumour-specific methylation (methylated in normal tissue (n=1); methylated only in RCC cell lines (n=9) and not methylated in RCC cell lines (n=12)). Re-expression of CXCL16 reduced growth of an RCC cell line in vitro. In a summary, a functional epigenomic analysis of four RCC cell lines using microarrays representing 11 000 human genes yielded both known and novel candidate TSGs epigenetically inactivated in RCC, suggesting that this is valid strategy for the identification of novel TSGs and biomarkers.
    • Genome-wide methylation analysis identifies epigenetically inactivated candidate tumour suppressor genes in renal cell carcinoma.

      Morris, M R; Ricketts, C J; Gentle, D; McRonald, F; Carli, N; Khalili, H; Brown, Michael D; Kishida, T; Yao, M; Banks, R E; et al. (2011-03-24)
      The detection of promoter region hypermethylation and transcriptional silencing has facilitated the identification of candidate renal cell carcinoma (RCC) tumour suppressor genes (TSGs). We have used a genome-wide strategy (methylated DNA immunoprecipitation (MeDIP) and whole-genome array analysis in combination with high-density expression array analysis) to identify genes that are frequently methylated and silenced in RCC. MeDIP analysis on 9 RCC tumours and 3 non-malignant normal kidney tissue samples was performed, and an initial shortlist of 56 candidate genes that were methylated by array analysis was further investigated; 9 genes were confirmed to show frequent promoter region methylation in primary RCC tumour samples (KLHL35 (39%), QPCT (19%), SCUBE3 (19%), ZSCAN18 (32%), CCDC8 (35%), FBN2 (34%), ATP5G2 (36%), PCDH8 (58%) and CORO6 (22%)). RNAi knockdown for KLHL35, QPCT, SCUBE3, ZSCAN18, CCDC8 and FBN2 resulted in an anchorage-independent growth advantage. Tumour methylation of SCUBE3 was associated with a significantly increased risk of cancer death or relapse (P=0.0046). The identification of candidate epigenetically inactivated RCC TSGs provides new insights into renal tumourigenesis.
    • Investigating FTIR based histopathology for the diagnosis of prostate cancer.

      Baker, Matthew J; Gazi, Ehsan; Brown, Michael D; Shanks, Jonathan H; Clarke, Noel W; Gardner, Peter; Manchester Interdisciplinary Biocentre, Centre for Instrumentation and Analytical Science, School of Chemical Engineering and Analytical Science, The University of Manchester, 131 Princess Street, Manchester, UK. (2009-02)
      Prostate cancer is the most common gender specific cancer. The current gold standard for diagnosis, histopathology, is subjective and limited by variation between different pathologists. The diagnostic problems associated with the correct grading and staging of prostate cancer (CaP) has led to an interest in the development of spectroscopic based diagnostic techniques. FTIR microspectroscopy used in combination with a Principal Component Discriminant Function Analysis (PC-DFA) was applied to investigate FTIR based histopathology for the diagnosis of CaP. In this paper we report the results of a large patient study in which FTIR has been proven to grade CaP tissue specimens to a high degree of sensitivity and specificity.
    • Molecular mechanisms of metastasis in prostate cancer.

      Clarke, Noel W; Hart, Claire A; Brown, Michael D; 1Genito-Urinary Cancer Research Group, School of Cancer and Imaging Sciences, Paterson Institute for Cancer Research, Christie Hospital, University of Manchester, Manchester M20 4BX, UK. (2009-01)
      Prostate cancer (PCa) preferentially metastasizes to the bone marrow stroma of the axial skeleton. This activity is the principal cause of PCa morbidity and mortality. The exact mechanism of PCa metastasis is currently unknown, although considerable progress has been made in determining the key players in this process. In this review, we present the current understanding of the molecular processes driving PCa metastasis to the bone.Asian Journal of Andrology (2009) 11: 57-67. doi: 10.1038/aja.2008.29; published online 1 December 2008.