• 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.
    • Early hormonal data from a multicentre phase II trial using transdermal oestrogen patches as first-line hormonal therapy in patients with locally advanced or metastatic prostate cancer.

      Langley, Ruth E; Godsland, Ian F; Kynaston, Howard; Clarke, Noel W; Rosen, Stuart D; Morgan, Rachel C; Pollock, Philip; Kockelbergh, Roger; Lalani, El-Nasir; Dearnaley, David P; et al. (2008-08)
      OBJECTIVE: To assess the hormonal effects of Fem7 (Merck, KGaA, Darmstadt, Germany) 100 microg transdermal oestrogen patches on men undergoing first-line androgen-deprivation therapy for prostate cancer. PATIENTS AND METHODS: PATCH is a multicentre, randomized, phase II trial for men with locally advanced or metastatic prostate cancer, comparing luteinizing hormone-releasing hormone agonist therapy with oestrogen patches. To assess the dosing schedule for the patches, as this was the first time that this brand of patch had been used in men, and to reassure patients and participating clinicians, the Independent Data Monitoring Committee agreed to early release of hormonal data from this study. RESULTS: Oestradiol, testosterone and prostate-specific antigen (PSA) levels are presented for the first group of 14 patients who received the patches (with 1 withdrawal) and for whom there were > or =12 weeks of follow-up by March 2007. After 12 weeks, testosterone levels (nmol/L) in eight of the 13 patients were <1.7, two were 1.7-2 and three were >2. The median (range) serum oestradiol levels was 442 (52.1-1542) pmol/L and all patients had a PSA response, with eight having a PSA level of <4 ng/mL. CONCLUSION: These results confirm that oestrogen patches produce castrate levels of testosterone and concomitant PSA responses. They also highlighted the potential differences between different brands of oestrogen patches, and the need to monitor hormonal response, toxicity and efficacy until more experience with oestrogen patches for this clinical indication is obtained. The number of patches recommended in the PATCH study has now been increased.
    • Histological outcome of delayed orchidectomy after primary chemotherapy for metastatic germ cell tumour of the testis.

      Ramani, Vijay A C; Grey, Benjamin R; Addla, Sanjai K; Dunham, Mark P; Sangar, Vijay K; Clarke, Noel W; Christie Hospital NHS Foundation Trust, Manchester, UK. (2008-04)
      AIMS: To identify the incidence of viable local tumour in the testis of patients undergoing delayed orchidectomy after initial presentation with advanced germ cell tumour (GCT) treated by primary chemotherapy. PATIENTS AND METHODS: Thirty-three patients presenting with advanced metastatic GCT were reviewed. The median age at presentation was 34 years. All received chemotherapy without previous orchidectomy. The decision to initiate chemotherapy without orchidectomy was based on a heavy tumour load and the patient's condition at initial presentation. A histological diagnosis was available from a biopsy of metastases in 23 patients; treatment in the remaining 10 patients was initiated after diagnosis based on a combination of elevated serum tumour markers, testicular findings and the presence of a retroperitoneal mass. RESULTS: Seminomatous GCT (SGCT) was diagnosed in 13 patients, non-seminomatous GCT (NSGCT) in 17 patients and mixed GCT (MGCT) in the remaining three patients. Bleomycin/etoposide/cisplatin-based chemotherapy was the principle regimen. After initial chemotherapy, all patients with pure SGCT had only scar tissue in the orchidectomy specimen, with no residual tumour. Nine of 17 patients (52.9%) with NSGCT had viable tumour remaining in the orchidectomy specimen. All three cases of MGCT had persistent viable invasive seminoma. Twenty-seven patients (81.8%) were recurrence free and alive after a median of 49 months of follow-up. CONCLUSIONS: Thirty-six per cent of patients had residual tumour locally in the testis after primary chemotherapy for metastatic GCT of the testis. However, in the cases with pure seminomatous disease, there was no residual tumour present. It may not be necessary to undertake delayed orchidectomy in these patients.
    • 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.
    • 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.