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Biomedical subjects

N W Clarke

Publications and source records attributed to N W Clarke.

At least 19 recordsLinked to original sources
PubMed2006

Promotion of prostatic metastatic migration towards human bone marrow stoma by Omega 6 and its inhibition by Omega 3 PUFAs.

Epidemiological studies have shown not only a relationship between the intake of dietary lipids and an increased risk of developing metastatic prostate cancer, but also the type of lipid intake that influences the risk of metastatic prostate cancer. The Omega-6 poly-unsaturated fatty acid, Arachidonic acid, has been shown to enhance the proliferation of malignant prostate epithelial cells and increase the risk of advanced prostate cancer. However, its role in potentiating the migration of cancer cells is unknown. Here we show that arachidonic acid at concentrations <or=5 microM is a potent stimulator of malignant epithelial cellular invasion, which is able to restore invasion toward hydrocortisone-deprived adipocyte-free human bone marrow stroma completely. This observed invasion is mediated by the arachidonic acid metabolite prostaglandin E2 and is inhibited by the Omega-3 poly-unsaturated fatty acids eicosapentaenoic acid and docosahexaenoic acid at a ratio of 1 : 2 Omega-3 : Omega-6, and by the COX-2 inhibitor NS-398. These results identify a mechanism by which arachidonic acid may potentiate the risk of metastatic migration and secondary implantation in vivo, a risk which can be reduced with the uptake of Omega-3 poly-unsaturated fatty acids.

Arachidonic Acid↗
PubMed2005

Expression of the 5T4 oncofoetal antigen in renal cell carcinoma: a potential target for T-cell-based immunotherapy.

The 5T4 oncofoetal antigen is a heavily glycosylated cell surface protein found on human placental trophoblast and on diverse types of human cancer but is not expressed at significant levels on adult human tissues in health. It therefore satisfies the criteria for a tumour-associated antigen and is an ideal target for the immunotherapy of cancer. We report here that 5T4 is strongly expressed on the majority of renal cell carcinomas and therefore this population of patients is suitable for trials of 5T4-targeted therapies. In particular, we have shown that T cells from renal cell carcinoma patients can be genetically modified to kill 5T4 expressing renal cancer cell lines by introduction of a chimeric-signalling protein. This protein consists of a single chain antibody fragment capable of binding antigen directly at the cell surface and then activating the T cell by virtue of a CD3zeta-signalling domain. This is a powerful tool that bypasses a number of mechanisms that allow tumours to escape T-cell killing and can be readily scaled up for clinical use.

Adenocarcinoma, Clear Cell↗
PubMed2005

Invasive characteristics of human prostatic epithelial cells: understanding the metastatic process.

Prostate cancer has a predilection to metastasise to the bone marrow stroma (BMS) by an as yet uncharacterised mechanism. We have defined a series of coculture models of invasion, which simulate the blood/BMS boundary and allow the elucidation of the signalling and mechanics of trans-endothelial migration within the complex bone marrow environment. Confocal microscopy shows that prostate epithelial cells bind specifically to bone marrow endothelial-to-endothelial cell junctions and initiate endothelial cell retraction. Trans-endothelial migration proceeds via an epithelial cell pseudopodial process, with complete epithelial migration occurring after 232+/-43 min. Stromal-derived factor-1 (SDF-1)/CXCR4 signalling induced PC-3 to invade across a basement membrane although the level of invasion was 3.5-fold less than invasion towards BMS (P=0.0007) or bone marrow endothelial cells (P=0.004). Maximal SDF-1 signalling of invasion was completely inhibited by 10 microM of the SDF-1 inhibitor T140. However, 10 microM T140 only reduced invasion towards BMS and bone marrow endothelial cells by 59% (P=0.001) and 29% (P=0.011), respectively. This study highlights the need to examine the potential roles of signalling molecules and/or inhibitors, not just in single-cell models but in coculture models that mimic the complex environment of the bone marrow.

Bone Marrow Cells↗
PubMed2005

Differential radiosensitisation by ZD1839 (Iressa), a highly selective epidermal growth factor receptor tyrosine kinase inhibitor in two related bladder cancer cell lines.

The epidermal growth factor receptor (EGFR) is expressed in a wide variety of epithelial tumours including carcinoma of the bladder. Stimulation of the EGFR pathway is blocked by ZD1839 (Iressa), a highly selective EGFR tyrosine kinase inhibitor. Radical radiotherapy is an established organ sparing treatment option for muscle invasive bladder cancer and this study has explored the possibility for the use of ZD1839 as a radiosensitiser in this scenario. The effect of combination treatment with ZD1839 (0.01 microM) and ionising radiation in the established bladder cancer cell lines MGH-U1 and its radiosensitive mutant clone S40b was measured by clonogenic assays. A highly significant radiosensitising effect was seen in both cell lines (P < 0.001 for MGH-U1 and S40b cell lines). This effect was independent of the concentration of the drug and the duration of exposure prior to treatment with ionising radiation. Cell cycle kinetics of both cell lines was not significantly altered with ZD1839 (0.01 microM) as a single agent. A modest induction of apoptosis was observed with ZD1839 (0.01 microM) as a single agent, but a marked induction was observed with the combination treatment of ZD1839 and ionising radiation. These results suggest a potentially important role for ZD1839 in combination with radiotherapy in the treatment of muscle invasive bladder cancer.

Antineoplastic Agents↗
PubMed2005

Fixation protocols for subcellular imaging by synchrotron-based Fourier transform infrared microspectroscopy.

Synchrotron-based Fourier transform infrared (SR-FTIR) microspectroscopy is a powerful bioanalytical technique for the simultaneous analysis of lipids, proteins, carbohydrates, and a variety of phosphorylated molecules within intact cells. SR-FTIR microspectroscopy can be used in the imaging mode to generate biospectroscopic maps of the distribution and intensity profiles of subcellular biomolecular domains at diffraction-limited spatial resolution. However, the acquisition of highly spatially resolved IR images of cells is not only a function of instrumental parameters (source brightness, sampling aperture size) but also the cell preparation method employed. Additionally, for the IR data to be biochemically relevant the cells must be preserved in a life-like state without introducing artefacts. In the present study we demonstrate, for the first time, the differences in biomolecular localizations observed in SR-FTIR images of cells fixed by formalin, formalin-critical point drying (CPD), and glutaraldehyde-osmium tetroxide-CPD, using the PC-3 prostate cancer cell line. We compare these SR-FTIR images of fixed cells to unfixed cells. The influence of chemical fixatives on the IR spectrum is discussed in addition to the biological significance of the observed localizations. Our experiments reveal that formalin fixation at low concentration preserves lipid, phosphate, and protein components without significantly influencing the IR spectrum of the cell.

Animals↗
PubMed2005

Adjuvant and salvage treatment after radical prostatectomy: current practice in the UK.

OBJECTIVE: To survey UK urologists and radiation oncologists in the evaluation and treatment of localised prostate cancer in the adjuvant and salvage setting. METHODS: Postal questionnaires were mailed to 292 urologists and 98 radiation oncologists in the UK. RESULTS: In all, 188 (48%) questionnaires were returned. In total, 72/128 (56%) of the urologist respondents and 58/60 (97%) of the oncologist respondents perform routine radical prostate treatment. Among 43 (60%) of the urologist, 40 (69%) recommended adjuvant treatment, which could be radiotherapy, hormonal treatment or combined hormonal and radiation treatment. There is no significant difference between the modality of treatment recommended. The poor prognostic factors that would influence the decision to offer adjuvant treatment include a detectable postoperative PSA, seminal vesicle involvement, positive margins, Gleason score>8 and pathological T3. With regard to the choice of hormonal treatment, most urologists preferred antiandrogens, whereas most oncologists prefer lutienising hormone releasing hormone (LHRH) analogue (P=0.03). Regarding salvage treatment, there is a wide variation in the PSA threshold and number of PSA rises before initiation of investigations and treatment. Significantly more urologists recommended salvage radiotherapy (P=0.02), whereas oncologists recommended combined hormonal radiation therapy (P=0.03). There is a wide variation of practice regarding the duration of hormonal treatment, the type of investigations initiated, range of radiotherapy doses and treatment volumes. CONCLUSION: There is a wide variation in practice among UK clinicians.

Chemotherapy, Adjuvant↗
PubMed2005

National review of urology outpatient practice in the UK.

OBJECTIVE: To audit the current UK outpatient workload and compare this to the national standards as set out by the British Association of Urological Surgeons (BAUS) in A Quality Urological Service for Patients in the New Millennium published in October 2000. PARTICIPANTS: 520 UK (NHS) and 21 Republic of Ireland (non-NHS) consultant urologists registered with BAUS in 2000. MAIN OUTCOME MEASURES: Extent to which consultant urologists are able to comply with guidelines set out by their specialist association, the BAUS and by the Royal College of Surgeons of England. RESULTS: The questionnaire return rate was 61% (318/520; regional range 42%-75%). The median "routine" clinics/week was two (1-5) with a mean of 13 (1-40) new and 26 (7-80) follow ups. Fifteen percent (49/318) of consultants worked alone in clinic; of the remainder assistance included specialist registrar 67% (212/318), staff grade/associate specialist 32% (102/318), senior house officer 53% (172/318), and pre-registration house officer 2% (7/318). Only 21% (66/318; regional range 0%-46%) of responding consultants followed the BAUS recommendations for outpatient workload/manpower. CONCLUSIONS: A minority of consultants are able to adhere to the outpatient workload guidelines as set out by BAUS council in 2000. In addition, there appears to be significant variations within and between training regions. Development of this project into a regional audit tool may allow intraregional guideline formation governing hospital outpatient workload.

Guideline Adherence↗
PubMed2004

An evaluation of gemcitabines differential radiosensitising effect in related bladder cancer cell lines.

The aim of this study was to establish the radiosensitising properties of gemcitabine in a pair of related bladder tumour cell lines with differential radiosensitivity. The radioresistant bladder tumour cell line MGH-U1 and its radiosensitive mutant clone, S40b (both p53 mutant), had SF(2) values (surviving fraction at 2 Gy) of 0.98 and 0.64, respectively (P<0.001). Colony-forming assays showed that at 0.01 microM gemcitabine radiosensitisation occurred only in the S40b cell line (dose-modifying factor (DMF)=1.4). At 0.3 microM (killing 50% of cells), both cell lines were radiosensitised; DMF=2.25 and 1.2 for MGH-U1 and S40b, respectively. These data suggest that gemcitabine is an effective radiosensitiser in bladder cancer cell lines, with greater sensitisation in the radioresistant parental line-a feature that should be useful in a clinical setting.

Antimetabolites, Antineoplastic↗
PubMed2003

Applications of Fourier transform infrared microspectroscopy in studies of benign prostate and prostate cancer. A pilot study.

Fourier transform infrared (FTIR) microspectroscopy has been applied to a study of prostate cancer cell lines derived from different metastatic sites and to tissue from benign prostate and Gleason-graded malignant prostate tissue. Paraffin-embedded tissue samples were analysed by FTIR, after mounting onto a BaF(2) plate and subsequent removal of wax using Citroclear followed by acetone. Cell lines were analysed as aliquots of cell suspension held between two BaF(2) plates. It was found that the ratio of peak areas at 1030 and 1080 cm(-1), corresponding to the glycogen and phosphate vibrations respectively, suggests a potential method for the differentiation of benign from malignant cells. The use of this ratio in association with FTIR spectral imaging provides a basis for estimating areas of malignant tissue within defined regions of a specimen. Initial chemometric treatment of FTIR spectra, using the linear discriminant algorithm, demonstrates a promising method for the classification of benign and malignant tissue and the separation of Gleason-graded CaP spectra. Using the principle component analysis, this study has achieved for the first time the separation of FTIR spectra of prostate cancer cell lines derived from different metastatic sites.

Adenocarcinoma↗
PubMed2002

Role of proteolytic enzymes in human prostate bone metastasis formation: in vivo and in vitro studies.

Prostate cancers ability to invade and grow in bone marrow stroma is thought to be due in part to degradative enzymes. The formation of prostate skeletal metastases have been reproduced in vitro by growing co-cultures of prostatic epithelial cells in bone marrow stroma. Expression of urokinase plasminogen activator, matrix metalloproteinase 1 and 7 by prostatic epithelial cells were identified using immunocytochemistry. Also, in vivo tissue sections from human prostatic bone marrow metastases were stained. To establish the role of these enzymes on colony formation, inhibitory antibodies directed against urokinase plasminogen activator, matrix metalloproteinase 1 and matrix metalloproteinase 7 were added into primary prostatic epithelial cells and bone marrow stroma co-cultures. All prostatic epithelial cell cultures stained positively for matrix metalloproteinase 1, matrix metalloproteinase 7 and urokinase plasminogen activator. Generally prostatic epithelial cells derived from malignant tissues showed increased staining in comparison to epithelia derived from non-malignant tissue. In agreement with in vitro co-cultures, the in vivo tissue sections of prostate bone marrow metastases showed positive staining for all three enzymes. Inhibition studies demonstrated that blocking matrix metalloproteinase 1, matrix metalloproteinase 7 and urokinase plasminogen activator function reduced the median epithelial colony area significantly in bone marrow stroma co-cultures in vitro. Using a human ex-vivo model we have shown that matrix metalloproteinase 1, matrix metalloproteinase 7 and urokinase plasminogen activator play an important role in the establishment of prostatic epithelial cells within bone marrow.

Adult↗
PubMed2002

Parathyroid hormone related peptide and receptor expression in paired primary prostate cancer and bone metastases.

Parathyroid hormone-related peptide is a regulatory protein implicated in the pathogenesis of bone metastases, particularly in breast carcinoma. Parathyroid hormone-related peptide is widely expressed in primary prostate cancers but there are few reports of its expression in prostatic metastases. The aim of this study was to examine the expression of parathyroid hormone-related peptide and its receptor in matched primary and in bone metastatic tissue from patients with untreated adenocarcinoma of the prostate. Eight-millimetre trephine iliac crest bone biopsies containing metastatic prostate cancer were obtained from 14 patients from whom matched primary tumour tissue was also available. Histological grading was performed by an independent pathologist. The cellular location of mRNA for parathyroid hormone-related peptide and parathyroid hormone-related peptide receptor was identified using in situ hybridization with (35)S-labelled probe. Expression of parathyroid hormone-related peptide and its receptor was described as uniform, heterogenous or negative within the tumour cell population. Parathyroid hormone-related peptide expression was positive in 13 out of 14 primary tumours and in all 14 metastases. Receptor expression was evident in all 14 primaries and 12 out of 14 metastases. Co-expression of parathyroid hormone-related peptide and parathyroid hormone-related peptide receptor was common (13 primary tumours, 12 metastases). The co-expression of parathyroid hormone-related peptide and its receptor suggest that autocrine parathyroid hormone-related peptide mediated stimulation may be a mechanism of escape from normal growth regulatory pathways. The high frequency of parathyroid hormone-related peptide expression in metastases is consistent with a role in the pathogenesis of bone metastases.

Autoradiography↗
PubMed2002

Development of a novel rapid assay to assess the fidelity of DNA double-strand-break repair in human tumour cells.

Cellular survival following ionising radiation-mediated damage is primarily a function of the ability to successfully detect and repair DNA double-strand breaks (DSBs). Previous studies have demonstrated that radiosensitivity, determined as a reduction in colony forming ability in vitro, may be related to the incorrect repair (misrepair) of DSBs. The novel rapid dual fluorescence (RDF) assay is a plasmid-based reporter system that rapidly assesses the correct rejoining of a restriction-enzyme produced DSBs within transfected cells. We have utilised this novel assay to determine the fidelity of DSB repair in the prostate tumour cell line LNCaP, the bladder tumour cell line MGH-U1 and a radiosensitive subclone S40b. The two bladder cell lines have been shown in previous studies to differ in their ability to correctly repair plasmids containing a single DSB. Using the RDF assay we found that a substantial portion of LNCaP cells [80.4 +/- 5.3(standard error)%] failed to reconstitute reporter gene expression; however, there was little difference in this measure of DSB repair fidelity between the two bladder cell lines (48.3 +/- 3.5% for MGH-U1; 39.9 +/- 8.2% for S40b). The RDF assay has potential to be developed to study the relationship between DSB repair fidelity and radiosensitivity as well as the mechanisms associated with this type of repair defect.

Cell Survival↗
PubMed2002

Non-surgical interventions for late radiation cystitis in patients who have received radical radiotherapy to the pelvis.

BACKGROUND: Chronic radiation cystitis occurs a minimum of three months after completion of pelvic radiotherapy and represents a range of clinical symptoms for which there is as yet no recommended standard management. OBJECTIVES: The aim of this review was to identify the various non-surgical treatment options for the management of late chronic radiation cystitis and evaluate the evidence. SEARCH STRATEGY: Synonyms for radiation therapy and for the spectrum of radiation toxicity to the bladder in both text and MeSH terms were combined and applied to a range of databases without restriction of year of publication, methodology or language. SELECTION CRITERIA: The inclusion criteria included studies of interventions for the non-surgical management of all grades of late radiation cystitis. DATA COLLECTION AND ANALYSIS: Out of 80 relevant studies, there were no RCTs that met the inclusion criteria, but there were three prospective case series and two non-randomised studies which assessed different interventions and were not comparable. MAIN RESULTS: Sixty-three reports met the stated inclusion criteria. The majority were predominantly retrospective case series with the exception of two trials which were unrandomised and unblinded studies with a control group for comparison of effect. Although these two trials, Micic 1988, (intravesical placental extract) and Milani 1988, (flavoxate) provided the strongest evidence they were not randomised and were essentially isolated controlled studies. REVIEWER'S CONCLUSIONS: In such a relatively rare condition there are obvious difficulties in identifying sufficient patients to participate in a randomised controlled trial. The number of published reports is a reflection of the degree of medical interest that exists in providing therapeutic solutions for late radiation cystitis. However, in spite of the two studies of level IIA evidence, the absence of randomised controlled trials makes it impossible to draw any firm conclusions.

Chronic Disease↗
PubMed2002

Parathyroid hormone-related peptide: expression in prostate cancer bone metastases.

Parathyroid hormone-related peptide (PTHrP) is a regulatory protein associated with cell growth in non-osseous tissues and with osteoclast stimulation in bone. It has been implicated in the pathogenesis of bone metastases, particularly in breast carcinoma. PTHrP is widely expressed in primary prostate cancers, but there are few reports of its expression in prostatic metastases. The aim of this study was to examine the expression of PTHrP in bone metastases from patients with untreated adenocarcinoma of the prostate. Ten bone biopsies containing metastatic deposits of untreated prostatic cancer were identified. These were immunohistochemically stained for PTHrP using a murine monoclonal antibody (PTHLP[Ab1]) and the streptavidin-biotin complex technique. Intensity of staining for PTHrP was graded by two observers. In total, PTHrP expression was positive in 5/10 specimens. This was graded as moderate in four and weak in one. In those specimens with positive staining, the expression varied between cells. There was no obvious association between expression of PTHrP and tumour differentiation. PTHrP is expressed in prostatic bone metastases and may have a role in their pathogenesis and pathophysiology. However, expression is not universal.

Adenocarcinoma↗
PubMed2002

E-cadherin and beta-catenin are down-regulated in prostatic bone metastases.

OBJECTIVE: To determine the E-cadherin and beta-catenin expression phenotype in untreated primary prostate cancer and corresponding bone metastases. MATERIALS AND METHODS: Paired bone metastasis and primary prostate specimens were obtained from 14 men with untreated metastatic prostate carcinoma. The tumours were histologically graded by an independent pathologist. Expression of mRNA for E-cadherin and beta-catenin was detected within the tumour cells using in-situ hybridization with a 35S-labelled cDNA probe. The expression of E-cadherin and beta-catenin were graded as uniform, heterogeneous or negative. RESULTS: The mRNA for E-cadherin was expressed in 13 of 14 primary carcinomas and 11 bone metastases; beta-catenin was expressed by 13 and nine, respectively. Of the primary tumours, nine expressed E-cadherin and beta-catenin uniformly; in contrast, all metastases had down-regulated E-cadherin and/or beta-catenin. CONCLUSIONS: The down-regulation of E-cadherin and beta-catenin are a feature of the metastatic phenotype, which may be a significant factor in the genesis of bone metastases. However, this does not appear to be reflected in the expression of these molecules in the primary tumours.

Bone Neoplasms↗
PubMed2002

Photodynamic therapy for superficial bladder cancer under local anaesthetic.

OBJECTIVES: To evaluate the use of local anaesthesia (LA) in 5-aminolaevulinic acid (ALA) photodynamic therapy (PDT) for superficial transitional cell carcinoma (TCC) of the bladder, and to provide further toxicity and tolerability data on this new method within the context of a phase 1 trial. PATIENTS AND METHODS: ALA PDT was administered to 19 patients with recurrent superficial TCC (stage Ta/carcinoma in situ, grades 1-3) using escalating doses of ALA (3-6%) and 633 nm laser light (25-50 J/cm2) under various LA (lignocaine) protocols. Pain was assessed using a linear analogue scale from 0 to 10. The endpoints of tolerability and toxicity were assessed for the different LA, light and ALA doses, with lignocaine levels. RESULTS: ALA PDT is painful and requires some form of anaesthesia. The discomfort was immediate, associated with bladder spasm, and was a function of the ALA concentration rather than the total light dose given. Simple passive diffusion (PD) of 2% lignocaine instilled for 40 min before PDT gave adequate anaesthesia with 3% ALA (n=8; median pain score 1, range 0-2). With 6% ALA the pain was dramatically increased using PD (n=6; median pain score 8, range 5-10) and therefore the more potent LA technique of electromotive drug administration (EMDA) of 2% lignocaine was used, with excellent results (n=3; median pain score 1, range 0-2). All patients had transient bladder irritability that typically lasted 9-12 days, with no subjective/objective change in long-term bladder function. No other toxicity was reported. Serum lignocaine levels were minimal. CONCLUSION: Bladder ALA PDT is both safe and feasible under LA. At a dose of 3% ALA, the procedure was well-tolerated using PD of lignocaine. At higher doses (6% ALA) more effective anaesthesia is required and this can be obtained satisfactorily with EMDA of lignocaine. With refinement, ALA PDT may be feasible as an outpatient treatment for superficial bladder TCC.

Aged↗
PubMed2002

Comparison of the cellular molecular stress responses after treatments used in bladder cancer.

OBJECTIVE: To investigate the molecular stress responses related to the quality of recovery of normal tissue after various treatments for bladder cancer, i.e. hyperthermia, ionizing radiation, mitomycin-C and 5-aminolaevulinic acid photodynamic therapy (ALA-PDT). MATERIALS AND METHODS: The study focused particularly on intracellular fibroblast levels of heat-shock protein-47 (HSP47) and HSP72, which are associated with collagen metabolism and the development of tolerance to repeated treatment, respectively. Iso-effective treatment doses (50% clonogenic cell survival) of each method were delivered to a 3T6 murine fibroblast model. Intracellular extracts were analysed at 3, 6, 9, 12 and 24 h after treatment, using Western blot analysis to compare the levels of HSP47 and HSP72. Time-matched treatment and control groups were quantified by comparison with actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression using appropriate software. RESULTS: There were various changes in levels of HSP expression with treatment method; HSP47 levels were significantly higher after hyperthermia and radiation but not with mitomycin-C or ALA-PDT. HSP72 levels were significantly higher with all methods except ALA-PDT. CONCLUSIONS: Hyperthermia and ionizing radiation are associated with early increases in levels of HSP47 (a marker of collagen metabolism), in contrast to ALA-PDT and mitomycin-C. These findings are compatible with clinical findings where fibrosis/scarring is common with the first two but not the last two methods. In addition, all methods except ALA-PDT are associated with an increase in HSP 72 (a protein associated with cellular tolerance) and this may help to explain, at a cellular level, why resistance to repeated ALA-PDT treatments does not seem to occur.

Animals↗