Analysis of the distal urinary tract in larval and adult zebrafish reveals homology to the human system.

Little is known about the distal excretory component of the urinary tract in Danio rerio (zebrafish). This component is affected by many human diseases and disorders of development. Here, we have undertaken multi-level analyses to determine the structure and composition of the distal urinary tract in the zebrafish. In silico searches identified uroplakin 1a (ukp1a), uroplakin 2 (upk2) and uroplakin 3b (upk3b) genes in the zebrafish genome (orthologues to genes that encode urothelium-specific proteins in humans). In situ hybridization demonstrated ukp1a expression in the zebrafish pronephros and cloaca from 96 h post-fertilization. Haematoxylin and Eosin staining of adult zebrafish demonstrated two mesonephric ducts uniting into a urinary bladder that leads to a distinct urethral opening. Immunohistochemistry identified Uroplakin 1a, Uroplakin 2 and GATA3 expression in zebrafish urinary bladder cell layers that match human urothelial expression. Fluorescent dye injections demonstrated zebrafish urinary bladder function, including urine storage and intermittent micturition, and a urethral orifice separate from the larger anal canal and rectum. Our findings reveal homology between the urinary tracts of zebrafish and humans, and offer the former as a model system to study disease.

Results Of Immediate Facial Nerve Reconstruction In Patients Undergoing Parotid Tumour Resection.

BACKGROUND: Facial nerve is usually sacrificed in total parotidectomy. The objective of this study is to present results of immediate reconstruction of facial nerve in total parotidectomy cases where facial nerve is sacrificed. METHODS: This is a prospective study done in patients who had total parotidectomy including facial nerve and immediate reconstruction was done with inter-positional nerve grafts (sural n=12 and greater auricular n=10) from December 2017 till February 2018 by single surgeon (MR). Wounds were closed primarily (n=15), local flap (n=2) and free flap (n=5). Clinical evaluation was done at four months minimum follow up (those operated in January to February 2018) and eight months maximum follow up (those operated in December 2017), for facial nerve functional recovery using House and Brackmann grading system by single author (MR). RESULTS: Total of 22 (male n=7, female n=15) patients included in study from December 2017 till February 2018. Sural nerve grafts were used in 54% (n=12) and greater auricular nerve grafts in 45% (n=10) patients for reconstruction of facial nerve. On clinical evaluation using House and Brackmann grading system, showed grade V (n=4), grade IV (n=7), grade III (n=8) and grade II (n=3) repairs. CONCLUSIONS: Although primary end to end facial nerve repair is ideal but in situation where a significant segment of nerve is lost or where the repair is under tension, inter-positional nerve grafting is a simple and reliable reconstructive technique with good outcomes.

Comparison Of Microvascular Free Tissue Transfer In Adult And Paediatric Patients.

BACKGROUND: Free tissue transfer is a routine practice in adults with good success rates. Further advances in techniques and microsurgical skills have proved that free tissue transfer in paediatric population is feasible, reliable and safe. METHODS: This study is conducted to compare anastomosis duration, total general anaesthesia duration, hospital stay and outcomes of flaps (survival, partial loss, complete loss, complications) in paediatric group (age <15 years) and adult group (15-70 years age). All patients with large soft tissue defects, congenital defects, traumatic defects and post tumour extirpation were included in this study from December 1st 2017 to May 30th 2018. These patients underwent different microsurgical procedures, the reconstructive armamentarium included use of Latissimus dorsi flap, Anterolateral thigh flap, Fibula flap, Radial forearm flap, functioning Gracillis muscle, iliac crest flap, deep inferior epigastric artery perforator flap and Rectus abdominis muscle flap. Post-traumatic defects were the commonest indication of free tissue transfer in Paediatric population while post tumour extirpation defects were commonest defects encountered in adult population.. RESULTS: On average the total anaesthesia duration is slightly shorter in paediatric group than in adult patients while anastomosis duration is slightly shorter in adults then in paediatric patients. The overall complication rate is comparable in both groups and all the flaps survived well. CONCLUSIONS: Microsurgical free tissue transfer can be confidently attempted in children and their results are comparable with those of adult group.

Can The Anterolateral Thigh Flap Replace The Rectus Abdominis Free Flap In The Reconstruction Of Complex Maxillary Defects?

BACKGROUND: Maxilla is perhaps the most essential and visible part of the mid-face. It is a threedimensional structure and when reconstructing maxillectomy defects the principles of aesthetics as well as the best functional outcomes are taken into account. The aim of this study is to compare the Anterolateral Thigh Flap (ALTF) to the standard option like the Rectus Abdominis Free Flap (RAMFF) for the reconstruction of complex maxillary defects. METHODS: This descriptive case series was conducted at the Department of Plastic and Reconstructive Surgery, Shifa International Hospital Islamabad, Pakistan from 2009 to 2016. Patients of all age groups with complex maxillectomy defects, (Type III and IV according to Cordeiro classification) resulting from tumour resection, trauma, osteoradionecrosis or infection, underwent reconstruction with the free anterolateral thigh flap and the rectus abdominis free flap. RESULTS: Over a period of 8 years, 49 Rectus Abdominis free flaps and 32 Anterolateral thigh free flaps were performed for reconstruction of Type III and IV maxillectomy defects. The follow up was weekly for 1 month and then 3 monthly for the 1st year, 6 monthly for 2nd year and then yearly. All the patients had an uneventful immediate recovery. CONCLUSIONS: ALTF has advantages over the RAMFF in terms of the donor site morbidity, operative time and postoperative recovery in the reconstruction of complex maxillectomy defects.

Alzheimer's disease pathogenesis: Is there a role for folate?

Epigenetic modifications, including changes in DNA methylation, have been implicated in a wide range of diseases including neurological diseases such as Alzheimer's. The role of dietary folate in providing methyl groups required for maintenance and modulation of DNA methylation makes it a nutrient of interest in Alzheimer's. Late onset Alzheimer's disease is the most common form of dementia and at present its aetiology is largely undetermined. From epidemiological studies, the interactions between folate, B-vitamins and homocysteine as well as the long latency period has led to difficulties in interpretation of the data, thus current evidence exploring the role of dietary folate in Alzheimer's is contradictory and unresolved. Therefore, examining the effects at a molecular level and exploring potential epigenetic mechanisms could increase our understanding of the disease and aetiology. The aim of this review is to examine the role that folate could play in Alzheimer's disease neuropathology and will focus on the effects of folate on DNA methylation which link to disease pathology, initiation and progression.

Identification and diagnostic performance of a small RNA within the PCA3 and BMCC1 gene locus that potentially targets mRNA.

BACKGROUND: PCA3 is a long noncoding RNA (lncRNA) with unknown function, upregulated in prostate cancer. LncRNAs may be processed into smaller active species. We hypothesized this for PCA3. METHODS: We computed feasible RNA hairpins within the BMCC1 gene (encompassing PCA3) and searched a prostate transcriptome for these. We measured expression using qRT-PCR in three cohorts of prostate cancer tissues (n = 60), exfoliated urinary cells (n = 484 with cancer and n = 166 controls), and in cell lines (n = 22). We used in silico predictions and RNA knockup to identify potential mRNA targets of short transcribed RNAs. RESULTS: We predicted 13 hairpins, of which PCA3-shRNA2 was most abundant within the prostate transcriptome. PCA3-shRNA2 is located within intron 1 of PCA3 and appears regulated by androgens. Expression of PCA3-shRNA2 was upregulated in malignant prostatic tissues, exfoliated urinary cells from men with prostate cancer (13-273 fold change; t test P < 0.003), and closely correlated to PCA3 expression (r = 0.84-0.93; P < 0.001). Urinary PCA3-shRNA2 (C-index, 0.75-0.81) and PCA3 (C-index, 0.78) could predict the presence of cancer in most men. PCA3-shRNA2 knockup altered the expression of predicted target mRNAs, including COPS2, SOX11, WDR48, TEAD1, and Noggin. PCA3-shRNA2 expression was negatively correlated with COPS2 in patient samples (r = -0.32; P < 0.001). CONCLUSION: We identified a short RNA within PCA3, whose expression is correlated to PCA3, which may target mRNAs implicated in prostate biology. IMPACT: This short RNA is stable ex vivo, suggesting a role as a robust biomarker. We identify cytoplasmic enrichment of this RNA and potential targeting of mRNAs implicated in prostate carcinogenesis.

NMR-based evaluation of the metabolic profile and response to dichloroacetate of human prostate cancer cells.

The aim of this study was to evaluate the metabolic profile of human prostate cancer cells that have different metastatic potential and to determine their response to dichloroacetate (DCA) using NMR technology. Two isogenic human prostate cancer cell lines, differing in their metastatic potential [LNCaP (poorly metastatic) and LNCaP-LN3 (highly metastatic)], were studied. Metabolite ratios from NMR spectral integrals acquired at a field strength of 9.4 T using a 5-mm broadband probe with an NMR-compatible bioreactor were compared in the presence and absence of the pyruvate dehydrogenase kinase inhibitor DCA. Lactate dehydrogenase (LDH) isoenzymes were assessed by zymography. Following the treatment of cells with 50 mm DCA, there was a significant reduction in the lactate/choline, lactate/creatine, lactate/alanine and the combined lactate/(choline + creatine + alanine) ratios in LNCaP-LN3 cells relative to LNCaP cells. No significant changes in metabolite ratios were found in LNCaP cells following DCA treatment. As expected, LDH zymography assays showed an absence of the LDH-B subunit in LNCaP-LN3 cells, whereas both LDH-A and LDH-B subunits were present in LNCaP cells. DCA was shown to significantly modify the metabolite ratios in highly metastatic LNCaP-LN3 cells, but not in poorly metastatic LNCaP cells. This effect was probably related to the absence of the LDH-B subunit in LNCaP-LN3 cells, and could have a bearing on cancer treatment with DCA and related compounds.

Lactate dehydrogenase-B is silenced by promoter methylation in a high frequency of human breast cancers.

OBJECTIVE: Under normoxia, non-malignant cells rely on oxidative phosphorylation for their ATP production, whereas cancer cells rely on Glycolysis; a phenomenon known as the Warburg effect. We aimed to elucidate the mechanisms contributing to the Warburg effect in human breast cancer. EXPERIMENTAL DESIGN: Lactate Dehydrogenase (LDH) isoenzymes were profiled using zymography. LDH-B subunit expression was assessed by reverse transcription PCR in cells, and by Immunohistochemistry in breast tissues. LDH-B promoter methylation was assessed by sequencing bisulfite modified DNA. RESULTS: Absent or decreased expression of LDH isoenzymes 1-4, were seen in T-47D and MCF7 cells. Absence of LDH-B mRNA was seen in T-47D cells, and its expression was restored following treatment with the demethylating agent 5'Azacytadine. LDH-B promoter methylation was identified in T-47D and MCF7 cells, and in 25/25 cases of breast cancer tissues, but not in 5/5 cases of normal breast tissues. Absent immuno-expression of LDH-B protein (<10% cells stained), was seen in 23/26 (88%) breast cancer cases, and in 4/8 cases of adjacent ductal carcinoma in situ lesions. Exposure of breast cancer cells to hypoxia (1% O(2)), for 48 hours resulted in significant increases in lactate levels in both MCF7 (14.0 fold, p = 0.002), and T-47D cells (2.9 fold, p = 0.009), but not in MDA-MB-436 (-0.9 fold, p = 0.229), or MCF10AT (1.2 fold, p = 0.09) cells. CONCLUSIONS: Loss of LDH-B expression is an early and frequent event in human breast cancer occurring due to promoter methylation, and is likely to contribute to an enhanced glycolysis of cancer cells under hypoxia.

iTRAQ identification of candidate serum biomarkers associated with metastatic progression of human prostate cancer.

A major challenge in the management of patients with prostate cancer is identifying those individuals at risk of developing metastatic disease, as in most cases the disease will remain indolent. We analyzed pooled serum samples from 4 groups of patients (n = 5 samples/group), collected prospectively and actively monitored for a minimum of 5 yrs. Patients groups were (i) histological diagnosis of benign prostatic hyperplasia with no evidence of cancer 'BPH', (ii) localised cancer with no evidence of progression, 'non-progressing' (iii) localised cancer with evidence of biochemical progression, 'progressing', and (iv) bone metastasis at presentation 'metastatic'. Pooled samples were immuno-depleted of the 14 most highly abundant proteins and analysed using a 4-plex iTRAQ approach. Overall 122 proteins were identified and relatively quantified. Comparisons of progressing versus non-progressing groups identified the significant differential expression of 25 proteins (p<0.001). Comparisons of metastatic versus progressing groups identified the significant differential expression of 23 proteins. Mapping the differentially expressed proteins onto the prostate cancer progression pathway revealed the dysregulated expression of individual proteins, pairs of proteins and 'panels' of proteins to be associated with particular stages of disease development and progression. The median immunostaining intensity of eukaryotic translation elongation factor 1 alpha 1 (eEF1A1), one of the candidates identified, was significantly higher in osteoblasts in close proximity to metastatic tumour cells compared with osteoblasts in control bone (p = 0.0353, Mann Whitney U). Our proteomic approach has identified leads for potentially useful serum biomarkers associated with the metastatic progression of prostate cancer. The panels identified, including eEF1A1 warrant further investigation and validation.

Eight-plex iTRAQ analysis of variant metastatic human prostate cancer cells identifies candidate biomarkers of progression: An exploratory study.

BACKGROUND: Due to the heterogeneity in the biological behavior of prostate cancer, biomarkers that can reliably distinguish indolent from aggressive disease are urgently needed to inform treatment choices. METHODS: We employed 8-plex isobaric Tags for Relative and Absolute Quantitation (iTRAQ), to profile the proteomes of two distinct panels of isogenic prostate cancer cells with varying growth and metastatic potentials, in order to identify novel biomarkers associated with progression. The LNCaP, LNCaP-Pro5, and LNCaP-LN3 panel of cells represent a model of androgen-responsive prostate cancer, while the PC-3, PC-3M, and PC-3M-LN4 panel represent a model of androgen-insensitive disease. RESULTS: Of the 245 unique proteins identified and quantified (>or=95% confidence; >or=2 peptides/protein), 17 showed significant differential expression (>or=+/-1.5), in at least one of the variant LNCaP cells relative to parental cells. Similarly, comparisons within the PC-3 panel identified 45 proteins to show significant differential expression in at least one of the variant PC-3 cells compared with parental cells. Differential expression of selected candidates was verified by Western blotting or immunocytochemistry, and corresponding mRNA expression was determined by quantitative real-time PCR (qRT-PCR). Immunostaining of prostate tissue microarrays for ERp5, one of the candidates identified, showed a significant higher immunoexpression in pre-malignant lesions compared with non-malignant epithelium (P < 0.0001, Mann-Whitney U-test), and in high Gleason grade (4-5) versus low grade (2-3) cancers (P < 0.05). CONCLUSIONS: Our study provides proof of principle for the application of an 8-plex iTRAQ approach to uncover clinically relevant candidate biomarkers for prostate cancer progression.

Evaluation of the frequency of putative prostate cancer stem cells in primary and metastatic prostate cancer.

BACKGROUND: Tumour cells with a stem cell-like phenotype have recently been identified in prostate tumors and it has been suggested that this population may be responsible for the diversity of cell types within tumors and also for the initiation of metastases. These cells carry a number of defined markers: they are cd133 and cd44+ve and express high levels of alpha2beta1 integrin. In this study we have, for the first time, assessed matched primary and bone marrow biopsies from prostate cancer patients for the distribution of cells carrying these and a number of other putative stem cell markers. METHODS: Eleven matched (primary and bone metastasis) specimens from prostate cancer patients were assessed for the presence of cd133, cd44, alpha2beta1 integrin, CXCR4, c-met, alpha6 integrin, and nestin using immunohistochemistry and stain intensity and distribution scored. RESULTS: In the bone metastases, tumor cells staining positively for cd133 were detected at low frequency in approximately 50% of samples. Staining for nestin was confined to endothelium. Positive staining of tumor cells for the other antigens was present at variable frequency in >70% of metastases with the exception of CXCR4 which was absent from all but 2 specimens. Where positive staining of tumor cells was present in the metastasis, cells staining for each antigen were present in the matched primary with the exception of cd44 which was absent in all but 2/11 matched primary tissues. CONCLUSIONS: In established metastases no single or combination of marker expression profiles identify the established metastatic phenotype, although cd44 expression was shown to be more frequent in metastases that in primary cancers.

The application of artificial intelligence to microarray data: identification of a novel gene signature to identify bladder cancer progression.

BACKGROUND: New methods for identifying bladder cancer (BCa) progression are required. Gene expression microarrays can reveal insights into disease biology and identify novel biomarkers. However, these experiments produce large datasets that are difficult to interpret. OBJECTIVE: To develop a novel method of microarray analysis combining two forms of artificial intelligence (AI): neurofuzzy modelling (NFM) and artificial neural networks (ANN) and validate it in a BCa cohort. DESIGN, SETTING, AND PARTICIPANTS: We used AI and statistical analyses to identify progression-related genes in a microarray dataset (n=66 tumours, n=2800 genes). The AI-selected genes were then investigated in a second cohort (n=262 tumours) using immunohistochemistry. MEASUREMENTS: We compared the accuracy of AI and statistical approaches to identify tumour progression. RESULTS AND LIMITATIONS: AI identified 11 progression-associated genes (odds ratio [OR]: 0.70; 95% confidence interval [CI], 0.56-0.87; p=0.0004), and these were more discriminate than genes chosen using statistical analyses (OR: 1.24; 95% CI, 0.96-1.60; p=0.09). The expression of six AI-selected genes (LIG3, FAS, KRT18, ICAM1, DSG2, and BRCA2) was determined using commercial antibodies and successfully identified tumour progression (concordance index: 0.66; log-rank test: p=0.01). AI-selected genes were more discriminate than pathologic criteria at determining progression (Cox multivariate analysis: p=0.01). Limitations include the use of statistical correlation to identify 200 genes for AI analysis and that we did not compare regression identified genes with immunohistochemistry. CONCLUSIONS: AI and statistical analyses use different techniques of inference to determine gene-phenotype associations and identify distinct prognostic gene signatures that are equally valid. We have identified a prognostic gene signature whose members reflect a variety of carcinogenic pathways that could identify progression in non-muscle-invasive BCa.

Distinct microRNA alterations characterize high- and low-grade bladder cancer.

Urothelial carcinoma of the bladder (UCC) is a common disease that arises by at least two different molecular pathways. The biology of UCC is incompletely understood, making the management of this disease difficult. Recent evidence implicates a regulatory role for microRNA in cancer. We hypothesized that altered microRNA expression contributes to UCC carcinogenesis. To test this hypothesis, we examined the expression of 322 microRNAs and their processing machinery in 78 normal and malignant urothelial samples using real-time rtPCR. Genes targeted by differentially expressed microRNA were investigated using real-time quantification and microRNA knockdown. We also examined the role of aberrant DNA hypermethylation in microRNA downregulation. We found that altered microRNA expression is common in UCC and occurs early in tumorogenesis. In normal urothelium from patients with UCC, 11% of microRNAs had altered expression when compared with disease-free controls. This was associated with upregulation of Dicer, Drosha, and Exportin 5. In UCC, microRNA alterations occur in a tumor phenotype-specific manner and can predict disease progression. High-grade UCC were characterized by microRNA upregulation, including microRNA-21 that suppresses p53 function. In low-grade UCC, there was downregulation of many microRNA molecules. In particular, loss of microRNAs-99a/100 leads to upregulation of FGFR3 before its mutation. Promoter hypermethylation is partly responsible for microRNA downregulation. In conclusion, distinct microRNA alterations characterize UCC and target genes in a pathway-specific manner. These data reveal new insights into the disease biology and have implications regarding tumor diagnosis, prognosis and therapy.

iTRAQ underestimation in simple and complex mixtures: "the good, the bad and the ugly".

The increasing popularity of iTRAQ for quantitative proteomics applications makes it necessary to evaluate its relevance, accuracy, and precision for biological interpretation. Here, we have assessed (a) the accuracy and precision of iTRAQ quantification in a controlled experimental setup, using low- and high-complexity protein mixtures; and (b) the potential pitfalls that hamper the applicability and attainable dynamic range of iTRAQ: isotopic contamination, background interference, and signal-to-noise ratio. Our data suggest greater dynamic crosstalk between interfering factors affecting underestimations, and that these interferences were largely scenario-specific, dependent on sample complexity. The good is the potential for iTRAQ to provide accurate quantification spanning 2 orders of magnitude. This potential is however limited by two factors. (1) The bad: the existence of isotopic impurities that can be corrected for; provided accurate isotopic factors are at one's disposal. (2) The ugly: we demonstrate here the interference of mixed MS/MS contribution occurring during precursor selection, an issue that is currently very difficult to minimize. In light of our results, we propose a list of advice for iTRAQ data analysis that could routinely ameliorate quantitative interpretation of proteomic data sets.

FGFR3 mutations indicate better survival in invasive upper urinary tract and bladder tumours.

BACKGROUND: Promoter hypermethylation and microsatellite instability are frequent in tumours of the upper urinary tract (UTT) and infrequent in bladder tumours. FGFR3 mutations are common findings in bladder tumours and are associated with a good prognosis. OBJECTIVE: To investigate the occurrence of FGFR3 mutations in UTT and determine the prognostic effect of these genetic changes. DESIGN, SETTING, AND PARTICIPANTS: Tissue from the initial tumour was obtained from 280 patients (117 bladder tumours and 163 UTT). Patients were selected from pathologic archives to represent the disease spectrum of UCC throughout the urinary tract. Following UCC excision, patients underwent surveillance for a median of 56 mo (range 1-216 mo) or until death. MEASUREMENTS: FGFR3 mutation analysis was successfully performed on 252 of the 280 primary tumours using the SNaPshot method. Two-tailed statistical analyses were done using the chi(2), Fisher exact tests, and log rank tests. Cox proportional hazard ratios were estimated to obtain risks of recurrence, progression, and death, and to find independent prognostic factors in a multivariate model. RESULTS AND LIMITATIONS: FGFR3 mutations occurred with the same frequency in bladder and upper tract tumours. Mutations were associated with low-stage tumours and a milder disease course in bladder, ureter, and renal pelvis tumours. Strikingly, our data suggest that these mutations indicate a better survival in patients with invasive tumours from the bladder and upper urinary tract. CONCLUSIONS: FGFR3 mutation status might be used to select patients with invasive UCC who have a lower risk of death.

Prostate cancer proteomics: The urgent need for clinically validated biomarkers.

Prostate cancer (PCa) is the most common cancer diagnosis and the second most common cause of cancer-related deaths in men. Currently, serum prostate-specific antigen (PSA) is the only biomarker widely used in the diagnosis and management of patients with PCa. However, PSA lacks diagnostic sensitivity and specificity, leading to false-negative and false-positive test results. PSA cannot distinguish indolent from aggressive disease, leading to many patients being over-treated with associated side-effects. Furthermore, PSA is unable to identify which tumors are likely to become unresponsive to treatment at an early stage. Thus, there is an urgent need for clinically validated biomarkers which will improve the diagnosis and management of PCa. Given the heterogeneity of PCa it is likely that a panel of biomarkers will be required. In the quest for PCa biomarkers, a wide range of samples including urine, serum, tissues, and cell lines have been studied using proteomic approaches such as 2-DE, SELDI-TOF, SILAC, ICAT, iTRAQ, and MALDI-IMS. The value of these technologies, and other emerging platforms such as selected reaction monitoring (SRM) and multiple reaction monitoring (MRM), are discussed in the context of biomarker discovery, validation and addressing the "bottle-necks" that exist prior to clinical translation.

iTRAQ-facilitated proteomic analysis of human prostate cancer cells identifies proteins associated with progression.

The unpredictable behavior of prostate cancer presents a major clinical challenge during patient management. In order to gain an insight into the molecular mechanisms associated with prostate cancer progression, we employed the shot-gun proteomic approach of isobaric tags for relative and absolute quantitation (iTRAQ), followed by 2D-LC-MS/MS, using the poorly metastatic LNCaP cell line and its highly metastatic variant LNCaP-LN3 cell line as a model. A total number of 280 unique proteins were identified (> or =95% confidence), and relative expression data was obtained for 176 of these. Ten proteins were found to be significantly up-regulated (> or =1.50 fold), while 4 proteins were significantly down-regulated (> or = -1.50 fold), in LNCaP-LN3 cells. Differential expression of brain creatine kinase (CKBB), soluble catechol-O-methyltransferase (S-COMT), tumor rejection antigen (gp96), and glucose regulated protein, 78 kDa (grp78), was confirmed by Western blotting or independent 2D-PAGE analysis. Additionally, iTRAQ analysis identified absence of the lactate dehydrogenase-B (LDH-B) subunit in LNCaP-LN3 cells, confirming our published data. The clinical relevance of gp96 was assessed by immunohistochemistry using prostate tissues from benign ( n = 95), malignant ( n = 66), and metastatic cases ( n = 3). Benign epithelium showed absent/weak gp96 expression in the basal cells, in contrast to the moderate/strong expression seen in malignant epithelium. Furthermore, there was a statistically significant difference in the intensity of gp96 expression between benign and malignant cases ( p < 0.0005, Mann-Whitney U). Our study is the first to report the application of iTRAQ technology and its potential for the global proteomic profiling of prostate cancer cells, including the identification of absent protein expression.

Promoter hypermethylation in circulating blood cells identifies prostate cancer progression.

Promoter hypermethylation of circulating cell DNA has been advocated as a diagnostic marker for prostate cancer, but its prognostic use is currently unclear. To assess this role, we compared hypermethylation of circulating cell DNA from prostate cancer patients with (Group 1, n = 20) and without (Group 2, n = 22) disease progression and age-matched controls (benign prostatic hyperplasia, Group 3, n = 22). We measured hypermethylation of 10 gene promoters in 2 sequential venous samples, obtained at diagnosis and during disease progression (median time, 15 months later). Matched time samples were obtained in the nonprogressing patients. We found that more hypermethylation was detected in the diagnostic sample from the patients with cancer than in controls for GSTP1, RASSF1 alpha, APC and RAR beta (p < 0.0001). Patients undergoing disease progression had a significant increase in methylation levels of these 4 genes when compared to the other patients (p < 0.001). Patients at risk of disease progression have higher detectable concentrations of circulating cell hypermethylation, than those without progression. The extent of this hypermethylation increases during disease progression and can be used to identify the extent and duration of treatment response in prostate cancer.

Human prostate cancer cells express neuroendocrine cell markers PGP 9.5 and chromogranin A.

BACKGROUND: A proportion of men with prostate cancer will progress to develop metastatic disease involving the lymph-nodes and bone. To identify novel candidates associated with metastatic progression, we compared the proteomic profiles of LNCaP (lymph-node metastatic, androgen-dependant) and PC-3 (bone metastatic, androgen-independent), human prostate cancer cells. METHODS: Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), followed by electrospray ionisation tandem mass spectrometry (ESI-MS/MS), was used to identify differentially expressed proteins. Western blotting was used to validate the identity of any candidates. Immunohistochemistry was used to assess tissue expression. RESULTS: 2D-PAGE followed by ESI-MS/MS analyses identified the expression of glutathione S-transferase-pi (GST-pi) and protein gene product 9.5 (PGP 9.5) in PC-3 cells, but absent expression in LNCaP cells. PGP 9.5 expression in PC-3 cells was confirmed by Western blotting, in addition to expression in DU145 cells. Analysis of cell conditioned media showed that PGP 9.5 was secreted. Sequencing of the PGP 9.5 gene promoter region in bisulfite modified DNA, suggested that the regulation of expression involves promoter hypermethylation. RT-PCR analysis for Chromogranin A (ChA) mRNA (a marker of neuroendocrine cells), showed expression in PC-3 and DU145 cells but was undetectable in LNCaP cells. Immunohistochemistry localised PGP 9.5 expression exclusively within neuroendocrine cells and nerve fibres. CONCLUSIONS: Our unexpected finding that the neuroendocrine cell markers PGP 9.5 and ChA are expressed by PC-3 and DU145 cells, suggests that these cells may have been derived from metastatic adenocarcinomas which had undergone neuroendocrine differentiation or alternatively the expression occurred ectopically as a result of cell culture.

The antibody MAB8051 directed against osteoprotegerin detects carbonic anhydrase II: implications for association studies with human cancers.

A commonly used monoclonal antibody targeting osteoprotegerin (OPG), MAB8051, detects a truncated protein species in breast and prostate cancer cell lysates. OPG expression has been reported to contribute to cell survival of both of these cancers. We hypothesised that the truncated protein represented a unique tumour-associated OPG isoform. However, here we show that the truncated protein identified by MAB8051 in cancer cell lines is carbonic anhydrase II (CA II), also implicated in tumour biology. We clearly demonstrate cross-reactivity of this OPG antibody in western blots. OPG and CA II RNA-interference studies confirmed the identity of the bands. We show almost identical staining patterns between MAB8051 and CA II immunohistochemistry of different human tissue types and human tumour types using serial sections. We conclude that care should be exercised using this antibody for immunohistochemistry studies, without additional in situ hybridisation, or parallel use of other OPG-specific antibodies.