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1.  RNA-seq profiling of a radiation resistant and radiation sensitive prostate cancer cell line highlights opposing regulation of DNA repair and targets for radiosensitization 
BMC Cancer  2014;14(1):808.
Radiotherapy is a chosen treatment option for prostate cancer patients and while some tumours respond well, up to 50% of patients may experience tumour recurrence. Identification of functionally relevant predictive biomarkers for radioresponse in prostate cancer would enable radioresistant patients to be directed to more appropriate treatment options, avoiding the side-effects of radiotherapy.
Using an in vitro model to screen for novel biomarkers of radioresistance, transcriptome analysis of a radioresistant (PC-3) and radiosensitive (LNCaP) prostate cancer cell line was performed. Following pathway analysis candidate genes were validated using qRT-PCR. The DNA repair pathway in radioresistant PC-3 cells was then targeted for radiation sensitization using the PARP inhibitor, niacinimide.
Opposing regulation of a DNA repair and replication pathway was observed between PC-3 and LNCaP cells from RNA-seq analysis. Candidate genes BRCA1, RAD51, FANCG, MCM7, CDC6 and ORC1 were identified as being significantly differentially regulated post-irradiation. qRT-PCR validation confirmed BRCA1, RAD51 and FANCG as being significantly differentially regulated at 24 hours post radiotherapy (p-value =0.003, 0.045 and 0.003 respectively). While the radiosensitive LNCaP cells down-regulated BRCA1, FANCG and RAD51, the radioresistant PC-3 cell line up-regulated these candidates to promote cell survival post-radiotherapy and a similar trend was observed for MCM7, CDC6 and ORC1. Inhibition of DNA repair using niacinamide sensitised the radioresistant cells to irradiation, reducing cell survival at 2 Gy from 66% to 44.3% (p-value =0.02).
These findings suggest that the DNA repair candidates identified via RNA-seq hold potential as both targets for radiation sensitization and predictive biomarkers in prostate cancer.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-808) contains supplementary material, which is available to authorized users.
PMCID: PMC4233036  PMID: 25369795
Radiation; Prostate cancer; RNA-seq; DNA repair; Sensitization
2.  Gene expression of muscarinic, tachykinin, and purinergic receptors in porcine bladder: comparison with cultured cells 
Urothelial cells, myofibroblasts, and smooth muscle cells are important cell types contributing to bladder function. Multiple receptors including muscarinic (M3/M5), tachykinin (NK1/NK2), and purinergic (P2X1/P2Y6) receptors are involved in bladder motor and sensory actions. Using female pig bladder, our aim was to differentiate between various cell types in bladder by genetic markers. We compared the molecular expression pattern between the fresh tissue layers and their cultured cell counterparts. We also examined responses to agonists for these receptors in cultured cells. Urothelial, suburothelial (myofibroblasts), and smooth muscle cells isolated from pig bladder were cultured (10–14 days) and identified by marker antibodies. Gene (mRNA) expression level was demonstrated by real-time PCR. The receptor expression pattern was very similar between suburothelium and detrusor, and higher than urothelium. The gene expression of all receptors decreased in culture compared with the fresh tissue, although the reduction in cultured urothelial cells appeared less significant compared to suburothelial and detrusor cells. Cultured myofibroblasts and detrusor cells did not contract in response to the agonists acetylcholine, neurokinin A, and β,γ-MeATP, up to concentrations of 0.1 and 1 mM. The significant reduction of M3, NK2, and P2X1 receptors under culture conditions may be associated with the unresponsiveness of cultured suburothelial and detrusor cells to their respective agonists. These results suggest that under culture conditions, bladder cells lose the receptors that are involved in contraction, as this function is no longer required. The study provides further evidence that cultured cells do not necessarily mimic the actions exerted by intact tissues.
PMCID: PMC3842897  PMID: 24348420
smooth muscle; urothelium; suburothelium; myofibroblasts; muscarinic receptors; tachykinin receptors; purinergic receptors; cell culture
3.  Host-Pathogen Checkpoints and Population Bottlenecks in Persistent and Intracellular Uropathogenic E. coli Bladder Infection 
FEMS microbiology reviews  2012;36(3):616-648.
Bladder infections affect millions of people yearly, and recurrent symptomatic infections (cystitis) are very common. The rapid increase in infections caused by multi-drug resistant uropathogens threatens to make recurrent cystitis an increasingly troubling public health concern. Uropathogenic E. coli (UPEC) cause the vast majority of bladder infections. Upon entry into the lower urinary tract, UPEC face obstacles to colonization that constitute population bottlenecks, reducing diversity and selecting for fit clones. A critical mucosal barrier to bladder infection is the epithelium (urothelium). UPEC bypass this barrier when they invade urothelial cells and form intracellular bacterial communities (IBCs), a process which requires type 1 pili. IBCs are transient in nature, occurring primarily during acute infection. Chronic bladder infection is common and can be either latent, in the form of the Quiescent Intracellular Reservoir (QIR), or active, in the form of asymptomatic bacteriuria (ASB/ABU) or chronic cystitis. In mice, the fate of bladder infection: QIR, ASB, or chronic cystitis, is determined within the first 24 hours of infection and constitutes a putative host-pathogen mucosal checkpoint that contributes to susceptibility to recurrent cystitis. Knowledge of these checkpoints and bottlenecks is critical for our understanding of bladder infection and efforts to devise novel therapeutic strategies.
PMCID: PMC3675774  PMID: 22404313
cystitis; urinary tract infection; UPEC; type 1 pili; intracellular bacterial communities; antibiotic resistance
5.  Depletion of c-Rel from Cytokine Gene Promoters Is Required for Chromatin Reassembly and Termination of Gene Responses to T Cell Activation 
PLoS ONE  2012;7(7):e41734.
The role of the Nuclear Factor κB (NF-κB) transcription factor family in T cell function has been well described. The c-Rel family member is of particular importance in initiating T cell responses to antigen and regulating activation of inflammatory cytokine genes, including the Interleukin-2 (IL-2) and Granulocyte macrophage colony stimulating factor (GM-CSF) genes. c-Rel is required for chromatin remodeling of these gene promoters, which involves depletion of histones from the promoters in response to T cell activating signals. These chromatin remodeling events precede transcriptional activation of the genes. The subsequent down-regulation of cytokine gene expression is important in the termination of an immune response and here we examine this process at the murine GM-CSF and IL-2 genes. We show that the cytokine mRNA levels rapidly return to basal levels following stimulus removal and this is associated with reassembly of histones onto the promoter. Histone reassembly at the GM-CSF and IL-2 promoters occurs concomitantly with depletion of RelA, c-Rel and RNA polymerase II from the promoters. Furthermore we show that transcriptional down-regulation and chromatin reassembly is dependent on depletion of c-Rel from the nucleus, and that this is regulated by the nuclear translocation of the NF-κB inhibitor, IκBα. The nuclear activation of c-Rel therefore not only regulates the initiation of GM-CSF and IL-2 gene activation in response to T cell activation, but also the termination of these gene responses following the removal of the activating signal.
PMCID: PMC3408492  PMID: 22860011
6.  Porcine Bladder Urothelial, Myofibroblast, and Detrusor Muscle Cells: Characterization and ATP Release 
ATP is released from the bladder mucosa in response to stretch, but the cell types responsible are unclear. Our aim was to isolate and characterize individual populations of urothelial, myofibroblast, and detrusor muscle cells in culture, and to examine agonist-stimulated ATP release. Using female pig bladders, urothelial cells were isolated from bladder mucosa following trypsin-digestion of the luminal surface. The underlying myofibroblast layer was dissected, minced, digested, and cultured until confluent (10–14 days). A similar protocol was used for muscle cells. Cultures were used for immunocytochemical staining and/or ATP release investigations. In urothelial cultures, immunoreactivity was present for the cytokeratin marker AE1/AE3 but not the contractile protein α-smooth muscle actin (α-SMA) or the cytoskeletal filament vimentin. Neither myofibroblast nor muscle cell cultures stained for AE1/AE3. Myofibroblast cultures partially stained for α-SMA, whereas muscle cultures were 100% stained. Both myofibroblast and muscle stained for vimentin, however, they were morphologically distinct. Ultrastructural studies verified that the suburothelial layer of pig bladder contained abundant myofibroblasts, characterized by high densities of rough endoplasmic reticulum. Baseline ATP release was higher in urothelial and myofibroblast cultures, compared with muscle. ATP release was significantly stimulated by stretch in all three cell populations. Only urothelial cells released ATP in response to acid, and only muscle cells were stimulated by capsaicin. Tachykinins had no effect on ATP release. In conclusion, we have established a method for culture of three cell populations from porcine bladder, a well-known human bladder model, and shown that these are distinct morphologically, immunologically, and pharmacologically.
PMCID: PMC3113165  PMID: 21713125
ATP; porcine; bladder; urothelium; myofibroblast; detrusor; ultrastructure
7.  Immunocytochemical characterisation of cultures of human bladder mucosal cells 
BMC Urology  2011;11:5.
The functional role of the bladder urothelium has been the focus of much recent research. The bladder mucosa contains two significant cell types: urothelial cells that line the bladder lumen and suburothelial interstitial cells or myofibroblasts. The aims of this study were to culture these cell populations from human bladder biopsies and to perform immunocytochemical characterisation.
Primary cell cultures were established from human bladder biopsies (n = 10). Individual populations of urothelial and myofibroblast-like cells were isolated using magnetic activated cell separation (MACS). Cells were slow growing, needing 3 to 5 weeks to attain confluence.
Cytokeratin 20 positive cells (umbrella cells) were isolated at primary culture and also from patients' bladder washings but these did not proliferate. In primary culture, proliferating cells demonstrated positive immunocytochemical staining to cytokeratin markers (AE1/AE3 and A0575) as well fibroblasts (5B5) and smooth muscle (αSMA) markers. An unexpected finding was that populations of presumptive urothelial and myofibroblast-like cells, isolated using the MACS beads, stained for similar markers. In contrast, staining for cytokeratins and fibroblast or smooth muscle markers was not co-localised in full thickness bladder sections.
Our results suggest that, in culture, bladder mucosal cells may undergo differentiation into a myoepithelial cell phenotype indicating that urothelial cells have the capacity to respond to environmental changes. This may be important pathologically but also suggests that studies of the physiological function of these cells in culture may not give a reliable indicator of human physiology.
PMCID: PMC3104367  PMID: 21496348
urothelial cells; myofibroblasts; immunocytochemistry; human
8.  Correction: LINE Retrotransposon RNA Is an Essential Structural and Functional Epigenetic Component of a Core Neocentromeric Chromatin 
PLoS Genetics  2009;5(2):10.1371/annotation/3b497aec-b7d3-442e-9086-751251f649dd.
PMCID: PMC2665016
9.  LINE Retrotransposon RNA Is an Essential Structural and Functional Epigenetic Component of a Core Neocentromeric Chromatin 
PLoS Genetics  2009;5(1):e1000354.
We have previously identified and characterized the phenomenon of ectopic human centromeres, known as neocentromeres. Human neocentromeres form epigenetically at euchromatic chromosomal sites and are structurally and functionally similar to normal human centromeres. Recent studies have indicated that neocentromere formation provides a major mechanism for centromere repositioning, karyotype evolution, and speciation. Using a marker chromosome mardel(10) containing a neocentromere formed at the normal chromosomal 10q25 region, we have previously mapped a 330-kb CENP-A–binding domain and described an increased prevalence of L1 retrotransposons in the underlying DNA sequences of the CENP-A–binding clusters. Here, we investigated the potential role of the L1 retrotransposons in the regulation of neocentromere activity. Determination of the transcriptional activity of a panel of full-length L1s (FL-L1s) across a 6-Mb region spanning the 10q25 neocentromere chromatin identified one of the FL-L1 retrotransposons, designated FL-L1b and residing centrally within the CENP-A–binding clusters, to be transcriptionally active. We demonstrated the direct incorporation of the FL-L1b RNA transcripts into the CENP-A–associated chromatin. RNAi-mediated knockdown of the FL-L1b RNA transcripts led to a reduction in CENP-A binding and an impaired mitotic function of the 10q25 neocentromere. These results indicate that LINE retrotransposon RNA is a previously undescribed essential structural and functional component of the neocentromeric chromatin and that retrotransposable elements may serve as a critical epigenetic determinant in the chromatin remodelling events leading to neocentromere formation.
Author Summary
The centromere is an essential chromosomal structure for the correct segregation of chromosomes during cell division. Normal human centromeres comprise a 171-bp α-satellite DNA arranged into tandem and higher-order arrays. Neocentromeres are fully functional centromeres that form epigenetically on noncentromeric regions of the chromosomes, with recent evidence indicating an important role they play in centromere repositioning, karyotype evolution, and speciation. Neocentromeres contain fully definable DNA sequences and provide a tractable system for the molecular analysis of the centromere chromatin. Here, the authors investigate the role of epigenetic determinants in the regulation of neocentromere structure and function. They identify that a retrotransposable DNA element found within the neocentromere domain is actively transcribed and that the transcribed RNA is essential for the structural and functional integrity of the neocentromere. This study defines a previously undescribed epigenetic determinant that regulates the neocentromeric chromatin and provides insight into the mechanism of neocentromere formation and centromere repositioning.
PMCID: PMC2625447  PMID: 19180186
10.  GM-CSF promoter chromatin remodelling and gene transcription display distinct signal and transcription factor requirements 
Nucleic Acids Research  2005;33(1):225-234.
Granulocyte-macrophage colony stimulating factor (GM-CSF) plays a key role in myeloid cell function and is rapidly and transiently expressed in T cells in response to immune or inflammatory stimuli. Induction of GM-CSF gene expression is accompanied by changes in chromatin structure across the proximal promoter region of the gene. We show that the promoter remodelling and subsequent gene transcription occurs with distinct signal and transcription factor requirements. Activation of the protein kinase C (PKC) signalling pathway is sufficient to induce changes in chromatin structure across the promoter, but both the PKC and calcium signalling pathways are required for efficient gene transcription. Although NFAT transcription factors contribute to GM-CSF gene transcription, they are not required for promoter remodelling. However, the presence of the nuclear factor-κB transcription factor, c-Rel, in the nucleus is strongly correlated with and required for the events of chromatin remodelling.
PMCID: PMC546149  PMID: 15647505

Results 1-10 (10)