A novel method for the establishment of autologous skin cell suspensions: characterisation of cellular sub-populations, epidermal stem cell content and wound response-enhancing biological properties.

Introduction: Autologous cell suspension (ACS)-based therapy represents a highly promising approach for burns and chronic wounds. However, existing technologies have not achieved the desired clinical success due to several limitations. To overcome practical and cost-associated obstacles of existing ACS methods, we have established a novel methodology for rapid, enzymatic disaggregation of human skin cells and their isolation using a procedure that requires no specialist laboratory instrumentation and is performed at room temperature. Methods: Cells were isolated using enzymatic disaggregation of split-thickness human skin followed by several filtration steps for isolation of cell populations, and cell viability was determined. Individual population recovery was confirmed in appropriate culture medium types, and the presence of epidermal stem cells (EpSCs) within keratinocyte sub-populations was defined by flow cytometry via detection of CD49 and CD71. Positive mediators of wound healing secreted by ACS-derived cultures established on a collagen-based wound-bed mimic were detected by proteome arrays and quantified by ELISA, and the role of such mediators was determined by cell proliferation assays. The effect of ACS-derived conditioned-medium on myofibroblasts was investigated using an in-vitro model of myofibroblast differentiation via detection of α-SMA using immunoblotting and immunofluorescence microscopy. Results: Our methodology permitted efficient recovery of keratinocytes, fibroblasts and melanocytes, which remained viable upon long-term culture. ACS-derivatives comprised sub-populations with the CD49-high/CD71-low expression profile known to demarcate EpSCs. Via secretion of mitogenic factors and wound healing-enhancing mediators, the ACS secretome accelerated keratinocyte proliferation and markedly curtailed cytodifferentiation of myofibroblasts, the latter being key mediators of fibrosis and scarring. Discussion: The systematic characterisation of the cell types within our ACS isolates provided evidence for their superior cell viability and the presence of EpSCs that are critical drivers of wound healing. We defined the biological properties of ACS-derived keratinocytes, which include ability to secrete positive mediators of wound healing as well as suppression of myofibroblast cytodifferentiation. Thus, our study provides several lines of evidence that the established ACS isolates comprise highly-viable cell populations which can physically support wound healing and possess biological properties that have the potential to enhance not only the speed but also the quality of wound healing.

TRAF3/p38-JNK Signalling Crosstalk with Intracellular-TRAIL/Caspase-10-Induced Apoptosis Accelerates ROS-Driven Cancer Cell-Specific Death by CD40.

The capacity to induce tumour-cell specific apoptosis represents the most unique feature of the TNF receptor (TNFR) family member CD40. Recent studies on the signalling events triggered by its membrane-presented ligand CD40L (mCD40L) in normal and malignant epithelial cells have started to unravel an exquisite context and cell type specificity for the functional effects of CD40. Here, we demonstrate that, in comparison to other carcinomas, mCD40L triggered strikingly more rapid apoptosis in colorectal carcinoma (CRC) cells, underpinned by its ability to entrain two concurrently operating signalling axes. CD40 ligation initially activates TNFR-associated factor 3 (TRAF3) and subsequently NADPH oxidase (NOX)/Apoptosis signal-regulating kinase 1 (ASK1)-signalling and induction of reactive oxygen species (ROS) to mediate p38/JNK- and ROS-dependent cell death. At that point, p38/JNK signalling directly activates the mitochondrial pathway, and triggers rapid induction of intracellular TNF-related apoptosis-inducing ligand (TRAIL) that signals from internal compartments to initiate extrinsic caspase-10-asscociated apoptosis, leading to truncated Bid (tBid)-activated mitochondrial signalling. p38 and JNK are essential both for direct mitochondrial apoptosis induction and the TRAIL/caspase-10/tBid pathway, but their involvement follows functional hierarchy and temporally controlled interplay, as p38 function is required for JNK phosphorylation. By engaging both intrinsic and extrinsic pathways to activate apoptosis via two signals simultaneously, CD40 can accelerate CRC cell death. Our findings further unravel the multi-faceted properties of the CD40/mCD40L dyad, highlighted by the novel TNFR crosstalk that accelerates tumour cell-specific death, and may have implications for the use of CD40 as a therapeutic target.

Cooling-mediated protection from chemotherapy drug-induced cytotoxicity in human keratinocytes by inhibition of cellular drug uptake.

Chemotherapy-induced alopecia (CIA) represents the most distressing side-effect for cancer patients. Scalp cooling is currently the only treatment to combat CIA, yet little is known about its cytoprotective effects in human hair follicles (HF). We have previously established in vitro human keratinocyte models to study the effects of taxanes and anthracyclines routinely-used clinically and reported that cooling markedly-reduced or even completely-prevented cytotoxicity in a temperature dependent manner. Using these models (including HF-derived primary keratinocytes), we now demonstrate that cooling markedly attenuates cellular uptake of the anthracyclines doxorubicin and epirubicin to reduce or prevent drug-mediated human keratinocyte cytotoxicity. We show marked reduction in drug uptake and nuclear localization qualitatively by fluorescence microscopy. We have also devised a flow cytometry-based methodology that permitted semi-quantitative analysis of differences in drug uptake, which demonstrated that cooling can reduce drug uptake by up to ~8-fold in comparison to normal/physiological temperature, an effect that was temperature-dependent. Our results provide evidence that attenuation of cellular drug uptake represents at least one of the mechanisms underpinning the ability of cooling to rescue human keratinocytes from chemotherapy drug-cytotoxicity, thus supporting the clinical efficacy of scalp cooling.

CD40 induces renal cell carcinoma-specific differential regulation of TRAF proteins, ASK1 activation and JNK/p38-mediated, ROS-dependent mitochondrial apoptosis.

A unique feature of CD40 among the TNF receptor (TNFR) superfamily is its exquisitely contextual effects, as originally demonstrated in normal and malignant B-lymphocytes. We studied renal cell carcinoma (RCC) in comparison to normal (human renal proximal tubule) cells, as a model to better understand the role of CD40 in epithelial cells. CD40 ligation by membrane-presented CD40 ligand (mCD40L), but not soluble CD40 agonist, induced extensive apoptosis in RCC cells; by contrast, normal cells were totally refractory to mCD40L. These findings underline the importance of CD40 'signal-quality' on cell fate and explain the lack of pro-apoptotic effects in RCC cells previously, while confirming the tumour specificity of CD40 in epithelial cells. mCD40L differentially regulated TRAF expression, causing sustained TRAF2/TRAF3 induction in RCC cells, yet downregulation of TRAF2 and no TRAF3 induction in normal cells, observations strikingly reminiscent of TRAF modulation in B-lymphocytes. mCD40L triggered reactive oxygen species (ROS) production, critical in apoptosis, and NADPH oxidase (Nox)-subunit p40phox phosphorylation, with Nox blockade abrogating apoptosis thus implying Nox-dependent initial ROS release. mCD40L mediated downregulation of Thioredoxin-1 (Trx-1), ASK1 phosphorylation, and JNK and p38 activation. Although both JNK/p38 were essential in apoptosis, p38 activation was JNK-dependent, which is the first report of such temporally defined JNK-p38 interplay during an apoptotic programme. CD40-killing entrained Bak/Bax induction, controlled by JNK/p38, and caspase-9-dependent mitochondrial apoptosis, accompanied by pro-inflammatory cytokine secretion, the repertoire of which also depended on CD40 signal quality. Previous reports suggested that, despite the ability of soluble CD40 agonist to reduce RCC tumour size in vivo via immunocyte activation, RCC could be targeted more effectively by combining CD40-mediated immune activation with direct tumour CD40 signalling. Since mCD40L represents a potent tumour cell-specific killing signal, our work not only offers insights into CD40's biology in normal and malignant epithelial cells, but also provides an avenue for a 'double-hit' approach for inflammatory, tumour cell-specific CD40-based therapy.

An in vitro Co-culture System for the Activation of CD40 by Membrane-presented CD40 Ligand versus Soluble Agonist.

One fundamental property of the TNR receptor (TNFR) family relates to how 'signal quality' (the extent of receptor ligation or cross-linking) influences the outcome of receptor ligation, for instance the induction of death in tumour cells. It is unequivocal that membrane-presented ligand (delivered to target cells via cell-surface presentation by co-culture with ligand-expressing third-party cells) induces a greater extent of carcinoma cell death in vitro in comparison to non-cross-linked agonists (agonistic antibodies and/or recombinant ligands). The CD40 receptor epitomises this fundamental property of TNF receptor-ligand interactions, as the extent of CD40 cross-linking dictates cell fate. Membrane-presented CD40 ligand (mCD40L), but not soluble agonists (e.g., agonistic anti-CD40 antibody), induces high level of pro-inflammatory cytokine secretion and causes extensive cell death (apoptosis) in malignant (but not normal) epithelial cells. In this article, we describe a co-culture system for the activation of CD40 by mCD40L and subsequent detection of various features of apoptosis (including cell membrane permeabilisation, DNA fragmentation, caspase activation) as well as detection of intracellular mediators of cell death (including adaptor proteins, pro-apoptotic kinases and reactive oxygen species, ROS).

Preclinical anti-cancer activity and multiple mechanisms of action of a cationic silver complex bearing N-heterocyclic carbene ligands.

Organometallic complexes offer the prospect of targeting multiple pathways that are important in cancer biology. Here, the preclinical activity and mechanism(s) of action of a silver-bis(N-heterocyclic carbine) complex (Ag8) were evaluated. Ag8 induced DNA damage via several mechanisms including topoisomerase I/II and thioredoxin reductase inhibition and induction of reactive oxygen species. DNA damage induction was consistent with cytotoxicity observed against proliferating cells and Ag8 induced cell death by apoptosis. Ag8 also inhibited DNA repair enzyme PARP1, showed preferential activity against cisplatin resistant A2780 cells and potentiated the activity of temozolomide. Ag8 was substantially less active against non-proliferating non-cancer cells and selectively inhibited glycolysis in cancer cells. Ag8 also induced significant anti-tumour effects against cells implanted intraperitoneally in hollow fibres but lacked activity against hollow fibres implanted subcutaneously. Thus, Ag8 targets multiple pathways of importance in cancer biology, is less active against non-cancer cells and shows activity in vivo in a loco-regional setting.

Reactive oxygen species (ROS) and wound healing: the functional role of ROS and emerging ROS-modulating technologies for augmentation of the healing process.

Reactive oxygen species (ROS) play a pivotal role in the orchestration of the normal wound-healing response. They act as secondary messengers to many immunocytes and non-lymphoid cells, which are involved in the repair process, and appear to be important in coordinating the recruitment of lymphoid cells to the wound site and effective tissue repair. ROS also possess the ability to regulate the formation of blood vessels (angiogenesis) at the wound site and the optimal perfusion of blood into the wound-healing area. ROS act in the host's defence through phagocytes that induce an ROS burst onto the pathogens present in wounds, leading to their destruction, and during this period, excess ROS leakage into the surrounding environment has further bacteriostatic effects. In light of these important roles of ROS in wound healing and the continued quest for therapeutic strategies to treat wounds in general and chronic wounds, such as diabetic foot ulcers, venous and arterial leg ulcers and pressure ulcers in particular, the manipulation of ROS represents a promising avenue for improving wound-healing responses when they are stalled. This article presents a review of the evidence supporting the critical role of ROS in wound healing and infection control at the wound site, and some of the new emerging concepts associated with ROS modulation and its potential in improving wound healing are discussed.

Ketamine-Induced Apoptosis in Normal Human Urothelial Cells: A Direct, N-Methyl-d-Aspartate Receptor-Independent Pathway Characterized by Mitochondrial Stress.

Recreational abuse of ketamine has been associated with the emergence of a new bladder pain syndrome, ketamine-induced cystitis, characterized by chronic inflammation and urothelial ulceration. We investigated the direct effects of ketamine on normal human urothelium maintained in organ culture or as finite cell lines in vitro. Exposure of urothelium to ketamine resulted in apoptosis, with cytochrome c release from mitochondria and significant subsequent caspase 9 and 3/7 activation. The anesthetic mode-of-action for ketamine is mediated primarily through N-methyl d-aspartate receptor (NMDAR) antagonism; however, normal (nonimmortalized) human urothelial cells were unresponsive to NMDAR agonists or antagonists, and no expression of NMDAR transcript was detected. Exposure to noncytotoxic concentrations of ketamine (≤1 mmol/L) induced rapid release of ATP, which activated purinergic P2Y receptors and stimulated the inositol trisphosphate receptor to provoke transient release of calcium from the endoplasmic reticulum into the cytosol. Ketamine concentrations >1 mmol/L were cytotoxic and provoked a larger-amplitude increase in cytosolic Ca(2+) concentration that was unresolved. The sustained elevation in cytosolic Ca(2+) concentration was associated with pathological mitochondrial oxygen consumption and ATP deficiency. Damage to the urinary barrier initiates bladder pain and, in ketamine-induced cystitis, loss of urothelium from large areas of the bladder wall is a reported feature. This study offers first evidence for a mechanism of direct toxicity of ketamine to urothelial cells by activating the intrinsic apoptotic pathway.

Regulation of cell fate by lymphotoxin (LT) receptor signalling: Functional differences and similarities of the LT system to other TNF superfamily (TNFSF) members.

The role of TNFR family members in regulating cell fate both in the immune system and in non-lymphoid tissues has been under extensive research for decades. Moreover, the ability of several family members (death receptors) to induce death (mainly via apoptosis) represents a promising target for cancer therapy. Many studies have focused mostly on death receptors such as TNFRI, Fas and TRAIL-R due to their strong pro-apoptotic potential. Yet, cell death can be triggered via non-classical death receptors, and the lymphotoxin (LT) system represents a very good example of such a TNFR subfamily. Here we provide a comprehensive review of intracellular signalling pathways and cellular responses to LT-specific signalling, and compare for the first time the LT system to other TNFRs, such as CD40. Our aim is to highlight that non-classical TNFR-TNFL dyads such as the LT system demonstrate more complex, cell-type and context-specific capabilities. Understanding these complexities will permit a better understanding of the biological mechanisms via which non-death domain-containing TNFRs induce cell death, but may also allow the design of better therapeutic strategies.

Loss of AQP3 protein expression is associated with worse progression-free and cancer-specific survival in patients with muscle-invasive bladder cancer.

PURPOSE: Urothelial carcinoma has recently been shown to express several aquaporins (AQP), with AQP3 being of particular interest as its expression is reduced or lost in tumours of higher grade and stage. Loss of AQP3 expression was associated with worse progression-free survival (PFS) in patients with pT1 bladder cancer. The objective of this study was to investigate the prognostic value of AQP3 expression in patients with muscle-invasive bladder carcinoma (MIBC). METHODS: Retrospective single-centre analysis of the oncological outcome of patients following radical cystectomy (Cx) due to MIBC. Immunohistochemistry was used to assess AQP3 protein expression in 100 Cx specimens. Expression levels of AQP3 were related to clinicopathological variables. The impact of biomarker expression on progression-free, cancer-specific and overall survival was determined by multivariate Cox regression analysis (MVA). RESULTS: High expression of AQP3 by the tumour was associated with a statistically significantly improved PFS (75 vs. 19 %, p = 0.043) and CSS (75 vs. 18 %, p = 0.030) and, alongside lymph node involvement, was an independent predictor of PFS (HR 2.871, CI 1.066-7.733, p = 0.037), CSS (HR 3.325, CI 1.204-8.774, p = 0.019) and OS (HR 2.001, CI 1.014-3.947) in MVA. CONCLUSIONS: Although the results of the study would be strengthened by a larger, more appropriately powered, prospective, multi-institutional study, our findings strongly suggest that AQP3 expression status may represent an independent predictor of PFS and CSS in MIBC and may help select patients in need for (neo-)adjuvant chemotherapy.

Use of in vitro human keratinocyte models to study the effect of cooling on chemotherapy drug-induced cytotoxicity.

A highly distressing side-effect of cancer chemotherapy is chemotherapy-induced alopecia (CIA). Scalp cooling remains the only treatment for CIA, yet there is no experimental evidence to support the cytoprotective capacity of cooling. We have established a series of in vitro models for the culture of human keratinocytes under conditions where they adopt a basal, highly-proliferative phenotype thus resembling the rapidly-dividing sub-population of native hair-matrix keratinocytes. Using a panel of chemotherapy drugs routinely used clinically (docetaxel, doxorubicin and the active metabolite of cyclophosphamide 4-OH-CP), we demonstrate that although these drugs are highly-cytotoxic, cooling can markedly reduce or completely inhibit drug cytotoxicity, in agreement with clinical observations. By contrast, we show that cytotoxicity caused by specific combinatorial drug treatments cannot be adequately attenuated by cooling, supporting data showing that such treatments do not always respond well to cooling clinically. Importantly, we provide evidence that the choice of temperature may be critical in determining the efficacy of cooling in rescuing cells from drug-mediated toxicity. Therefore, despite their reductive nature, these in vitro models have provided experimental evidence for the clinically-reported cytoprotective role of cooling and represent useful tools for future studies on the molecular mechanisms of cooling-mediated cytoprotection.

A novel bidirectional positive-feedback loop between Wnt-ß-catenin and EGFR-ERK plays a role in context-specific modulation of epithelial tissue regeneration.

By operating as both a subunit of the cadherin complex and a key component of Wnt signalling, ß-catenin acts as the lynchpin between cell-cell contact and transcriptional regulation of proliferation, coordinating epithelial tissue homeostasis and regeneration. The integration of multiple growth-regulatory inputs with ß-catenin signalling has been observed in cancer-derived cells, yet the existence of pathway crosstalk in normal cells is unknown. Using a highly regenerative normal human epithelial culture system that displays contact inhibition, we demonstrate that the receptor tyrosine kinase (RTK)-driven MAPK and Wnt-ß-catenin signalling axes form a bidirectional positive-feedback loop to drive cellular proliferation. We show that ß-catenin both drives and is regulated by proliferative signalling cues, and its downregulation coincides with the switch from proliferation to contact-inhibited quiescence. We reveal a novel contextual interrelationship whereby positive and negative feedback between three major signalling pathways - EGFR-ERK, PI3K-AKT and Wnt-ß-catenin - enable autocrine-regulated tissue homeostasis as an emergent property of physical interactions between cells. Our work has direct implications for normal epithelial tissue homeostasis and provides insight as to how dysregulation of these pathways could drive excessive and sustained cellular growth in disease.

Expression of aquaporin water channels in human urothelial carcinoma: correlation of AQP3 expression with tumour grade and stage.

PURPOSE: To study the expression, localization and potential clinical significance of aquaporin water channels both in well-established urothelial cancer (UC) cell lines and in human bladder carcinoma specimens of different stages and grades and to discuss the clinical relevance of the findings. METHODS: AQP transcript and protein expression by RT4, RT112 and T24 UC cell lines was investigated using reverse transcriptase polymerase chain reaction and immunofluorescence labelling. Immunohistochemistry (IHC) was used to assess AQP protein expression in 94 UC specimens of various grades and stages. RESULTS: AQP3 and 9 transcripts were expressed in low-grade RT4 and RT112, but not in high-grade T24 cells. By contrast, AQP4 mRNA was absent in RT4, but expressed by RT112 and T24. Transcripts for AQP7 and 11 were detected in all three UC cell lines. Immunofluorescence microscopy confirmed the expression of AQP3, 4 and 7 at the protein level. By IHC, AQP3 was shown to be intensely expressed by 86 %, 66 % and 33 % of specimens of stage pTa, pT1 and pT2 tumours, respectively (p < 0.001). Whereas 100 % of G1 tumours were positive, only 73 % and 55 % of G2 and G3 tumours were found to express AQP3 (p = 0.004). CONCLUSIONS: This is the first study to demonstrate that several AQPs are expressed in UC. Our results indicate that there is a correlation between AQP3 protein expression and tumour stage and grade, with AQP3 expression being reduced or lost in tumours of higher grade and stage. Taken together with the available evidence from other studies, we conclude that AQPs may play a role in the progression of UC and, in particular, that this could be of prognostic value.

Aquaporin expression contributes to human transurothelial permeability in vitro and is modulated by NaCl.

It is generally considered that the bladder is impervious and stores urine in unmodified form on account of the barrier imposed by the highly-specialised uro-epithelial lining. However, recent evidence, including demonstration of aquaporin (AQP) expression by human urothelium, suggests that urothelium may be able to modify urine content. Here we have we applied functional assays to an in vitro-differentiated normal human urothelial cell culture system and examined both whether AQP expression was responsive to changes in osmolality, and the effects of blocking AQP channels on water and urea transport. AQP3 expression was up-regulated by increased osmolality, but only in response to NaCl. A small but similar effect was seen with AQP9, but not AQP4 or AQP7. Differentiated urothelium revealed significant barrier function (mean TER 3862 Ω.cm(2)), with mean diffusive water and urea permeability coefficients of 6.33×10(-5) and 2.45×10(-5) cm/s, respectively. AQP blockade with mercuric chloride resulted in decreased water and urea flux. The diffusive permeability of urothelial cell sheets remained constant following conditioning in hyperosmotic NaCl, but there was a significant increase in water and urea flux across an osmotic gradient. Taken collectively with evidence emerging from studies in other species, our results support an active role for human urothelium in sensing and responding to hypertonic salt concentrations through alterations in AQP protein expression, with AQP channels providing a mechanism for modifying urine composition. These observations challenge the traditional concept of an impermeable bladder epithelium and suggest that the urothelium may play a modulatory role in water and salt homeostasis.

Immortalisation of normal human urothelial cells compromises differentiation capacity.

BACKGROUND: The development of urothelial malignancy is not solely a consequence of loss of proliferation constraints but also involves loss of cellular differentiation, defined histopathologically as grade. Although tumour grade is an independent prognostic marker for urothelial carcinoma (UC), the molecular events underpinning the loss of urothelial differentiation are poorly understood. OBJECTIVE: To examine the effect of gene alterations implicated in UC development on the ability of human urothelial cells to undergo molecular differentiation and form a functional urothelial barrier. DESIGN, SETTING, AND PARTICIPANTS: Laboratory study. INTERVENTION: Normal human urothelial (NHU) cell cultures were transduced with recombinant retroviruses to produce stable sublines overexpressing wild-type or oncogenic mutated fibroblast growth factor receptor 3 or human telomerase reverse transcriptase (hTERT). Previously generated NHU sublines carrying dominant-negative CDK4 and p53 mutant genes or immortalised with the human papillomavirus 16 E6 oncoprotein were included. MEASUREMENTS: The activity of introduced transgenes was demonstrated by comparing phenotypes of transgene-expressing and isogenic control NHU cells. Modified and control sublines were compared for changes in generational potential (life span) and capacity to respond to differentiation-inducing signals by transcript expression of uroplakins 2 and 3. The ability to form a barrier epithelium was assessed by measuring the transepithelial electrical resistance. RESULTS AND LIMITATIONS: By contrast to tumour suppressor loss of function or oncogene overactivation, hTERT overexpression alone led to life span extension and immortalisation. The hTERT immortalised cells carried no gross genomic alterations but became progressively insensitive to differentiation signals and lost the ability to form an epithelial barrier. Further characterisation of hTERT cells revealed a downregulation of p16 cyclin-dependent kinase inhibitor expression and loss of responsiveness to peroxisome proliferator-activated receptor γ, providing mechanistic explanations for the subjugation of senescence constraints and the abrogation of differentiation capability, respectively. Although immortalised urothelial cell lines without karyotypic aberrations may be generated, such cell lines are compromised in terms of differentiation and functional capacity. CONCLUSIONS: Overexpression of hTERT promotes development of an immortalised differentiation-insensitive urothelial cell phenotype. Although such cells offer a useful insight into the grade/stage paradigm of UC, they have limited value for investigating normal urothelial cell/tissue biology and physiology.

Differential regulation of growth-promoting signalling pathways by E-cadherin.

BACKGROUND: Despite the well-documented association between loss of E-cadherin and carcinogenesis, as well as the link between restoration of its expression and suppression of proliferation in carcinoma cells, the ability of E-cadherin to modulate growth-promoting cell signalling in normal epithelial cells is less well understood and frequently contradictory. The potential for E-cadherin to co-ordinate different proliferation-associated signalling pathways has yet to be fully explored. METHODOLOGY/PRINCIPAL FINDINGS: Using a normal human urothelial (NHU) cell culture system and following a calcium-switch approach, we demonstrate that the stability of NHU cell-cell contacts differentially regulates the Epidermal Growth Factor Receptor (EGFR)/Extracellular Signal-Regulated Kinase (ERK) and Phosphatidylinositol 3-Kinase (PI3-K)/AKT pathways. We show that stable cell contacts down-modulate the EGFR/ERK pathway, whilst inducing PI3-K/AKT activity, which transiently enhances cell growth at low density. Functional inactivation of E-cadherin interferes with the capacity of NHU cells to form stable calcium-mediated contacts, attenuates E-cadherin-mediated PI3-K/AKT induction and enhances NHU cell proliferation by allowing de-repression of the EGFR/ERK pathway and constitutive activation of ß-catenin-TCF signalling. CONCLUSIONS/SIGNIFICANCE: Our findings provide evidence that E-cadherin can differentially and concurrently regulate specific growth-related signalling pathways in a context-specific fashion, with direct, functional consequences for cell proliferation and population growth. Our observations not only reveal a novel, complex role for E-cadherin in normal epithelial cell homeostasis and tissue regeneration, but also provide the basis for a more complete understanding of the consequences of E-cadherin loss on malignant transformation.

Expression and localisation of aquaporin water channels in human urothelium in situ and in vitro.

BACKGROUND: Urothelium is generally considered to be impermeable to water and constituents of urine. The possibility that human urothelium expresses aquaporin (AQP) water channels as the basis for water and solute transport has not previously been investigated. OBJECTIVE: To investigate the expression of AQP water channels by human urothelium in situ, in proliferating urothelial cell cultures and in differentiated tissue constructs. DESIGN, SETTING, AND PARTICIPANTS: AQP expression by human urothelium in situ and cultured urothelial cells was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunolabelling. Expression screening was carried out on samples of freshly isolated urothelia from multiple surgical (bladder and ureteric) specimens and on proliferating and differentiated normal human urothelial (NHU) cells in culture. Urothelial tissue constructs were established and investigated for expression of urothelial differentiation markers and AQPs. MEASUREMENTS: Qualitative study. RESULTS AND LIMITATIONS: Transcripts for AQP3, AQP4, AQP7, AQP9, and AQP11 were expressed consistently by freshly isolated urothelia as well as by cultured NHU cells. AQP0, AQP1, AQP2, AQP5, AQP6, AQP8, AQP10, and AQP12 were not expressed. Immunochemistry confirmed expression of AQP3, AQP4, AQP7, and AQP9 at the protein level. AQP3 was shown to be intensely expressed at cell borders in the basal and intermediate layers in both urothelium in situ and differentiated tissue constructs in vitro. CONCLUSIONS: This is the first study to demonstrate that AQPs are expressed by human urothelium, suggesting a potential role in transurothelial water and solute transport. Our findings challenge the traditional concept of the urinary tract as an impermeable transit and storage unit and provide a versatile platform for further investigations into the biological and clinical relevance of AQPs in human urothelium.

OK432-activated human dendritic cells kill tumor cells via CD40/CD40 ligand interactions.

In vivo, dendritic cells (DC) are programmed to orchestrate innate and adaptive immunity in response to pathogen-derived "danger" signals. Under particular circumstances, DC can also be directly cytotoxic against tumor cells, potentially allowing them to release tumor associated Ags from dying cells and then prime antitumor immunity against them. In this study, we describe the innate characteristics of DC (OK-DC) generated in vitro after exposure of immature human myeloid-derived DC to OK432, a penicillin-inactivated and lyophilized preparation of Streptococcus pyrogenes. OK-DC produced proinflammatory cytokines, stimulated autologous T cell proliferation and IFN-gamma secretion, expressed CCR7, and migrated in response to MIP-3beta. Moreover, OK-DC displayed strong, specific cytotoxicity toward tumor cell targets. This cytotoxicity was associated with novel, OK432-induced up-regulation of CD40L on the cell surface of OK-DC, and was absolutely dependent on expression of CD40 on the tumor targets. These data demonstrate that maturation of human DC with OK432, an adjuvant suitable for clinical use, induces direct tumor cell killing by DC, and describes a novel CD40/CD40L-mediated mechanism for specific DC antitumor cytotoxicity.

CD40-mediated death and cytokine secretion in colorectal cancer: a potential target for inflammatory tumour cell killing.

CD40, a member of the tumour necrosis factor family, is expressed in a variety of epithelial cells. Although soluble CD40 agonists are growth-inhibitory, membrane-presented CD40 ligand (CD40L) induces extensive apoptosis in carcinoma cells. This study investigated whether CD40 is expressed in human colorectal carcinoma (CRC) cells and explored the functional consequences of CD40 ligation. CD40 expression in a panel of CRC lines was assessed by flow cytometry and in resected human CRCs by immunohistochemistry. CRC cells were treated in vitro with soluble CD40 agonists or cocultured with fibroblasts expressing membrane-bound CD40 ligand. Apoptosis was determined by flow cytometry using Annexin V/propidium iodide labelling and by a DNA fragmentation assay. Cytokine secretion induced by CD40 ligation was quantified by a multiplex-bead array approach. We show that CD40 is expressed in a proportion of established CRC lines in culture and that receptor expression is functional. Activation of CD40 by membrane-presented CD40L, but not soluble agonists, causes high levels of death in CD40-positive CRC cells and induces secretion of proinflammatory cytokines. In agreement with our in vitro observations, immunohistochemical studies demonstrated that CD40 is highly expressed in a proportion of colorectal cancer specimens. The high level of susceptibility of CRC cells to CD40-killing combined with the ability of CD40 to induce concomitant secretion of proinflammatory cytokines suggest that CD40 ligation may represent a novel mechanism for elimination of CRC cells and render CD40 a promising therapeutic target for the eradication of colorectal tumours.