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Purpose Electrochemotherapy (ECT) is the application of electric pulses to tumour tissue to render the cell membranes permeable to usually impermeant hydrophilic anti-cancer drugs, thereby enhancing cytotoxic effects. We sought to ascertain whether ECT can be an effective palliative treatment for cutaneous metastases of breast cancer.

Background and study aims: Targeted delivery of specific chemotherapeutic drugs into tumors can be achieved by delivering electrical pulses directly to the tumor tissue. This causes a transient formation of pores in the cell membrane that enables passive diffusion of normally impermeant drugs. A novel device has been developed to enable the endoscopic delivery of this tumor permeabilizing treatment. The aim of the preclinical studies described here was to investigate the efficacy and safety of this nonthermal ablation system in the treatment of gastrointestinal cancer models. Methods: Murine, porcine, and canine gastrointestinal tumors and tissues were used to assess the efficacy and safety of electroporation delivered through the special device in combination with bleomycin. Tumor cell death, volume, and overall survival were recorded. Results: Murine tumors treated with electrochemotherapy showed excellent responses, with cell death being induced rapidly, mainly via an apoptotic-type mechanism. Use of the system in canine gastrointestinal cancers demonstrated successful local endoluminal tumor resolution, with no safety or adverse effects noted. Conclusions: Electroporation via the new device in combination with bleomycin offers a nonthermal tumor ablative approach, and presents clinicians with a new option for the management of gastrointestinal cancers.

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers in both men and women, posing a serious demographic and economic burden worldwide. In the United Kingdom, CRC affects 1 in every 20 people and it is often detected once well established and after it has spread beyond the bowel (Stage IIA-C and Stage IIIA-C). A diagnosis at such advanced stages is associated with poor treatment response and survival. However, studies have identified two sub-groups of post-treatment CRC patients--those with good outcome (reactive disease) and those with poor outcome (non-reactive disease). We aim to review the state-of-the-art for CRC with respect to the expression of cancer-testis antigens (CTAs) and their identification, evaluation and correlation with disease progression, treatment response and survival. We will also discuss the relationship between CTA expression and regulatory T-cell (Treg) activity to tumorigenesis and tumor immune evasion in CRC and how this could account for the clinical presentation of CRC. Understanding the molecular basis of reactive CRC may help us identify more potent novel immunotherapeutic targets to aid the effective treatment of this disease. In this review, based on our presentation at the 2012 International Society for the Cell and Gene Therapy of Cancer annual meeting, we will summarize some of the most current advances in CTA and CRC research and their influence on the development of novel immunotherapeutic approaches for this common and at times difficult to treat disease.

Background: The establishment of assessment reliability at the level of the individual trainee is an important attribute of assessment methodologies, particularly for doctors who have been failed. This issue is of particular importance for the process of competence assessment in the USA, UK, Australia and New Zealand. Methods: We use data from 19 applicants for higher surgical training in 2008 at the Royal College of Surgeons in Ireland to compare: (i) the objective structured assessment of technical skills (OSATS) method; and (ii) a procedure-specific checklist to assess surgical technical skills in the excision of a sebaceous cyst task by two experienced senior surgeons. Results: The overall interrater reliability (IRR) of the OSATS assessment as determined by a correlation coefficient was 0.507 (P < 0.03) and 0.67 with coefficient alpha, considerably below the accepted 0.8 level of IRR. The checklist’s overall IRR was 0.89. Individually, only five (26%) of the OSATS assessments reached the 0.8 level of IRR in contrast to 18 (95%) of the checklist assessments. Discussion: We propose binary procedure-based assessment checklists as more reliable assessment instruments with more robust reproducibility.

BACKGROUND: Laparoscopic skills are difficult to learn. We, therefore, assessed the factors involved in skill acquisition, maintenance, and loss in 2 prospective, randomized studies. METHODS: In study 1, 24 laparoscopic novices were randomly assigned to a control condition who performed the laparoscopic assessment task; Massed condition who trained on virtual reality (VR) simulation during 1 day or Interval condition who had the same amount of VR training distributed over 3 consecutive days. All groups also completed a novel laparoscopic box-trainer task on 5 consecutive days. In study 2, 16 laparoscopic novices were randomly assigned to a Practice or a No-practice condition. All subjects were required to train on a VR simulation curriculum for the same duration and skill attainment level. The week after completion of training, subjects in the Practice condition were allowed 1 complete practice trial on the simulator. Both groups completed the same tasks 2 weeks after completion of the training. RESULTS: In study 1, the Interval trained group showed the fastest rate of learning and on completion of training significantly outperformed both the Massed and Control groups (P < 0.0001). In study 2, both groups showed significant skills improvement from training trial T1 to T3 (P < 0.0001). The subjects in the Practice group maintained or improved their skills at 1 week but those in the No practice group showed significant decline of skills at 2 weeks after training completion (P < 0.0001). CONCLUSIONS: Laparoscopic skills are optimally acquired on an Interval training schedule. They significantly decline with 2 weeks of nonuse.

The prognosis of oesophageal cancer remains poor because of the absence of molecularly targeted therapies and resistance to conventional DNA-damaging chemotherapeutics. This resistance to therapy has been associated with a failure to induce apoptosis in response to DNA damage (O'Donovan et al, 2011). Disruption of apoptotic signalling can be achieved by loss of tumour-suppressor activity and upregulation of survival signalling pathways (Adams and Cory, 2007). Ultimately, many signalling pathways that determine cell fate converge at the Bcl-2 family. In cancer, the balance between Bcl-2-negative regulators (Bcl-2/Bcl-xL/Mcl-1/A1/BCL-w) and positive regulators (Bax/Bak/BH3-only proteins) are disturbed such that initiation of apoptosis is difficult to achieve. Strategies to restore apoptosis have led to the development of BH3 mimetics (a novel class of therapeutics – now in clinical trials) designed to inhibit the interaction between Bcl-2 family members at BH3 domains (Zhang et al, 2007; Lessene et al, 2008; Kang and Reynolds, 2009). Although this strategy is designed to engage the apoptosis pathway in otherwise resistant cells, both Bcl-xL and Bcl-2 have also been implicated in the autophagy process. Both proteins can bind Beclin 1 (an autophagy regulator with a BH3 domain). Disruption of this interaction with ABT-737 (BH3 mimetic) induces autophagy (Maiuri et al, 2007). The BH3-only proteins Bid, Bad and BNIP3 have also been reported to induce autophagy in cancer cells (Lamparska-Przybysz et al, 2005; Hamacher-Brady et al, 2007; Maiuri et al, 2007). ApoL1, an autophagy regulator has a BH3 domain and its overexpression induces autophagic cell death (AuCD) (Wan et al, 2008). Autophagy is a survival mechanism that enables cells to tolerate adverse conditions such as starvation or withdrawal of survival signalling. However, it also has the potential to drive type II cell death/AuCD (Berry and Baehrecke, 2007; Wan et al, 2008). It is possible therefore, that the cytotoxic effects of BH3 mimetics may not be limited to apoptosis induction. In this study, we evaluated the effects of HA14-1 (BH3 mimetic) on three human squamous oesophageal cancer cell lines. Two of these cell lines fail to undergo apoptosis in response to 5-fluorouracil (5-FU) but instead induce an autophagic response, which is accompanied by type II cell death. Despite the presence of type II cell death morphologies, these populations will retain a substantial number of surviving autophagic cells that will re-populate, thus rendering treatment ineffective (O'Donovan et al, 2011). In this study, we evaluated the potential of HA14-1 to reduce this survival and assessed the involved death mechanism.

Infiltration of tumors by regulatory T cells confers growth and metastatic advantages by inhibiting antitumor immunity and by production of receptor activator of NF-κB (RANK) ligand, which may directly stimulate metastatic propagation of RANK-expressing cancer cells. Modulation of regulatory T cells can enhance the efficacy of cancer immunotherapy. Strategies include depletion, interference with function, inhibition of tumoral migration, and exploitation of T-cell plasticity. Problems with these strategies include a lack of specificity, resulting in depletion of antitumor effector T cells or global interruption of regulatory T cells, which may predispose to autoimmune diseases. Emerging technologies, such as RNA interference and tetramer-based targeting, may have the potential to improve selectivity and efficacy. Cancer Res; 71(22); 6915–20. ©2011 AACR.

Cancer is one of the most wide-spread diseases of modern times, with an estimated increase in the number of patients diagnosed worldwide, from 11.3 million in 2007 to 15.5 million in 2030 (www.who.int). In many cases, due to the delay in diagnosis and high increase of relapse, survival rates are low. Current therapies, including surgery, radiation and chemotherapy, have made significant progress, but they have many limitations and are far from ideal. Although immunotherapy has recently offered great promise as a new approach in cancer treatment, it is still very much in its infancy and more information on this approach is required before it can be widely applied. For these reasons effective, safe and patient-acceptable cancer therapy is still largely an unmet clinical need. Recent knowledge of the genetic basis of the disease opens up the potential for cancer gene therapeutics based on siRNA. However, the future of such gene-based therapeutics is dependent on achieving successful delivery. Extensive research is ongoing regarding the design and assessment of non-viral delivery technologies for siRNA to treat a wide range of cancers. Preliminary results on the first human Phase I trial for solid tumours, using a targeted non-viral vector, illustrate the enormous therapeutic benefits once the issue of delivery is resolved. In this review the genes regulating cancer will be discussed and potential therapeutic targets will be identified. The physiological and biochemical changes caused by tumours, and the potential to exploit this knowledge to produce bio-responsive 'smart' delivery systems, will be evaluated. This review will also provide a critical and comprehensive overview of the different non-viral formulation strategies under investigation for siRNA delivery, with particular emphasis on those designed to exploit the physiological environment of the disease site. In addition, a section of the review will be dedicated to pre-clinical animal models used to evaluate the stability, safety and efficacy of the delivery systems.

We investigated the cell-death mechanisms induced in esophageal cancer cells in response to the chemotherapeutic drugs, 5-fluorouracil (5-FU) and cisplatin. Chemosensitive cell lines exhibited apoptosis whereas chemoresistant populations exhibited autophagy and a morphology resembling type II programmed cell death (PCD). Cell populations that respond with autophagy are more resistant and will recover following withdrawal of the chemotherapeutic agents. Specific inhibition of early autophagy induction with siRNA targeted to Beclin 1 and ATG7 significantly enhanced the effect of 5-FU and reduced the recovery of drug-treated cells. Pharmacological inhibitors of autophagy were evaluated for their ability to improve chemotherapeutic effect. The PtdIns 3-kinase inhibitor 3-methyladenine did not enhance the cytotoxicity of 5-FU. Disruption of lysosomal activity with bafilomycin A 1 or chloroquine caused extensive vesicular accumulation but did not improve chemotherapeutic effect. These observations suggest that an autophagic response to chemotherapy is a survival mechanism that promotes chemoresistance and recovery and that selective inhibition of autophagy regulators has the potential to improve chemotherapeutic regimes. Currently available indirect inhibitors of autophagy are, however, ineffective at modulating chemosensitivity in these esophageal cancer cell lines.

Preclinical results with various gene therapy strategies indicate significant potential for new cancer treatments. However, many therapeutics fail at clinical trial, often due to differences in tissue physiology between animal models and humans, and tumor phenotype variation. Clinical data relevant to treatment strategies may be generated prior to clinical trial through experimentation using intact patient tissue ex vivo. We developed a novel tumor slice model culture system that is universally applicable to gene delivery methods, using a realtime luminescence detection method to assess gene delivery. Methods investigated include viruses (adenovirus [Ad] and adeno-associated virus), lipofection, ultrasound (US), electroporation and naked DNA. Viability and tumor populations within the slices were well maintained for seven days, and gene delivery was qualitatively and quantitatively examinable for all vectors. Ad was the most efficient gene delivery vector with transduction efficiency >50%. US proved the optimal non-viral gene delivery method in human tumor slices. The nature of the ex vivo culture system permitted examination of specific elements. Parameters shown to diminish Ad gene delivery included blood, regions of low viability and secondary disease. US gene delivery was significantly reduced by blood and skin, while tissue hyperthermia improved gene delivery. US achieved improved efficacy for secondary disease. The ex vivo model was also suitable for examination of tissue-specific effects on vector expression, with Ad expression mediated by the CXCR4 promoter shown to provide a tumor selective advantage over the ubiquitously active cytomegalovirus promoter. In conclusion, this is the first study incorporating patient tissue models in comparing gene delivery from various vectors, providing knowledge on cell-type specificity and examining the crucial biological factors determining successful gene delivery. The results highlight the importance of in-depth preclinical assessment of novel therapeutics and may serve as a platform for further testing of current, novel gene delivery approaches.

Cancer remains a leading cause of morbidity and mortality. Despite advances in understanding, detection, and treatment, it accounts for almost one-fourth of all deaths per year in Western countries. Prostate cancer is currently the most commonly diagnosed noncutaneous cancer in men in Europe and the United States, accounting for 15% of all cancers in men. As life expectancy of individuals increases, it is expected that there will also be an increase in the incidence and mortality of prostate cancer. Prostate cancer may be inoperable at initial presentation, unresponsive to chemotherapy and radiotherapy, or recur following appropriate treatment. At the time of presentation, patients may already have metastases in their tissues. Preventing tumor recurrence requires systemic therapy; however, current modalities are limited by toxicity or lack of efficacy. For patients with such metastatic cancers, the development of alternative therapies is essential. Gene therapy is a realistic prospect for the treatment of prostate and other cancers, and involves the delivery of genetic information to the patient to facilitate the production of therapeutic proteins. Therapeutics can act directly (eg, by inducing tumor cells to produce cytotoxic agents) or indirectly by upregulating the immune system to efficiently target tumor cells or by destroying the tumor's vasculature. However, technological difficulties must be addressed before an efficient and safe gene medicine is achieved (primarily by developing a means of delivering genes to the target cells or tissue safely and efficiently). A wealth of research has been carried out over the past 20 years, involving various strategies for the treatment of prostate cancer at preclinical and clinical trial levels. The therapeutic efficacy observed with many of these approaches in patients indicates that these treatment modalities will serve as an important component of urological malignancy treatment in the clinic, either in isolation or in combination with current approaches.

Development of gene-based therapies for the treatment of inherited and acquired diseases, including cancer, has seen renewed interest in the use of nonviral vectors coupled to physical delivery modalities. Low-frequency ultrasound (US), with a well-established record in a clinical setting, has the potential to deliver DNA efficiently, accurately and safely. Optimal in vivo parameters for US-mediated delivery of naked plasmid DNA were established using the firefly luciferase reporter gene construct. Optimized parameters were used to administer a therapeutic gene construct, coding for granulocyte-macrophage colony-stimulating factor (GM-CSF) and B7-1 costimulatory molecule, to growing murine fibrosarcoma tumors. Tumor progression and animal survival was monitored throughout the study and the efficacy of the US-mediated gene therapy determined and compared with an electroporation-based approach. Optimal parameters for US-mediated delivery of plasmid DNA to tumors were deduced to be 1.0 W/cm 2 at 20% duty cycle for 5 min (60 J/cm 2 ). In vivo US-mediated gene therapy resulted in a 55% cure rate in tumor-bearing animals. The immunological response invoked was cell mediated, conferring resistance against re-challenge and resistance to tumor challenge after transfer of splenocytes to naive animals. US treatment was noninjurious to treated tissue, whereas therapeutic efficacy was comparable to an electroporation-based approach. US-mediated delivery of an immune-gene construct to growing tumors was therapeutically effective. Sonoporation has the potential to be a major factor in the development of nonviral gene delivery approaches. (E-mail: geraldc@iol.ie )

Challenges for oncology practitioners and researchers include specific treatment and detection of tumours. The ideal anti-cancer therapy would selectively eradicate tumour cells, whilst minimising side effects to normal tissue. Bacteria have emerged as biological gene vectors with natural tumour specificity, capable of homing to tumours and replicating locally to high levels when systemically administered. This property enables targeting of both the primary tumour and secondary metastases. In the case of invasive pathogenic species, this targeting strategy can be used to deliver genes intracellularly for tumour cell expression, while non-invasive species transformed with plasmids suitable for bacterial expression of heterologous genes can secrete therapeutic proteins locally within the tumour environment (cell therapy approach). Many bacterial genera have been demonstrated to localise to and replicate to high levels within tumour tissue when intravenously (IV) administered in rodent models and reporter gene tagging of bacteria has permitted real-time visualisation of this phenomenon. Live imaging of tumour colonising bacteria also presents diagnostic potential for this approach. The nature of tumour selective bacterial colonisation appears to be tumour origin- and bacterial species- independent. While originally a correlation was drawn between anaerobic bacterial colonisation and the hypoxic nature of solid tumours, it is recently becoming apparent that other elements of the unique microenvironment within solid tumours, including aberrant neovasculature and local immune suppression, may be responsible. Here, we consider the pre-clinical and clinical data supporting the use of bacteria as a tumour-targeting tool, recent advances in the area, and future work required to develop a beneficial clinical treatment.

The International Society for Cell and Gene Therapy (ISCGT) of Cancer annual meeting was held from September 2 through September 4, 2009, in Cork, Ireland (www.iscgt2009.com). The conference was held in conjunction with the Irish Society for Gene and Cell Therapy third annual meeting, and brought together scientists and clinicians from around the world in a country developing its knowledge economy. Next year's ISCGT meeting will be held in Doha, the capital of Qatar (www.iscgt.net), from September 27 through September 29, 2010.

Background: Oesophageal cancer incidence is increasing and survival rates remain extremely poor. Natural agents with potential for chemoprevention include the phytochemical curcumin (diferuloylmethane). We have examined the effects of curcumin on a panel of oesophageal cancer cell lines. Methods: MTT (3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyl tetrazolium bromide) assays and propidium iodide staining were used to assess viability and DNA content, respectively. Mitotic catastrophe (MC), apoptosis and autophagy were defined by both morphological criteria and markers such as MPM-2, caspase 3 cleavage and monodansylcadaverine (MDC) staining. Cyclin B and poly-ubiquitinated proteins were assessed by western blotting. Results: Curcumin treatment reduces viability of all cell lines within 24 h of treatment in a 5–50 μM range. Cytotoxicity is associated with accumulation in G2/M cell-cycle phases and distinct chromatin morphology, consistent with MC. Caspase-3 activation was detected in two out of four cell lines, but was a minor event. The addition of a caspase inhibitor zVAD had a marginal or no effect on cell viability, indicating predominance of a non-apoptotic form of cell death. In two cell lines, features of both MC and autophagy were apparent. Curcumin-responsive cells were found to accumulate poly-ubiquitinated proteins and cyclin B, consistent with a disturbance of the ubiquitin–proteasome system. This effect on a key cell-cycle checkpoint regulator may be responsible for the mitotic disturbances and consequent cytotoxicity of this drug. Conclusion: Curcumin can induce cell death by a mechanism that is not reliant on apoptosis induction, and thus represents a promising anticancer agent for prevention and treatment of oesophageal cancer.

Metastatic disease is dependent on tumor cell migration through the venous and lymphatic systems and requires dynamic rearrangement of adherens junctions. Endocytosis of cadherins is a key mechanism to dynamically arrange adherens junctions, signaling, and motility in tumor cells; however, the role of shear in regulating this process in metastatic cells is unknown. In this study, the role of shear in regulating cell surface expression of E-cadherin was investigated. We found that exposure to venous shear (shear rate, 200/s) induced internalization of E-cadherin in adherent metastatic oesophageal tumor cells (OC-1 tumor cell line). Internalized E-cadherin was found localized to Rab5-positive endosomes and was not present in lysosomes. As the Src family of tyrosine kinase have been implicated in regulating cadherin expression, we investigated the role of shear in regulating E-cadherin through Src activity. Pretreatment of OC-1 cells with the specific Src kinase inhibitor 4-amino-5- (4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) prevented shear-induced internalization of E-cadherin. Direct measurement of Src activity (phosphorylation on Y416) showed that Src is activated in sheared OC-1 cells and that the shear-induced increase in phospho-Src is inhibited by the presence of PP1. Moreover, we show that shear stress significantly increased the invasive capacity of OC-1 cells (P < 0.001), a process inhibited by the presence of PP1. These results indicate a novel role for shear in regulating the endocytosis of E-cadherin and invasiveness in metastatic cells.

We investigated if the range of efficacy of a gene-based immunotherapy of solid tumours against systemic disease could be extended when used as a neoadjuvant to surgery. Hundred percent mice whose subcutaneous tumours were surgically removed 4 days post-electroporation with GM-CSF and B7-1 plasmid were systemically resistance to tumour rechallenge. In mice bearing both subcutaneous and hepatic tumours, neoadjuvant treatment of the primary tumour resulted in significant reduction in, or elimination of, hepatic tumour growth, with a significant increase in survival, indicating that control of the primary tumour by immunotherapy was not a prerequisite for containment of systemic disease.

Gene therapy involves the transfer of genetic information to a target cell to facilitate the production of therapeutic proteins and is now a realistic prospect as a cancer treatment. Gene transfer may be achieved through the use of both viral and non-viral delivery methods and the role of this method in the gene therapy of cancer has been demonstrated. Viruses represent an attractive vehicle for cancer gene therapy due to their high efficiency of gene delivery. Many viruses can mediate long term gene expression, while some are also capable of infecting both dividing and non-dividing cells. Given the broadly differing capabilities of various viral vectors, it is imperative that the functionality of the virus meets the requirements of the specific treatment. A number of immunogene therapy strategies have been undertaken, utilising a range of viral vectors, and studies carried out in animal models and patients have demonstrated the therapeutic potential of viral vectors to carry genes to cancer cells and induce anti-tumour immune responses. This review critically discusses the advances in the viral vector mediated delivery of immunostimulatory molecules directly to tumour cells, the use of viral vectors to modify tumour cells, the creation of whole cell vaccines and the direct delivery of tumour antigens in animal models and clinical trials, specifically in the context of the suitability of vector types for specific strategies.

The aim of this review article is to provide a concise overview of the pre-clinical development of electrochemotherapy (ECT), its present utility in clinical practice and to examine its potential application to therapeutic modalities in the future.Results from the ESOPE trial demonstrate an 85% objective response rate (ORR) in solid cutaneous and subcutaneous tumours of varying histologies, that would previously have been recalcitrant to conventional therapies. Experimentally, neoadjuvant immunogene therapy of primary cancers has been found to be effective against minimal residual disease in metastatic models. As such, combinations of electrogene delivery and electrochemothearpy offer exciting possibilities for both local and systemic control of heretofore incurable cancers.Electrochemotherapy is a quick, safe, inexpensive treatment modality that has been shown to give consistently reproducible results in the treatment of solid cutaneous and subcutaneous malignant tumours. To date, its clinical license has limited its application to a palliative setting. Future work includes looking to extend this therapeutic profile to the management of primary tumours and earlier stage disease, as well as examining the potential for combining electrochemotherapy with gene and immunotherapies and developing novel electrode designs to facilitate the application of this treatment to internal cancers.

In rectal cancer variation in lymph node recovery influences the detection of nodal metastases and prognosis among Dukes B (Stage II) cases. However, the possible prognostic importance of node size and inherent patient/tumor characteristics in determining node recovery has not been studied. We examined 269 Dukes B (Stage II) rectal tumors, with a mean of 12 nodes per case. Primary tumor characteristics were correlated with the number and size of recovered nodes. Clinical follow-up permitted determination of long-term survival. The five-year survival of 94 Dukes B cases with nine or fewer nodes was 69.4 percent vs. 87.6 percent in 175 cases with ten or more nodes (P = 0.001). Lymph nodes were smaller in patients dying of recurrence; among 130 Dukes B patients whose mean node diameter was

The p21-activated kinase 1 (Pak1) is a serine/threonine kinase whose activity is regulated by both Rho GTPases and AGC kinase family members. It plays a role in cytoskeletal remodeling and cell motility as well as cell proliferation, angiogenesis, tumorigenesis and metastasis. An involvement of Pak1 in renal cell carcinoma (RCC), which remains highly refractory to chemotherapy and radiotherapy, remains to be investigated. Pak1 expression, phosphorylation and kinase activity were examined in RCC cell lines and human tissue from normal and renal carcinoma. We report increased Pak1 expression and constitutive activity in the membrane and nucleus but not the cytoplasm of resected human RCC. To study a role for Pak1 in RCC, we developed 786-0 clones that expressed either a kinase-active Pak1L83,L86 2 different Pak1 dominant negative mutants, Pak1R299 and Pak1L83,L86,R299 or Pak1 siRNA. The expression of Pak1L83,L86 increased 786-0 proliferation, motility and anchorage independent growth, while the dominant negative mutants and Pak1 siRNA abrogated these effects. In addition, Pak1L83,L86 conferred resistance to 5-fluorouracil with a 40%+/-10% increase in cell viability. Conversely, Pak1L83,L86,R299, Pak1R299 and Pak1 siRNA conferred sensitivity with a 65.2%+/-5.5%, 69.2%+/-3.3% and 73.0%+/-8.4% loss in viability, respectively. Finally, Pak1 plays a role in renal tumor growth in vivo. Only 33% of mice developed tumors in the Pak1L83,L86,R299 group and no tumors developed from Pak1R299 cell challenge. Together these findings point to Pak1 as an exciting target for therapy of renal cancer, which remains highly refractory to existing treatments.

An improved system for luciferase tagging Listeria monocytogenes was developed by constructing a highly active, constitutive promoter. This construct gave 100-fold-higher activity in broth than any native promoter tested and allowed for imaging of lux -tagged L. monocytogenes in food products, during murine infections, and in tumor targeting studies.

Lluis M. Mir*, Julie Gehl, Gregor Sersa, Christopher G. Collins , Jean-Remi Garbay, Valerie Billard, Poul F. Geertsen, Z. Rudolf , Gerald C. O’Sullivan, Michel Marty Institut Gustave-Roussy, 39 Rue Camille Desmoulins, F-94805 Villejuif Cedex, France CNRS UMR 8121, Institut Gustave-Roussy, F-94805 Villejuif Cedex, France Univ Paris-Sud, UMR 8121, France Department of Oncology, 54B1, University of Copenhagen at Herlev Hospital, Herlev Ringvej 75, DK-2730 Herlev, Denmark Institute of Oncology Ljubljana, Zaloska 2, SI-1000 Ljubljana, Slovenia Cork Cancer Research Centre, Mercy Hospital, Greenville Place, Cork, Ireland

Purpose: To evaluate and confirm efficacy and safety of electrochemotherapy with bleomycin or cisplatin on cutaneous and subcutaneous tumour nodules of patients with malignant melanoma and other malignancies in a multicenter study. Patients and methods: This was a two year long prospective non-randomised study on 41 patients evaluable for response to treatment and 61 evaluable for toxicity. Four cancer centers enrolled patients with progressive cutaneous and subcutaneous metastases of any histologically proven cancer. The skin lesions were treated by electrochemotherapy, using application of electric pulses to the tumours for increased bleomycin or cisplatin delivery into tumour cells. The treatment was performed using intravenous or intratumoural drug injection, followed by application of electric pulses generated by a Cliniporator TM using plate or needle electrodes. Tumour response to electrochemotherapy as well as possible sideeffects with respect to the treatment approach, tumour histology and location of the tumour nodules and electrode type were evaluated. Results: An objective response rate of 85% (73.7% complete response rate) was achieved on the electrochemotherapy treated tumour nodules, regardless of tumour histology, and drug used or route of its administration. At 150 days after the treatment (median follow up was 133 days and range 60‐380 days) local tumour control rate for electrochemotherapy was 88% with bleomycin given intravenously, 73% with bleomycin given intratumourally and 75% with cisplatin given intratumourally, demonstrating that all three approaches were

Colorectal cancers that adhere to the urinary bladder require en bloc partial or total cystectomy to achieve negative tumor margins.This prospective study evaluated the outcome of combined bladder resection for carcinoma of the colon or rectum at a unit specializing in gastrointestinal cancer.Patients (n = 63) with colorectal tumors adherent to the bladder at operation and without distal metastases were followed. Fifty-eight patients (92%) had tumors of the sigmoid colon or upper rectum. Operative morbidity and mortality rates were 18% and 1.5%, respectively. Histological staging demonstrated bladder adherence in 46% (29/63) and invasion in 54% (34/63). Overall disease-specific survival was 54%, with a mean follow-up of 7.6 (range 5-12) years. Five-year survival for margin negative patients was 72% (26/36) and 27% (4/15) for node negative and positive tumors, respectively. The bladder was closed primarily in 48 patients and reconstructed by enterocystoplasty in five, with ten patients requiring urinary diversion.En bloc bladder resection for adherent or invading tumors of the colon and rectum achieves good local control, but an infiltrative extravesical margin denotes poor prognosis. The potential for cure in completely excised node negative tumors is good. Bladder reconstruction is achievable in most patients.

To metastasize, tumor cells must adopt different morphological responses to resist shear forces encountered in circulating blood and invade through basement membranes. The Rho and Ras GTPases play a critical role in regulating this dynamic behavior. Recently, we demonstrated shear-induced activation of adherent esophageal metastatic cells, characterized by formation of dynamic membrane blebs. Although membrane blebbing has only recently been characterized as a rounded mode of cellular invasion promoted through Rho kinase (ROCK), the role of shear forces in modulating membrane blebbing activity is unknown. To further characterize membrane blebbing in esophageal metastatic cells (OC-1 cell line), we investigated the role of shear in cytoskeletal remodeling and signaling through ROCK and Ras. Our results show that actin and tubulin colocalize to the cortical ring of the OC-1 cell under static conditions. However, under shear, actin acquires a punctuate distribution and tubulin localizes to the leading edge of the OC-1 cell. We show for the first time that dynamic bleb formation is induced by shear alone independent of integrin-mediated adhesion ( P < 0.001, compared with OC-1 cells). Y-27632, a specific inhibitor of ROCK, causes a significant reduction in shear-induced bleb formation and inhibits integrin αvβ3-Ras colocalization at the leading edge of the cell. Direct measurement of Ras activation shows that the level of GTP-bound Ras is elevated in sheared OC-1 cells and that the shear-induced increase in Ras activity is inhibited by Y-27632. Finally, we show that shear stress significantly increases OC-1 cell invasion ( P < 0.007), an effect negated by the presence of Y-27632. Together our findings suggest a novel physiological role for ROCK and Ras in metastatic cell behavior.

The Bcr-Abl oncoprotein plays a major role in the development and progression of chronic myeloid leukemia (CML). Several studies have suggested that the expression levels of Bcr-Abl are elevated at disease progression to blast crisis and that this plays a significant role in the achievement of drug resistance. We have established cell lines expressing low and high levels of Bcr-Abl to study the molecular mechanisms involved in disease progression and drug resistance. It is now known that the endoplasmic reticulum (ER) can play a major role in the regulation of apoptosis. We therefore investigated whether Bcr-Abl expression modulates ER homeostasis and interferes with ER-mediated apoptotic pathways to promote survival. Bcr-Abl–expressing cells exhibit a decreased amount of free releasable calcium in the ER as well as a weaker capacitative calcium entry response, relative to parental cells. This effect is independent of Bcl-2, which is a known modulator of ER calcium homeostasis. The reduction in ER releasable calcium results in inhibition of the ER/mitochondria-coupling process and mitochondrial calcium uptake. This study demonstrates a novel downstream consequence of Bcr-Abl signaling. The ability to negate calcium-dependent apoptotic signaling is likely to be a major prosurvival mechanism in Bcr-Abl–expressing cells.

Lactobacillus salivarius subsp. salivarius strain UCC118 is a bacteriocin-producing strain with probiotic characteristics. The 2.13-Mb genome was shown by sequencing to comprise a 1.83 Mb chromosome, a 242-kb megaplasmid (pMP118), and two smaller plasmids. Megaplasmids previously have not been characterized in lactic acid bacteria or intestinal lactobacilli. Annotation of the genome sequence indicated an intermediate level of auxotrophy compared with other sequenced lactobacilli. No single-copy essential genes were located on the megaplasmid. However, contingency amino acid metabolism genes and carbohydrate utilization genes, including two genes for completion of the pentose phosphate pathway, were megaplasmid encoded. The megaplasmid also harbored genes for the Abp118 bacteriocin, a bile salt hydrolase, a presumptive conjugation locus, and other genes potentially relevant for probiotic properties. Two subspecies of L. salivarius are recognized, salivarius and salicinius , and we detected megaplasmids in both subspecies by pulsed-field gel electrophoresis of sizes ranging from 100 kb to 380 kb. The discovery of megaplasmids of widely varying size in L. salivarius suggests a possible mechanism for genome expansion or contraction to adapt to different environments.

Many patients with various types of cancers have already by the time of presentation, micrometastases in their tissues and are left after treatment in a minimal residual disease state [Am J Gastroenterol 95(12), 2000]. To prevent tumour recurrence these patients require a systemic based therapy, but current modalities are limited by toxicity or lack of efficacy. We have previously reported that immune reactivity to the primary tumour is an important regulator of micrometastases and determinant of prognosis. This suggests that recruitment of specific anti-tumour mechanisms within the primary tumour could be used advantageously for tumour control as either primary or neo-adjuvant treatments. Recently, we have focused on methods of stimulating immune eradication of solid tumours and minimal residual disease using gene therapy approaches. Gene therapy is now a realistic prospect and a number of delivery approaches have been explored, including the use of viral and non-viral vectors. Non-viral vectors have received significant attention since, in spite of their relative delivery inefficiency, they may be safer and have greater potential for delivery of larger genetic units. By in vivo electroporation of the primary tumour with plasmid expressing GM-CSF and B7-1, we aim to stimulate immune eradication of the treated tumour and associated metastases. In this symposium report, we describe an effective gene based approach for cancer immunotherapy by inducing cytokine and immune co-stimulatory molecule expression by the growing cells of the primary tumour using a plasmid electroporation gene delivery strategy. We discuss the potential for enhancement of this therapy by its application as a neoadjuvant to surgical excision and by its use in combination with suppressor T cell depletion.

Renal cell carcinoma (RCC) is the most common malignancy of the kidney. Unfortunately, RCCs are highly refractory to conventional chemotherapy, radiation therapy, and even immunotherapy. Thus, novel therapeutic targets need to be sought for the successful treatment of RCCs. We now report that 6-anilino-5,8-quinolinequinone (LY83583), an inhibitor of cyclic GMP production, induced growth arrest and apoptosis of the RCC cell line 786-0. It did not prove deleterious to normal renal epithelial cells, an important aspect of chemotherapy. To address the cellular mechanism(s), we used both genetic and pharmacological approaches. LY83583 induced a time- and dose-dependent increase in RCC apoptosis through dephosphorylation of mitogen-activated protein kinase kinase 1/2 and its downstream extracellular signal-regulated kinases (ERK) 1 and -2. In addition, we observed a decrease in Elk-1 phosphorylation and cyclooxygenase-2 (COX-2) down-regulation. We were surprised that we failed to observe an increase in either c-Jun NH(2)-terminal kinase or p38alpha and -beta mitogen-activated protein kinase activation. In contradiction, reintroduction of p38delta by stable transfection or overexpression of p38gamma dominant negative abrogated the apoptotic effect. Cell death was associated with a decrease and increase in Bcl-x(L) and Bax expression, respectively, as well as release of cytochrome c and translocation of apoptosis-inducing factor. These events were associated with an increase in reactive oxygen species formation. The antioxidant N-acetyl l-cysteine, however, opposed LY83583-mediated mitochondrial dysfunction, ERK1/2 inactivation, COX-2 down-regulation, and apoptosis. In conclusion, our results suggest that LY83583 may represent a novel therapeutic agent for the treatment of RCC, which remains highly refractory to antineoplastic agents. Our data provide a molecular basis for the anticancer activity of LY83583.

Lactobacillus salivarius subsp. salivarius UCC118 is a probiotic bacterium that was originally isolated from human intestinal tissues and was subsequently shown in a pilot study to alleviate symptoms associated with mild-moderate Crohn's disease. Strain UCC118 can adhere to animal and human intestinal tissue, and to both healthy and inflamed ulcerative colitis mucosa, irrespective of location in the gut. In this study, an enzymatic technique has been combined with proteomic analysis to correlate bacterial growth phase with the presence of factors present in the cell wall of the bacterium. Using PAGE electrophoresis, it was determined that progression from lag to log to stationary growth phases in vitro correlated with increasing prominence of an 84 kD protein associated with in vitro adherence ability. Isolated proteins from the 84 kD band region were further separated by two-dimensional electrophoresis, resolving this band into 20 individual protein spots at differing isoelectric points. The protein moieties were excised, trypsin digested and subjected to tandem mass spectrometry. The observed proteins are analogous to those reported to be associated with the Listeria monocytogenes cell-wall proteome, and include DnaK, Ef-Ts and pyruvate kinase. These data suggest that at least some of the beneficial attributes of probiotic lactobacilli, and in particular this strain, may be due to nonpathogenic mimicry of pathogens and potentially be mediated through a form of attenuated virulence.

There is considerable clinical interest in the utility of probiotic therapy--the feeding of (live) non-pathogenic bacteria, originally derived from the alimentary tract, for disease treatment or health promotion. The microflora of the gastrointestinal tract is essential for mucosal protection, for immune education and for metabolism of fecal residue. Physiological disturbances of these processes, when they occur, result from: i) alteration of a microbial ecosystem, originally conserved by evolution; ii) reduced consumption of microorganisms; iii) invasion of pathogens; or iv) modern interventions. Recent data support the use of proven probiotic organisms in prevention and treatment of flora-related gastrointestinal disorders including inflammatory bowel disease, infectious and antibiotic related diarrheas, and post-resection disorders including pouchitis. Therapeutic activity of probiotic bacteria can be due to competition with pathogens for nutrients and mucosal adherence, production of antimicrobial substances, and modulation of mucosal immune functions. Although a promising treatment, controlled clinical trials are necessary to validate the benefit of probiotics.

In a pilot-scale, open-label study to determine the ability of well-characterized probiotic Lactobacillus salivarius UCC118 cells to adhere to human epithelial cells in situ , the bacterial strain was administered to ulcerative colitis patients at approximately 10 9 CFU/day for 12 days. Microbiological analysis of biopsy specimens demonstrated that the ingested bacteria effectively adhered to both inflamed and non-inflamed mucosa of the large bowel in significant numbers. In previous reports, we have described the ability of the lactobacilli to adhere to enterocytic epithelial cells in vitro . In this study, we found that the bacteria adhered at higher levels to differentiated rather than undifferentiated epithelial monolayers; and that stationary phase lactobacilli were found to adhere to eukaryotic HT-29 and Caco-2 epithelial cells at greater levels than log phase bacterial cells. Pretreatment of the Lactobacillus cells with proteolytic enzymes abolished attachment, indicating the potential involvement of surface/exposed protein(s) as bacterial adhesin(s). SDS-PAGE (denaturing) techniques determined that the proteolytic treatment resulted in degradation of a cell wall-associated protein of approximately 84 kDa. The proteinaceous factor was purified by both anion-exchange chromatography and by gel extraction after SDS-PAGE electrophoresis, and under in vitro assay conditions proved capable of adherence and significant inhibition of bacterial attachment to enterocytic epithelial cells. Key words: probiotic, Lactobacillus, adhesin, cell-borne, proteinaceous.

The process of therapeutic “electroporation” involves application of electric fields to target tumour cells, thereby rendering them transiently porous such that uptake of any deliberately introduced therapeutic agent present will be enhanced. The objective of this research programme is the development of flexible electrode arrays for incorporation into microsystem devices, and assessment of their efficacy in delivering selected genetic and pharmaceutical anti-cancer therapies. Gold electrodes were fabricated on flexible polyimide substrates following predictive modelling and simulation of electric fields using FEMLAB™ software. Subsequent assessment of electroporation efficiency involved enumeration of viable tumour cells and, where appropriate, quantification of emitted fluorescence by genetically altered cells.

Flow cytometry has been validated as an objective method of demonstrating and quantifying micrometastases. Micrometastases within bone marrow indicate a poor prognosis in patients with upper gastrointestinal, colorectal and breast epithelial tumours. We prospectively sought to assess the feasibility of testing rib marrow for bone marrow micrometastases in head and neck cancer and to report their frequency in a cohort of patients. Nine patients were enrolled in the study. Bone marrow was obtained before manipulation of the primary tumour. Micrometastatic cells were detected by staining contaminant cytokeratin-18 positive cells and using the twin techniques of immuncytochemistry and flow cytometry. Cellular marrow was retrieved in 100% of cases. Micrometastases were detected in one out of nine epithelial tumours on both flow cytometry and immunocytochemistry. The detection rate appeared to be independent of TN staging. We were unable to culture the cells. Preoperative detection of bone marrow micrometastases may reflect transient shedding of cells, metastatic potential or residual disease. This prospective study confirms the feasibility of using rib marrow in future studies investigating micrometastases in head and neck cancer.

Background Bone marrow micrometastases are present in a high proportion of patients undergoing curative resection for esophagogastric cancer. The incorporation of preoperative systemic therapies into these patients’ treatment is widely practiced. This study investigates the effect of neoadjuvant chemoradiotherapy (CRT) on the incidence of micrometastases and the viability of detected tumor cells. Materials and methods Rib bone marrow was obtained from patients (n = 106) in three centers, who were selected for potentially curative resection. Patients received neoadjuvant CRT plus surgery (n = 55), or surgery alone (n = 51). To detect micrometastases, mononuclear cells were isolated from fresh marrow and immediately stained immunohistochemically with an anti-cytokeratin-18 antibody using the APAAP technique. Tumor cell viability was assessed by immunohistochemical staining of marrow cell cultures for cytokeratin-positive cells. Results Micrometastases were detected in fresh marrow in 42% (23/55) of patients who received neoadjuvant CRT plus surgery, and in 67% (34/51) of patients treated with surgery alone. Viable tumor cells were demonstrated in 10 of 18 marrow cultures from CRT plus surgery cases. In this patient subset, combination of results of staining fresh and cultured marrow significantly increased micromet detection to 78%. Conclusions A significant proportion of patients with esophagogastric cancer have disseminated viable tumor cells at time of surgery, irrespective of pre-operative treatment. The use of marrow culture in parallel with fresh marrow staining may increase the detection of micrometastases. The persistence of tumor cells resistant to systemic therapy may explain why these regimens fail in a majority of patients.

Many cancers express Fas ligand (FasL/CD95L) in vivo, and can kill lymphoid cells by Fas-mediated apoptosis in vitro. However, overexpression of recombinant FasL in murine tumour allografts revealed a potential antitumour effect of FasL, via recruitment of neutrophils. Transforming growth factor-beta1 (TGF-beta1) could inhibit these neutrophil-stimulatory effects of FasL. In the present study, we sought to determine directly whether FasL contributes to immune privilege or tumour rejection in human colon cancers in vivo, and whether TGF-beta1 regulates FasL function. Serial tumour sections were immunostained for FasL and TGF-beta1. Neutrophils and tumour infiltrating lymphocytes (TILs) were detected by immunohistochemistry for lactoferrin and CD45, respectively. Apoptotic TIL were identified by dual staining for TUNEL/CD45. FasL expression by nests of tumour cells was associated with a mean four-fold depletion of TILs (range 1.8-33-fold, n=16, P

Expression of Fas ligand (FasL/CD95L) may help to maintain colon cancers in a state of immune privilege by inducing apoptosis of antitumor immune effector cells. Colon tumor-derived cell lines appear to be relatively insensitive to apoptosis mediated by their own or exogenous FasL in vitro, despite expression of cell surface Fas. In our present study, we sought to investigate if FasL upregulated in human colon cancers leads to any increase in apoptosis of the tumor cells in vivo. FasL and Fas receptor (APO-1/CD95) expression by tumor cells were detected immunohistochemically. Apoptotic tumor cell death was detected by immunohistochemistry for caspase-cleaved cytokeratin-18. FasL expression did not correlate with the extent of apoptosis of tumor cells. There was no significant local difference in the frequency of apoptosis of tumor cells between tumor nests that expressed FasL (mean = 2.4%) relative to those that did not (mean = 2.6%) (p = 0.625, n = 10; Wilcoxon signed rank). FasL expressed by the tumor cells appeared to be functional, since FasL expression in tumor nests correlated with diminished infiltration of tumor-infiltrating lymphocytes (TILs). TILs were detected using immunohistochemistry for CD45. Expression of FasL by tumor nests was associated with a mean 4-fold fewer TILs relative to FasL-negative nests (range 2.4–33-fold, n = 10, p < 0.003). Together, our results indicate that colon tumors are insensitive to FasL-mediated apoptosis in vivo. © 2003 Wiley-Liss, Inc.

Background and aims: The uncertainty surrounding the role of Mycobacterium avium subsp paratuberculosis (Map) in Crohn’s disease has been compounded by possible contamination from Map present in the lumen microflora. This study used laser capture microdissection (LCM) and polymerase chain reaction (PCR) to detect Map DNA in subepithelial granulomas, isolated from 15 surgically resected, formalin fixed specimens of granulomatous Crohn’s disease and from 12 granulomatous disease controls (10 bowel, 2 non-bowel). Methods: The effect of amplicon size on reliability of PCR from formalin fixed samples was examined by amplifying 435 bp and 133 bp sequences of the human APC gene. After this, nested primers were designed to detect a small fragment (155 bp) of the Map specific IS900 gene in Crohn’s granulomas. LCM isolated granulomas from Map culture positive bovine intestine was used as positive control. PCR product specificity was confirmed by direct DNA sequencing. Results: The smaller, but not the larger, fragment of the APC gene amplified reliably in all samples. Amplification of the 155 bp fragment of the IS900 gene detected Map DNA in microdissected Crohn’s granulomas in 6 of 15 cases, and in 0 of 12 disease control granulomas. Conclusions: LCM can be used to detect Map DNA in granulomas in a proportion of patients with Crohn’s disease. However, formalin fixation requires that comparatively short DNA fragments of the Map specific IS900 gene be targeted, to permit consistent detection. Detection of Map DNA within granulomas might suggest an infectious aetiology in a subset of patients; alternatively, a transmissible agent may not be involved but mycobacterial DNA may influence pathogenesis by modifying the local cytokine responses.

Oral administration of tumour cells induces an immune hypo-responsiveness known as oral tolerance. We have previously shown that oral tolerance to a cancer is tumour antigen specific, non-cross-reactive and confers a tumour growth advantage. We investigated the utilisation of regulatory T cell (Treg) depletion on oral tolerance to a cancer and its ability to control tumour growth. Balb/C mice were gavage fed homogenised tumour tissue - JBS fibrosarcoma (to induce oral tolerance to a cancer), or PBS as control. Growth of subcutaneous JBS tumours were measured; splenic tissue excised and flow cytometry used to quantify and compare systemic Tregs and T effector (Teff) cell populations. Prior to and/or following tumour feeding, mice were intraperitoneally administered anti-CD25, to inactivate systemic Tregs, or given isotype antibody as a control. Mice which were orally tolerised prior to subcutaneous tumour induction, displayed significantly higher systemic Treg levels (14% vs 6%) and faster tumour growth rates than controls (p

Background: Primary hyperparathyroidism affects 1 in 700 individuals in the United States. A single adenoma is responsible in over 85% of cases. Surgery remains the most effective treatment. This study was designed to assess the feasibility of minimally invasive radioguided parathyroidectomy MIRP with confirmation of excision by ex vivo radioactivity alone. Methods: Seventy-five consecutive patients with primary hyperparathyroidism were prospectively studied. Following sestamibi scan, patients underwent unilateral neck exploration guided by a handheld gamma probe, which was also used to measure ex vivo radioactivity of excised tissue. Results: The sestamibi scan was positive in 88% of the patients. A small incision mean, 3.2 ± 0.3 cm was sufficient. Ectopic gland sites were localized in five patients with positive scans and single adenomas. Mean operative time was 48 minutes range, 15–125 minutes, with shorter procedures after the initial 20 cases mean, 24 vs. 72 minutes; P < .01. Radioguided parathyroidectomy was successful in 97%, with a mean follow-up of 11 months range, 1–26 months. As noted previously, adenomatous parathyroid glands contained more than 20% of the background radioactivity. Conclusions: MIRP is a feasible alternative to bilateral dissection with the advantages of guided dissection and rapid confirmation, and may become the procedure of choice for primary hyperparathyroidism.

Background: The enteric bacterial flora has been implicated in the pathogenesis of enterocolitis and colon cancer in C57BL/6 IL-10 knockout mice. Probiotic Lactobacilli modify the enteric flora and are thought to have a beneficial effect on enterocolitis. We conducted a controlled feeding trial in IL-10 knockout mice using the probiotic Lactobacillus salivarius ssp. salivarius UCC118. Aim: To determine the effect of probiotic consumption on the gastrointestinal microflora, tumour development and colitis in IL-10 knockout mice. Methods: Twenty IL-10 knockout mice were studied (10 consumed probiotic organisms in milk and 10 consumed unmodified milk) for 16 weeks. Faecal microbial analysis was performed weekly to enumerate excretion of the probiotic UCC118, total lactobacilli, Clostridium perfringens, bacteroides, coliforms, bifidobacteria and enterococci. At sacrifice, the small and large bowel were microbiologically and histologically assessed. Results: L. salivarius UCC118 was detected in faeces from all mice in the probiotic fed group, but not the control group. Faecal coliform and enterococci levels were significantly reduced in probiotic fed animals compared to the controls (P

Fas ligand (FasL) has become an enigmatic molecule: some evidence indicates that it contributes to immune privilege in tissues and tumors, whereas other data demonstrates that FasL can elicit inflammation. New findings may begin to reconcile the paradoxical effects of FasL.

1. We investigated the effect of oestradiol on basolateral potassium channels in human colonic epithelium. 2. Ion transport was quantified using short circuit current (I:(sc)) measurements of samples mounted in Ussing chambers. Serosal K transport was studied using nystatin permeabilization of the apical membrane. Intracellular pH changes were quantified using spectroflouresence techniques. 3. Experiments were performed with either 10 nM or 1 microM Ca(2+) in the apical bathing solution. With 10 nM Ca(2+) in the apical bathing solution addition of oestradiol (1 nM) to the basolateral bath produced a rapid increase in current (delta I(K)=11.2+/-1.2 microA.cm(-2), n=6). This response was prevented by treatment of the serosal membrane with tolbutamide (1 microM). With 1 microM Ca(2+) in the apical bathing solution addition of oestradiol produced a rapid fall in current (delta I(K)=-12.8+/-1.4 microA.cm(-2)), this response was prevented by treatment of the basolateral membrane with tetra-pentyl-ammonium (TPeA). These responses were rapid and occurred independently of protein synthesis. 4. Inhibition of basolateral Na(+)/H(+) exchange with either amiloride or a low sodium bathing solution prevented this response. These responses were prevented by inhibition of protein kinase C (PKC) with bis-indolyl-maleimide. 5. Oestradiol (1 nM) produced a rapid intracellular alkanization (mean increase=0.11 pH units; n=6; P

Background Oestrogens are important mitogens in epithelial cancers, particularly where tumours express complementary receptors. While the traditional model of oestrogen action involves gene-directed (genomic) protein synthesis, it has been established that more rapid, non-genomic steroid hormone actions exist. This study investigated the hypothesis that oestrogen rapidly alters cell membrane activity, intracellular pH and nuclear kinetics in a mitogenic fashion. Methods Crypts isolated from human distal colon and colorectal cancer cell lines were used as robust models. DNA replication and intracellular pH were measured by radiolabelled thymidine incorporation (12 h) and spectrofluorescence imaging respectively. Genomic protein synthesis, sodium–hydrogen exchanger (NHE) and protein kinase C (PKC) activity were inhibited with cycloheximide, ethylisopropylamiloride and chelerythrine chloride respectively. Results Oestrogen induced a rapid (less than 5 min) cellular alkalinization of crypts and cancer cells that was sensitive to NHE blockade (P < 0·01) or PKC inhibition (P < 0·01). Oestrogen increased thymidine incorporation by 44 per cent in crypts and by up to 38 per cent in cancer cells (P < 0·01), and this was similarly reduced by inhibiting the NHE (P < 0·01) or PKC (P < 0·05). Conclusion Oestrogen rapidly activates cell membrane and nuclear kinetics by a non-genomic mechanism mediated by PKC but not gene-directed protein synthesis.