Hedgehog Signalling in Androgen Independent Prostate Cancer

Objectives: Androgen-deprivation therapy effectively shrinks hormone-naïve prostate cancer, both in the prostate and at sites of distant metastasis. However prolonged androgen deprivation generally results in relapse and androgen-independent tumour growth, which is inevitably fatal. The molecular events that enable prostate cancer cells to proliferate in reduced androgen conditions are poorly understood. Here we investigate the role of Hedgehog signalling in androgen-independent prostate cancer (AIPC). Methods: Activity of the Hedgehog signalling pathway was analysed in cultured prostate cancer cells, and circulating prostate tumour cells were isolated from blood samples of patients with AIPC. Results: AIPC cells were derived through prolonged culture in reduced androgen conditions, modelling hormone therapy in patients, and expressed increased levels of Hedgehog signalling proteins. Exposure of cultured AIPC cells to cyclopamine, which inhibits Hedgehog signalling, resulted in inhibition of cancer cell growth. The expression of the Hedgehog receptor PTCH and the highly prostate cancer-specific gene DD3PCA3 was significantly higher in circulating prostate cancer cells isolated from patients with AIPC compared with samples prepared from normal individuals. There was an association between PTCH and DD3PCA3 expression and the length of androgen-ablation therapy. Conclusions: Our data are consistent with reports implicating overactivity of Hedgehog signalling in prostate cancer and suggest that Hedgehog signalling contributes to the androgen-independent growth of prostate cancer cells. As systemic anti-Hedgehog medicines are developed, the Hedgehog pathway will become a potential new therapeutic target in advanced prostate cancer.Peer reviewedFinal Accepted Versio

Expression of Distinct Desmocollin Isoforms in Human Epidermis

Previous evidence suggested the presence of two distinct desmocollin isoforms in human epidermis. These isoforms have now been distinguished at the protein level using monoclonal and polyclonal antibodies against N-terminal fragments of desmosomal glycoprotein (DG) IV/V isolated from plantar callus and antibodies against a fusion protein containing the extracellular domain of DGII/III. Immune blotting of glycoprotein fractions from whole epidermis, plantar callus, psoriatic scales and cultured keratinocytes showed that intact DGIV/V and its proteolytic fragments consistently migrated faster than DGII/III during SDS-PAGE. The apparent Mr difference between the two isoforms was in the range 2-5 kD. DGIV/V was the predominant species in epidermal tissue but was much less prominent in cultured cells by immune-blotting and immune precipitation. This is consistent with the differentiation-related expression of desmocollins revealed by immunofluorescence. DGIV/V was strongly expressed in the upper spinous/granular layer of the epidermis whereas DGII/III was more prominent in the basal layers of he tissue. The DGIV/V monoclonal (LH50) recognized an N-terminal, Ca++-sensitive epitope, because its staining of unfixed epidermal tissue was makedly influenced by Ca++ levels. Ca++ inhibition was observed at concentrations as low as 50 μM, suggesting its possible physiologic significance. Ca++ inhibition of LH50 binding was also observed in an enzyme-linked immunosorbent assay system using denatured glycoproteins although higher concentrations were required. It remains to be seen whether direct effects of Ca++ on desmocollin conformation are involved in the regulations of keratinization by extracellular Ca++

Detection of TMPRSS2 : ERG fusion gene in circulating prostate cancer cells

Creative Commons Attribution-NonCommercial-Share Alike 3.0 license (CC BY-NC SA)Aim: To investigate the existence of TMPRSS2:ERG fusion gene in circulating tumor cells (CTC) from prostate cancer patients and its potential in monitoring tumor metastasis. Methods: We analyzed the frequency of TMPRSS2: ERG and TMPRSS2:ETV1 transcripts in 27 prostate cancer biopsies from prostatectomies, and TMPRSS2:ERG transcripts in CTC isolated from 15 patients with advanced androgen independent disease using reverse transcription polymerase chain reaction (RT-PCR). Fluorescence in situ hybridization (FISH) was applied to analyze the genomic truncation of ERG, which is the result of TMPRSS2:ERG fusion in 10 of the 15 CTC samples. Results: TMPRSS2: ERG transcripts were found in 44% of our samples, but we did not detect expression of TMPRSS2:ETV1. Using FISH analysis we detected chromosomal rearrangements affecting the ERG gene in 6 of 10 CTC samples, including 1 case with associated TMPRSS2:ERG fusion at the primary site. However, TMPRSS2:ERG transcripts were not detected in any of the 15 CTC samples, including the 10 cases analyzed by FISH. Conclusion: Although further study is required to address the association between TMPRSS2:ERG fusion and prostate cancer metastasis, detection of genomic truncation of the ERG gene by FISH analysis could be useful for monitoring the appearance of CTC and the potential for prostate cancer metastasis.Peer reviewedFinal Published versio

Binding of Integrin α6β4 to Plectin Prevents Plectin Association with F-Actin but Does Not Interfere with Intermediate Filament Binding

Hemidesmosomes are stable adhesion complexes in basal epithelial cells that provide a link between the intermediate filament network and the extracellular matrix. We have investigated the recruitment of plectin into hemidesmosomes by the α6β4 integrin and have shown that the cytoplasmic domain of the β4 subunit associates with an NH2-terminal fragment of plectin that contains the actin-binding domain (ABD). When expressed in immortalized plectin-deficient keratinocytes from human patients with epidermol- ysis bullosa (EB) simplex with muscular dystrophy (MD-EBS), this fragment is colocalized with α6β4 in basal hemidesmosome-like clusters or associated with F-actin in stress fibers or focal contacts. We used a yeast two-hybrid binding assay in combination with an in vitro dot blot overlay assay to demonstrate that β4 interacts directly with plectin, and identified a major plectin-binding site on the second fibronectin type III repeat of the β4 cytoplasmic domain. Mapping of the β4 and actin-binding sites on plectin showed that the binding sites overlap and are both located in the plectin ABD. Using an in vitro competition assay, we could show that β4 can compete out the plectin ABD fragment from its association with F-actin. The ability of β4 to prevent binding of F-actin to plectin explains why F-actin has never been found in association with hemidesmosomes, and provides a molecular mechanism for a switch in plectin localization from actin filaments to basal intermediate filament–anchoring hemidesmosomes when β4 is expressed. Finally, by mapping of the COOH-terminally located binding site for several different intermediate filament proteins on plectin using yeast two-hybrid assays and cell transfection experiments with MD-EBS keratinocytes, we confirm that plectin interacts with different cytoskeletal networks

An interdisciplinary team communication framework and its application to healthcare 'e-teams' systems design

<p>Abstract</p> <p>Background</p> <p>There are few studies that examine the processes that interdisciplinary teams engage in and how we can design health information systems (HIS) to support those team processes. This was an exploratory study with two purposes: (1) To develop a framework for interdisciplinary team communication based on structures, processes and outcomes that were identified as having occurred during weekly team meetings. (2) To use the framework to guide 'e-teams' HIS design to support interdisciplinary team meeting communication.</p> <p>Methods</p> <p>An ethnographic approach was used to collect data on two interdisciplinary teams. Qualitative content analysis was used to analyze the data according to structures, processes and outcomes.</p> <p>Results</p> <p>We present details for team meta-concepts of structures, processes and outcomes and the concepts and sub concepts within each meta-concept. We also provide an exploratory framework for interdisciplinary team communication and describe how the framework can guide HIS design to support 'e-teams'.</p> <p>Conclusion</p> <p>The structures, processes and outcomes that describe interdisciplinary teams are complex and often occur in a non-linear fashion. Electronic data support, process facilitation and team video conferencing are three HIS tools that can enhance team function.</p

Biallelic Mutations in p16(INK4a) Confer Resistance to Ras- and Ets-Induced Senescence in Human Diploid Fibroblasts

The INK4a/ARF tumor suppressor locus is implicated in the senescence-like growth arrest provoked by oncogenic Ras in primary cells. INK4a and ARF are distinct proteins encoded by transcripts in which a shared exon is decoded in alternative reading frames. Here we analyze dermal fibroblasts (designated Q34) from an individual carrying independent missense mutations in each copy of the common exon. Both mutations alter the amino acid sequence of INK4a and functionally impair the protein, although they do so to different degrees. Only one of the mutations affects the sequence of ARF, causing an apparently innocuous change near its carboxy terminus. Unlike normal human fibroblasts, Q34 cells are not permanently arrested by Ras or its downstream effectors Ets1 and Ets2. Moreover, ectopic Ras enables the cells to grow as anchorage-independent colonies, and in relatively young Q34 cells anchorage independence can be achieved without addition of telomerase or perturbation of the p53 pathway. Whereas ARF plays the principal role in Ras-induced arrest of mouse fibroblasts, our data imply that INK4a assumes this role in human fibroblasts