2007 Winter Meeting - London - The Pathological Society of Great ...

P9PKC ZETA ALTERNATIVE SPLICING IN PROSTATECANCER{P} S J Ireland, C S Foster, C M GosdenUniversity of Liverpool, Liverpool, United KingdomThe human Protein Kinase C zeta gene is located, at 1p36.32-1p36.3. The geneencodes isoforms of the enzyme that are regulators of many homeostaticsignalling mechanisms responsible for co-ordinating changes that modulatecellular phenotypes. Through the mechanism of alternative splicing, variants ofPKC zeta are believed to exert a range of physiological effects includingepithelial cell invasiveness and development of metastases.The data examine the splice variants of PKC zeta present in prostate cell linesand matched normal and tumour tissues as assessed by genome walking. RealTime PCR data shows the increase in expression of two PKC zeta splicevariants (a and r) as malignancy progresses.An Antisense knockdown of PKC zeta in PC3-M and Du145 cell lines showsthat cells with a reduced expression of PKC zeta exhibit reduced invasioncapacity and proliferation rate.IHC staining of 955 prostate tissues (TA-PG tissue microarray) shows splicevariants of PKC zeta have different subcellular localisation. Tumour samplesshow a higher level of positive PKC zeta staining than normal samples.Increase in expression of nuclear splice variant r was early event in theprogression of prostate cancer. The changes in cytoplasmic splice variant asuggest a change in function. Survival analysis showed that positive PKC zetastaining is related to poor survival compared with negative staining. Stainingfor PKC zeta is both diagnostic and prognostic.Splice variants of PKC zeta through their position in key signalling pathwayspresent themselves as new therapeutic targets.P10The Characterisation and Culture of Human Foetal Kidneys{P} D.K. Franklin, N.A. Hanley, P.S. Bass, J.E. CollinsSchool of Medicine University of Southampton, Southampton, UnitedKingdomRenal tubules differentiate from metanephric mesenchymal progenitor cells.The human kidney, at 7 to 10 weeks gestational age, is forming many newnephron units in rapidly enlarging developing kidneys. This important resourceallows mapping of foetal expression of many known stem cell markers,established from work mainly in mouse. The aim of this study was to stainhuman foetal kidneys with stem cell and differentiation markers and to assessthe capacity of tissue to survive and differentiate in culture. Staining waspositive for N-cadherin in the nephrogenic mesenchyme and in nascentproximal tubules which also stained with claudin 2. Epithelial membraneantigen (EMA) and E-cadherin were stained in nascent distal tubules. Dispasedisaggregated foetal tissue formed cell monolayers that were positive fordifferentiation markers, corresponding to fixed tissue. Cells were also positivefor stem cell markers CD133, Cdx1, Pax 2, Oct 4, TRA1-60, β-catenin andSSEA-4. Cultured explants of foetal tissue formed tubules in Matrigel, whilecollagenase disaggregated tissue formed cysts showing that these preparationshave developmental potential in vitro. Improved understanding of themechanisms that renal progenitor cells use to survive and differentiate may leadto new therapies aimed at promoting or enhancing the repair of diseased adultkidneys.P11The Effect of Cyclosporine A and TGF-β1 on Differentiationof Primary Human Renal Tubular Epithelial CellsAKirk,{P} D.K. Franklin, S.K. Campbell, M.A. Hardyman,P.S. Bass, J.C. Mason, J.E. CollinsSchool of Medicine, University of Southampton, Southampton, UnitedKingdomThe widely used immunosuppressant Cyclosporine A (CsA) stimulates theproduction of TGF-β in renal tubular epithelial cells (RTEC). The aim was tocharacterize the effects of CsA and TGF-β1 on the differentiation of culturedprimary RTEC. Antibody staining was observed on tissue sections, with N-cadherin and claudin 2 localised in subapical cell junctional areas of proximaltubules and E cadherin and epithelial membrane antigen (EMA) in distaltubules. Primary RTEC in serum-free medium were treated with CsA or TGFβ1for 72 hours and stained for N-cadherin, claudin 2, E-cadherin, alpha smoothmuscle actin (αSMA) and EMA. Most cells were N-cadherin and claudin 2positive suggesting that they were of proximal phenotype. Smaller percentagesof cells stained for E-cadherin and EMA. Cells lost their membrane staining forclaudin 2 in response to CsA and TGF- β1, whereas N-cadherin becameredistributed in the membrane. All cells retained cytokeratin, EMA and αSMAalthough their morphology became more flattened and elongated withalignment of actin filaments. These results suggest that tight and adherensjunctions of primary human RTEC are changed or lost upon treatment with CsAand TGF-β1. In vivo these effects would likely compromise the function ofexisting healthy tubulesP12This abstract is not availablefor publicationuntil after presentationat the Meeting30 Winter Meeting (191 st ) 3–5 January 2007 Scientific Programme