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EXPRESSION OF PBX1 AND UGT2B7 IN PROSTATE CANCER CELLS
29
CASTRATION-INDUCED EPITHELIAL CELL DEATH IN PROSTATE TISSUE IS RELATED TO LOCALLY REDUCED IGF-1 LEVEL AND ACTION Wikstr¨om P.1 , Ohlson N.1 , Bergh A.1 , Stattin P.2 1 Ume˚a University, Medical Biosciences, Pathology, Ume˚a, Sweden; 2 Ume˚a University, Surgical and Perioperative Sciences, Urology & Andrology, Ume˚a, Sweden
INTRODUCTION & OBJECTIVES: The mechanisms by which castration induces prostate involution are largely unknown. In a recent study we therefore explored early responses to castration in the mouse ventral prostate (VP) by cDNA array expression analysis (Ohlson et al, Prostate, in press). As several changes occurred in the insulinlike growth factor (IGF) system this was studied in more detail. Castration was shown to rapidly reduce stroma IGF-1 synthesis and epithelial action in the mouse VP. We now explore if similar changes are of importance for castration-induced prostate and prostate cancer regression also in humans. MATERIAL & METHODS: Prostate biopsies were obtained from patients with advanced prostate cancer before and within two weeks after castration therapy. Non-malignant and malignant epithelial cells and surrounding stroma cells were laser micro-dissected. IGF-1 mRNA levels were quantified using real time RTPCR and related to androgen receptor (AR) immunoreactivity and to epithelial cell proliferation and apoptosis. The IGF-1 and IGF-1 receptor proteins were studied by immunohistochemistry. RESULTS: IGF-1 mRNA was principally produced in the stroma in non-malignant and malignant prostate tissue, while IGF-R1 was predominately present in the glandular epithelium. Stroma IGF-1 mRNA levels were significantly decreased after castration in non-malignant, but not in malignant prostate tissue. Lack of stromal IGF-1 reduction in the tumor after castration was associated with low stromal AR expression before therapy. Castration-induced decrease of IGF-1 mRNA levels in the tumor stroma and/or epithelial cells were associated with high levels of epithelial apoptosis after therapy. The question whether castration therapy decreases IGF-1 responsiveness in human epithelial cells, as shown in normal epithelial cells in the mouse VP, remains to be examined. CONCLUSIONS: Rapid decrease of stromal IGF-1 levels and epithelial action after castration may thus mediate some of the key features of castration-induced normal prostate involution in both mice and men. Low AR expression in tumor stroma cells suggests androgen-independency and, consequently, lack of stroma IGF-1 reduction and limited tumor cell apoptosis after castration. The high levels of IGF-1 in bone may thus possibly limit castration response in skeletal metastases.
30 MULTI-COLOUR STAINING AND TISSUEFAXS ANALYSIS FOR VALIDATION OF PROSTATE CANCER TISSUE MARKERS AND THERAPY TARGETS EXPRESSION Pfeil K.1 , Seifarth C.1 , Sch¨afer G.2 , Steiner G.3 , Rogojanu R.3 , Gaillard G.3 , Nowak C.4 , Klocker H.1 1 Innsbruck Medical University, Department of Urology, Innsbruck, Austria; 2 Innsbruck Medical University, Department of Pathology, Innsbruck, Austria; 3 Tissue Gnostics, TG, Vienna, Austria; 4 Advanced Computer Vision, ACV, Vienna, Austria INTRODUCTION & OBJECTIVES: For validation of potential new prostate cancer markers and therapy targets analysis of protein expression in prostate tissue at cellular resolution is important. We developed multicolour immunohistochemistry (IHC) and immunofluorescence (IF) staining to assign protein co-expression levels in the different cell types. MATERIAL & METHODS: Multicolour IHC and IF staining techniques were established for paraffin-embedded prostate tissue specimens employing cell type specific and androgen receptor antibodies. Quantification of expression was done using image analysis by TissueFAXS. RESULTS: For IHC tetra labelling, a peroxidase detection system was used in conjunction with different colour substrates and sequential staining steps. The use of antibodies directed against cell markers of different cell types and the antigen in question (e.g. androgen receptor) allowed to determine the cellular composition and the relative expression levels in different cell types. The same principle was used for IF, employing primary and secondary antibodies from different species labelled with different fluorochromes in combination with DAPI for nuclear staining. Quantitative expression levels were determined by employing TissueFAXS analysis with newly developed computer algorithms for the automated identification of prostatic tubular ducts and individual cells within a section. Co-expression profile patterns can be used for the automated classification of normal and malignant glands and determination of expression in different cell types. As an example this classifier method was applied for the analysis of androgen receptor expression in benign as compared to matched tumor tissue. CONCLUSIONS: Using these techniques for expression analysis of known and potential new protein markers at cellular resolution is a step forward for using individual tumor signatures for a better assessment of the risk for tumor progression. Eur Urol Suppl 2006;5:794
EXPRESSION OF PBX1 AND UGT2B7 IN PROSTATE CANCER CELLS Berge V., Ramberg H., Eide T., Svindland A., Task´en K.A. Aker University Hospital, Oslo Urological University Clinic, Oslo, Norway
31
INTRODUCTION & OBJECTIVES: To study if expression of UGT2B and Pbx1 in LNCaP cells is an early marker for neuroendocrine differentiation and development of androgen-independent prostate cancer. In the absence of androgens, hormone-sensitive LNCaP-cells differentiate to neuroendocrine (NE)-like cells before they finally adapt to androgen-independent growth. We have previously characterized the transcriptosomes of normal and NE-like LNCaP-cells. Among the 200 genes regulated more than 2 fold in at least three independent experiments, was the gene Pbx1 (Pre-B cell leukaemia transcription factor), encoding a homeobox transcription factor involved in embryonic development and neuronal differentiation and UGT2B7 (UDP glucuronosyltransferase 2 family member 7), an enzyme mediating glucuronidation of several exogeneous and endogenous compunds. The UGT2B7 subfamily is present in the liver and several steroid target tissues and is an important member since it conjugates a large variety of compounds including androgens. UGT2B7 is a putative target gene for Pbx1 as it contains several putative Pbx binding sites within its promoter region MATERIAL & METHODS: Prostatic cell lines and human prostate cancer specimens were analyzed using real time RT-PCR, Western blot analysis and immunohistochemistry. The time-dependent regulation of Pbx1 and UGT2B7 mRNAs during NE-differentiation of LNCaP cells was determined by incubating the cells in charcoal-treated serum. RESULTS: Pbx1 is induced within 24 hours after induction of NE-differentiation of LNCaP-cells by incubating the cells in charcoal-stripped serum. Whereas Pbx1 was highly up-regulated in LNCaP-Rf cells, we observed reduced levels of Pbx1 in the other androgen-independent cell lines analyzed, LNCaP-C4, -C4-2B, PC-3 and DU145, relative to the hormone sensitive LNCaP-cells both at mRNA and protein level. A similar pattern of expression was observed for UGT2B7 mRNA. We are currently studying the expression of Pbx1 and UGT2B7 in prostate cancer specimens by real-time RT-PCR and immunohistochemistry. CONCLUSIONS: Our data indicate that Pbx1 and UGT2B7 expression can be early markers for neuroendocrine differentiation in prostate cancer although further studies are needed.
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ENGINEERING MAMMALIAN CELL TRANSCRIPTIONAL CONTROL SEQUENCES TO ACHIEVE IMPROVED TRANSCRIPTIONAL TARGETING OF PROSTATE TUMOURS Tieman K., Stanbridge L., Rippon H., Maitland N. University of York, Department of Biology, York, United Kingdom
INTRODUCTION & OBJECTIVES: To achieve transcriptional targeting for prostate cancer gene therapy there are two main issues affecting the use of prostate-specific promoters. Firstly most prostate-specific promoters are androgen regulated, which restricts their use for patients undergoing androgen ablation therapy. Secondly, most prostate-specific promoters are too weak to express therapeutic genes at active levels in a tissue setting. We are engineering the extended (4.5 kb) promoter sequence from a transglutaminase (hTGp) gene, whose expression in tissues (rather than cell cultures) is the most highly prostate-specific identified to date (Dubbink et al.,1998). In tissue cultures, powerful epigenetic silencing via methylation of an Sp1 site immediately upstream of the transcriptional start site, which results in the down-regulation of the native promoter in cancers has also been observed (Dubbink et al., 1999). Despite its extreme prostate-specificity, the hTGp promoter is probably not regulated directly by androgens, but is regulated by retinoic acid (RA) in normal tissues and cell lines (H. Rippon, 2003). AIM: Development of a strong androgen-independent and prostate-specific promoter construct. MATERIAL & METHODS: The transglutaminase expression pattern was analysed by RT-PCR in prostate cancer cell lines (LNCaP, PC3, P4E6 and PC346C) treated with demethylation agent (5azadC), atRA and DHT singly and in combination. To test the hypothesis that deletion of RA response elements (RARE) will eliminate an essential cell-specific control sequence we are carrying out a specific mutational analysis on the 3 RARE’s present in the hTGp 4.5 kb promoter. RESULTS: Single, double and triple RARE deletion mutants have been generated by PCR mutagenesis and fragment deletion. Promoter constructs, fused to an EGFP indicator gene, have been transfected into the range of prostate epithelial cell lines to assess the effects on expression levels. A deletion of 3.1 kbases, which eliminated the 3 predicted RARE’s within the hTGp 4.5 kb promoter, reduces specificity for prostate epithelial cells, but results in a high activity promoter. EMSA gelshift experiments have been carried out to confirm the binding of retinoic acid receptors to the 3 different RARE sequences found in the transglutaminase promoter under various expression conditions. CONCLUSIONS: Regulation of the hTGp promoter is complex and tightly regulated by cell differentiation state. Both specificity and promoter strength are located within a 3.1 kB region of the promoter sequence.
CASTRATION-INDUCED EPITHELIAL CELL DEATH IN PROSTATE TISSUE IS RELATED TO LOCALLY REDUCED IGF-1 LEVEL AND ACTION Wikstr¨om P.1 , Ohlson N.1 , Bergh A.1 , Stattin P.2 1 Ume˚a University, Medical Biosciences, Pathology, Ume˚a, Sweden; 2 Ume˚a University, Surgical and Perioperative Sciences, Urology & Andrology, Ume˚a, Sweden
INTRODUCTION & OBJECTIVES: The mechanisms by which castration induces prostate involution are largely unknown. In a recent study we therefore explored early responses to castration in the mouse ventral prostate (VP) by cDNA array expression analysis (Ohlson et al, Prostate, in press). As several changes occurred in the insulinlike growth factor (IGF) system this was studied in more detail. Castration was shown to rapidly reduce stroma IGF-1 synthesis and epithelial action in the mouse VP. We now explore if similar changes are of importance for castration-induced prostate and prostate cancer regression also in humans. MATERIAL & METHODS: Prostate biopsies were obtained from patients with advanced prostate cancer before and within two weeks after castration therapy. Non-malignant and malignant epithelial cells and surrounding stroma cells were laser micro-dissected. IGF-1 mRNA levels were quantified using real time RTPCR and related to androgen receptor (AR) immunoreactivity and to epithelial cell proliferation and apoptosis. The IGF-1 and IGF-1 receptor proteins were studied by immunohistochemistry. RESULTS: IGF-1 mRNA was principally produced in the stroma in non-malignant and malignant prostate tissue, while IGF-R1 was predominately present in the glandular epithelium. Stroma IGF-1 mRNA levels were significantly decreased after castration in non-malignant, but not in malignant prostate tissue. Lack of stromal IGF-1 reduction in the tumor after castration was associated with low stromal AR expression before therapy. Castration-induced decrease of IGF-1 mRNA levels in the tumor stroma and/or epithelial cells were associated with high levels of epithelial apoptosis after therapy. The question whether castration therapy decreases IGF-1 responsiveness in human epithelial cells, as shown in normal epithelial cells in the mouse VP, remains to be examined. CONCLUSIONS: Rapid decrease of stromal IGF-1 levels and epithelial action after castration may thus mediate some of the key features of castration-induced normal prostate involution in both mice and men. Low AR expression in tumor stroma cells suggests androgen-independency and, consequently, lack of stroma IGF-1 reduction and limited tumor cell apoptosis after castration. The high levels of IGF-1 in bone may thus possibly limit castration response in skeletal metastases.
30 MULTI-COLOUR STAINING AND TISSUEFAXS ANALYSIS FOR VALIDATION OF PROSTATE CANCER TISSUE MARKERS AND THERAPY TARGETS EXPRESSION Pfeil K.1 , Seifarth C.1 , Sch¨afer G.2 , Steiner G.3 , Rogojanu R.3 , Gaillard G.3 , Nowak C.4 , Klocker H.1 1 Innsbruck Medical University, Department of Urology, Innsbruck, Austria; 2 Innsbruck Medical University, Department of Pathology, Innsbruck, Austria; 3 Tissue Gnostics, TG, Vienna, Austria; 4 Advanced Computer Vision, ACV, Vienna, Austria INTRODUCTION & OBJECTIVES: For validation of potential new prostate cancer markers and therapy targets analysis of protein expression in prostate tissue at cellular resolution is important. We developed multicolour immunohistochemistry (IHC) and immunofluorescence (IF) staining to assign protein co-expression levels in the different cell types. MATERIAL & METHODS: Multicolour IHC and IF staining techniques were established for paraffin-embedded prostate tissue specimens employing cell type specific and androgen receptor antibodies. Quantification of expression was done using image analysis by TissueFAXS. RESULTS: For IHC tetra labelling, a peroxidase detection system was used in conjunction with different colour substrates and sequential staining steps. The use of antibodies directed against cell markers of different cell types and the antigen in question (e.g. androgen receptor) allowed to determine the cellular composition and the relative expression levels in different cell types. The same principle was used for IF, employing primary and secondary antibodies from different species labelled with different fluorochromes in combination with DAPI for nuclear staining. Quantitative expression levels were determined by employing TissueFAXS analysis with newly developed computer algorithms for the automated identification of prostatic tubular ducts and individual cells within a section. Co-expression profile patterns can be used for the automated classification of normal and malignant glands and determination of expression in different cell types. As an example this classifier method was applied for the analysis of androgen receptor expression in benign as compared to matched tumor tissue. CONCLUSIONS: Using these techniques for expression analysis of known and potential new protein markers at cellular resolution is a step forward for using individual tumor signatures for a better assessment of the risk for tumor progression. Eur Urol Suppl 2006;5:794
EXPRESSION OF PBX1 AND UGT2B7 IN PROSTATE CANCER CELLS Berge V., Ramberg H., Eide T., Svindland A., Task´en K.A. Aker University Hospital, Oslo Urological University Clinic, Oslo, Norway
31
INTRODUCTION & OBJECTIVES: To study if expression of UGT2B and Pbx1 in LNCaP cells is an early marker for neuroendocrine differentiation and development of androgen-independent prostate cancer. In the absence of androgens, hormone-sensitive LNCaP-cells differentiate to neuroendocrine (NE)-like cells before they finally adapt to androgen-independent growth. We have previously characterized the transcriptosomes of normal and NE-like LNCaP-cells. Among the 200 genes regulated more than 2 fold in at least three independent experiments, was the gene Pbx1 (Pre-B cell leukaemia transcription factor), encoding a homeobox transcription factor involved in embryonic development and neuronal differentiation and UGT2B7 (UDP glucuronosyltransferase 2 family member 7), an enzyme mediating glucuronidation of several exogeneous and endogenous compunds. The UGT2B7 subfamily is present in the liver and several steroid target tissues and is an important member since it conjugates a large variety of compounds including androgens. UGT2B7 is a putative target gene for Pbx1 as it contains several putative Pbx binding sites within its promoter region MATERIAL & METHODS: Prostatic cell lines and human prostate cancer specimens were analyzed using real time RT-PCR, Western blot analysis and immunohistochemistry. The time-dependent regulation of Pbx1 and UGT2B7 mRNAs during NE-differentiation of LNCaP cells was determined by incubating the cells in charcoal-treated serum. RESULTS: Pbx1 is induced within 24 hours after induction of NE-differentiation of LNCaP-cells by incubating the cells in charcoal-stripped serum. Whereas Pbx1 was highly up-regulated in LNCaP-Rf cells, we observed reduced levels of Pbx1 in the other androgen-independent cell lines analyzed, LNCaP-C4, -C4-2B, PC-3 and DU145, relative to the hormone sensitive LNCaP-cells both at mRNA and protein level. A similar pattern of expression was observed for UGT2B7 mRNA. We are currently studying the expression of Pbx1 and UGT2B7 in prostate cancer specimens by real-time RT-PCR and immunohistochemistry. CONCLUSIONS: Our data indicate that Pbx1 and UGT2B7 expression can be early markers for neuroendocrine differentiation in prostate cancer although further studies are needed.
32
ENGINEERING MAMMALIAN CELL TRANSCRIPTIONAL CONTROL SEQUENCES TO ACHIEVE IMPROVED TRANSCRIPTIONAL TARGETING OF PROSTATE TUMOURS Tieman K., Stanbridge L., Rippon H., Maitland N. University of York, Department of Biology, York, United Kingdom
INTRODUCTION & OBJECTIVES: To achieve transcriptional targeting for prostate cancer gene therapy there are two main issues affecting the use of prostate-specific promoters. Firstly most prostate-specific promoters are androgen regulated, which restricts their use for patients undergoing androgen ablation therapy. Secondly, most prostate-specific promoters are too weak to express therapeutic genes at active levels in a tissue setting. We are engineering the extended (4.5 kb) promoter sequence from a transglutaminase (hTGp) gene, whose expression in tissues (rather than cell cultures) is the most highly prostate-specific identified to date (Dubbink et al.,1998). In tissue cultures, powerful epigenetic silencing via methylation of an Sp1 site immediately upstream of the transcriptional start site, which results in the down-regulation of the native promoter in cancers has also been observed (Dubbink et al., 1999). Despite its extreme prostate-specificity, the hTGp promoter is probably not regulated directly by androgens, but is regulated by retinoic acid (RA) in normal tissues and cell lines (H. Rippon, 2003). AIM: Development of a strong androgen-independent and prostate-specific promoter construct. MATERIAL & METHODS: The transglutaminase expression pattern was analysed by RT-PCR in prostate cancer cell lines (LNCaP, PC3, P4E6 and PC346C) treated with demethylation agent (5azadC), atRA and DHT singly and in combination. To test the hypothesis that deletion of RA response elements (RARE) will eliminate an essential cell-specific control sequence we are carrying out a specific mutational analysis on the 3 RARE’s present in the hTGp 4.5 kb promoter. RESULTS: Single, double and triple RARE deletion mutants have been generated by PCR mutagenesis and fragment deletion. Promoter constructs, fused to an EGFP indicator gene, have been transfected into the range of prostate epithelial cell lines to assess the effects on expression levels. A deletion of 3.1 kbases, which eliminated the 3 predicted RARE’s within the hTGp 4.5 kb promoter, reduces specificity for prostate epithelial cells, but results in a high activity promoter. EMSA gelshift experiments have been carried out to confirm the binding of retinoic acid receptors to the 3 different RARE sequences found in the transglutaminase promoter under various expression conditions. CONCLUSIONS: Regulation of the hTGp promoter is complex and tightly regulated by cell differentiation state. Both specificity and promoter strength are located within a 3.1 kB region of the promoter sequence.