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614Decoy of androgen-responsive element induces apoptosis in LNCAP cells
613 DUAL SPECIFICITY PHOSPHATASE 1 AND SERUM/GLUCOCORTICOID REGULATED KINASE ARE DOWN REGULATED IN PROSTATE CANCER
614 DECOY OF ANDROGEN-RESPONSIVE APOPTOSIS IN LNCAP CELLS
ELEMENT
INDUCES
Su~imura K.. Kuratst&uri K., Harimoto K., Tanaka T., Kawashima H., Nakatani Y. Tammela T?, Rauhala H.2~ Tolonen T.3, Martikainen p.3, Porkka K.2~ Visakorpi T.:z Osaka City University, Department of Urology, Osaka, Japan ~Tampere University Hospital, Urology, Tampere, Finland, 2Institute of Medical Technotogy~ University o f Tampere, Cancer Genetics, Tampere, Finland, 3Tampere University Hospital, Pathology, Tampere, Finland INTRODUCTION & OBJECTIVES: Hypermethylation of the promoter is considered to be an alternative mechanism to mutation for inactivation of mmour suppressor genes. The aim of this study was to identify hypermethylated genes in prostate cancer. MATERIAL & METHODS: PC-3 prostate cancer cells were treated with DNA methylation and histone deacetylation inhibitors. Next, a combination of suppression subtraction hybridization (SSH) and cDNA microarrays were used to detect differentially expressed genes in the treated and non-treated cells. Real-time quantitatiye RT-PCR (Q-RT-PCR) and immunohistochemistry were then used to measure expression of selected genes in clinical material consisting of 51 frozen and 128 formalin-fixed paraffin-embedded specimens. RESULTS: 1! genes were found to be upreglulated by the treatments. Expression of 3 genes, dual specificity phosphatase 1 (DUSP1), serum/glucocorticoid regulated kinase (SGK) arid spermidine/spermine Nl-acetyltransferase (SAT), were subsequently studied in clinical sample material using Q-RT-PCR. The mRNA expression of DUSP1 and SGK was significantly lower in hormonerefractory prostate carcinomas compared to benign prostatic hyperplasia or unt~reated prostate carcinomas. Finally, the immunostaining demonstrated that 92% and 48% of the prostate carcinomas showed almost complete lack of DUSPI and SGK protein expression, respectively. CONCLUSIONS: Epigenetic down regulation of DUSPI and SGK could be important mechanisms for the tumourigenesis of prostate cancer.
INTRODUCTION & OBJECTIVES: The androgen receptor (AR) is activated by binding of androgens and binds to androgen responsive element (ARE) in the regulatory region of target genes as a transcriptional factor. We hypothesize that "ARE decoy", a double stranded oligonueleotide which includes the seine DNA sequence as ARE, can inhibit the proliferation of not only androgen dependent prostatic cancer but also androgen independent one due to AR gene mutations because it is assumed that the competitive inhibition of the transcriptional activity of AR occurs in both. MATERIAL & METHODS: We synthesized a 23-mer ARE decoy based on the deduced ARE sequence at the promoter region of the human prostate-specific antigen (PSA) gene. The nuclear extract was prepared from LNCaP cells, and DNA-protein interactions were examined by gel shift assay. Then the antiandrogen effect of the ARE decoy was studied in LNCaP cells transfected with the ARE decoy by lipofection. After 24-hr incubation with 10(-9) M dihydrotestosterone (DHT), induction of apoptosis was examined by DNA fragmentation. RESULTS: The gel shift assay demonstrated specific binding of the ARE decoy to the LNCaP nuclear protein which is most likely AR. The transfection experiment showed DNA fragmentation in the ARE decoy-transfected cells despite the presence of DHT, though not in the cells transfected with the control decoy. CONCLUSIONS: We demomstrated that ARE decoy had an anti-androgen effect attributed to the competitive inhibition of the activated AR. ARE decoy may become a potential therapeutic tool for both androgen dependent and independent prostatic cancers.
615
616
FINE STRUCTURAL ANALYSIS OF INVASIVE NEO-ANGIOGENESIS IN PROSTATE CARCINOMAS: H O W DO T U M O U R DUCTS B R E A K UP?
DETECTION OF PROSTATE CANCER USING SERUM PROTEOMICS PATTERN IN A HISTOLOGICAL CONFIRMED POPULATION
Debbage P.~, Wieser E. I, Rogatsch H.2~ Strohmeyer D?, Horninger W. 3, Bartsch G. 3
Feuerstein I. l, Rainer M?, Pelzer A. z, Hominger W. 2, Klocker H. 2, Kofler K. 2, Bartsch G. 2, Bernardo K. 1, Stecher G. 1, Huck C ) , Bonn G. 1
tMedical University of Innsbruck~ Department of Histology & Embryology, [nnsbruck, Austria, 2Medical University of Imasbruck, Department of Pathology, Innsbruck, Austria~ ~Medical University of Innsbruck, Depaxtment of Urology, Innsbrnck, Austria
1Medical University of Innsbruck, Dept. of Analytical Chemistry and Radiochemistry, Innsbruck, Austria, 2Medical University of Innsbmck, Dept. of Urology, Irmsbmck, Austria
INTRODUCTION & OBJECTIVES: Our previous work visualised the breakdown of tumour-blood barriers in highly malignant prostate carcinomas. Endothelial-tumour interactions with dynamic remodelling of the matrix documented reciprocal invasion as a striking feature of high grade malignancy. We analysed the forms this takes and assessed mutual attraction of vascular and tumour cells. MATERIAL & METHODS: The tumour bank we established previously was used, consisting of more than 40 epon-embedded prostate carcinomas of welldocumented malignant status. Semithin and ultrathin sections were viewed to locate sites of invasive neo-angiogenesis and these sites were imaged for context (magnification x1.000) and for fine structural detail (x5.000 to x20.000). RESULTS: Two forms of ductal breakdown were observed. Firstly, neoangiogenetic endothelial cells tunnelled into the basal side of the duct and extended endothelial tubes which broke through into the duct lumen. This was common in tumours of Gleason Score 9, much less so in Gleason Score 8. Early vascular penetration into the duct wall sometimes occurred at sites of epithelial cell death, associated with heavy local etching of the matrix. The endothelial cells were invariably flanked by ancillary cellular profiles, as yet unidentified. Secondly, and only at vascular hotspots, ductal epithelial cells assumed amoeboid forms and migrated out of the basal side of the duct towards neo-angiogenetic micro vessels. CONCLUSIONS: Normophysiologically regulated micro vessels in prostate glands lie aligned beneath the ducts. Neo-angiogenetic endothelial cells tunnelling into the duct wall indicates breakdown of normal regulation and suggests chemo attractive influences acting on the endothelial ceils. Emigration of tumour cells out of duct walls towards neo-angiogenetic endothelial cells likewise indicates breakdown of normal tissue regulation and suggests chemo attractive influences acting on turnout cells.
European Urology Supplements 4 (2005) No. 3, pp. 156
INTRODUCTION & OBJECTIVES: Although serum prostate specific antigen (PSA) and related isoforms provide some disease specificity of the prostate gland, PSA has not been proven specific enough for accurate prostate cancer detection. The objective of this study was to identify better biomarkers for early detection of prostate cancer using protein - profiling technologies that can simultaneously resolve and analyse multiple proteins. MATERIAL & METHODS: In the foilowing study we used derivatized cellulose particles for the direct analysis with MALDI-TOF-MS to generate serum profiles and an artificial intelligence learning algorithm to differentiate prostate cancer from non cancer cohorts. Protein profiles of sera from 435 men with either histologically confined prostate cancer, benign prostatic hyperplasia (BPH) (TZ volume > 30 g, negative prostate biopsy), histologically confirmed prostatitis or age matched tmaffected healthy men (PSA _<1.25 ng/ml) were used to train and develop a decision tree classification algorithm. RESULTS: Of the 435 men evaluated 56 had histologically confined prostate cancer, 51 had BPH, 56 prostatitis and 272 were unaffected, age matched men. With the above mentioned new protein profiling classification approach an overall sensitivity of 93% and a specificity of 100% was obtained in differentiating patients with prostate cancer from patients and volunteers without prostate cancer. CONCLUSIONS: The high sensitivity and -more important- the high specificity obtained by the serum protein profiling approach presented in this study demonstrate that this approach conthined with an artificial intelligence classification algorithm can facilitate the discovery of better biomarkers for prostate diseases and can be used for the early detection of prostate cancer.
614 DECOY OF ANDROGEN-RESPONSIVE APOPTOSIS IN LNCAP CELLS
ELEMENT
INDUCES
Su~imura K.. Kuratst&uri K., Harimoto K., Tanaka T., Kawashima H., Nakatani Y. Tammela T?, Rauhala H.2~ Tolonen T.3, Martikainen p.3, Porkka K.2~ Visakorpi T.:z Osaka City University, Department of Urology, Osaka, Japan ~Tampere University Hospital, Urology, Tampere, Finland, 2Institute of Medical Technotogy~ University o f Tampere, Cancer Genetics, Tampere, Finland, 3Tampere University Hospital, Pathology, Tampere, Finland INTRODUCTION & OBJECTIVES: Hypermethylation of the promoter is considered to be an alternative mechanism to mutation for inactivation of mmour suppressor genes. The aim of this study was to identify hypermethylated genes in prostate cancer. MATERIAL & METHODS: PC-3 prostate cancer cells were treated with DNA methylation and histone deacetylation inhibitors. Next, a combination of suppression subtraction hybridization (SSH) and cDNA microarrays were used to detect differentially expressed genes in the treated and non-treated cells. Real-time quantitatiye RT-PCR (Q-RT-PCR) and immunohistochemistry were then used to measure expression of selected genes in clinical material consisting of 51 frozen and 128 formalin-fixed paraffin-embedded specimens. RESULTS: 1! genes were found to be upreglulated by the treatments. Expression of 3 genes, dual specificity phosphatase 1 (DUSP1), serum/glucocorticoid regulated kinase (SGK) arid spermidine/spermine Nl-acetyltransferase (SAT), were subsequently studied in clinical sample material using Q-RT-PCR. The mRNA expression of DUSP1 and SGK was significantly lower in hormonerefractory prostate carcinomas compared to benign prostatic hyperplasia or unt~reated prostate carcinomas. Finally, the immunostaining demonstrated that 92% and 48% of the prostate carcinomas showed almost complete lack of DUSPI and SGK protein expression, respectively. CONCLUSIONS: Epigenetic down regulation of DUSPI and SGK could be important mechanisms for the tumourigenesis of prostate cancer.
INTRODUCTION & OBJECTIVES: The androgen receptor (AR) is activated by binding of androgens and binds to androgen responsive element (ARE) in the regulatory region of target genes as a transcriptional factor. We hypothesize that "ARE decoy", a double stranded oligonueleotide which includes the seine DNA sequence as ARE, can inhibit the proliferation of not only androgen dependent prostatic cancer but also androgen independent one due to AR gene mutations because it is assumed that the competitive inhibition of the transcriptional activity of AR occurs in both. MATERIAL & METHODS: We synthesized a 23-mer ARE decoy based on the deduced ARE sequence at the promoter region of the human prostate-specific antigen (PSA) gene. The nuclear extract was prepared from LNCaP cells, and DNA-protein interactions were examined by gel shift assay. Then the antiandrogen effect of the ARE decoy was studied in LNCaP cells transfected with the ARE decoy by lipofection. After 24-hr incubation with 10(-9) M dihydrotestosterone (DHT), induction of apoptosis was examined by DNA fragmentation. RESULTS: The gel shift assay demonstrated specific binding of the ARE decoy to the LNCaP nuclear protein which is most likely AR. The transfection experiment showed DNA fragmentation in the ARE decoy-transfected cells despite the presence of DHT, though not in the cells transfected with the control decoy. CONCLUSIONS: We demomstrated that ARE decoy had an anti-androgen effect attributed to the competitive inhibition of the activated AR. ARE decoy may become a potential therapeutic tool for both androgen dependent and independent prostatic cancers.
615
616
FINE STRUCTURAL ANALYSIS OF INVASIVE NEO-ANGIOGENESIS IN PROSTATE CARCINOMAS: H O W DO T U M O U R DUCTS B R E A K UP?
DETECTION OF PROSTATE CANCER USING SERUM PROTEOMICS PATTERN IN A HISTOLOGICAL CONFIRMED POPULATION
Debbage P.~, Wieser E. I, Rogatsch H.2~ Strohmeyer D?, Horninger W. 3, Bartsch G. 3
Feuerstein I. l, Rainer M?, Pelzer A. z, Hominger W. 2, Klocker H. 2, Kofler K. 2, Bartsch G. 2, Bernardo K. 1, Stecher G. 1, Huck C ) , Bonn G. 1
tMedical University of Innsbruck~ Department of Histology & Embryology, [nnsbruck, Austria, 2Medical University of Imasbruck, Department of Pathology, Innsbruck, Austria~ ~Medical University of Innsbruck, Depaxtment of Urology, Innsbrnck, Austria
1Medical University of Innsbruck, Dept. of Analytical Chemistry and Radiochemistry, Innsbruck, Austria, 2Medical University of Innsbmck, Dept. of Urology, Irmsbmck, Austria
INTRODUCTION & OBJECTIVES: Our previous work visualised the breakdown of tumour-blood barriers in highly malignant prostate carcinomas. Endothelial-tumour interactions with dynamic remodelling of the matrix documented reciprocal invasion as a striking feature of high grade malignancy. We analysed the forms this takes and assessed mutual attraction of vascular and tumour cells. MATERIAL & METHODS: The tumour bank we established previously was used, consisting of more than 40 epon-embedded prostate carcinomas of welldocumented malignant status. Semithin and ultrathin sections were viewed to locate sites of invasive neo-angiogenesis and these sites were imaged for context (magnification x1.000) and for fine structural detail (x5.000 to x20.000). RESULTS: Two forms of ductal breakdown were observed. Firstly, neoangiogenetic endothelial cells tunnelled into the basal side of the duct and extended endothelial tubes which broke through into the duct lumen. This was common in tumours of Gleason Score 9, much less so in Gleason Score 8. Early vascular penetration into the duct wall sometimes occurred at sites of epithelial cell death, associated with heavy local etching of the matrix. The endothelial cells were invariably flanked by ancillary cellular profiles, as yet unidentified. Secondly, and only at vascular hotspots, ductal epithelial cells assumed amoeboid forms and migrated out of the basal side of the duct towards neo-angiogenetic micro vessels. CONCLUSIONS: Normophysiologically regulated micro vessels in prostate glands lie aligned beneath the ducts. Neo-angiogenetic endothelial cells tunnelling into the duct wall indicates breakdown of normal regulation and suggests chemo attractive influences acting on the endothelial ceils. Emigration of tumour cells out of duct walls towards neo-angiogenetic endothelial cells likewise indicates breakdown of normal tissue regulation and suggests chemo attractive influences acting on turnout cells.
European Urology Supplements 4 (2005) No. 3, pp. 156
INTRODUCTION & OBJECTIVES: Although serum prostate specific antigen (PSA) and related isoforms provide some disease specificity of the prostate gland, PSA has not been proven specific enough for accurate prostate cancer detection. The objective of this study was to identify better biomarkers for early detection of prostate cancer using protein - profiling technologies that can simultaneously resolve and analyse multiple proteins. MATERIAL & METHODS: In the foilowing study we used derivatized cellulose particles for the direct analysis with MALDI-TOF-MS to generate serum profiles and an artificial intelligence learning algorithm to differentiate prostate cancer from non cancer cohorts. Protein profiles of sera from 435 men with either histologically confined prostate cancer, benign prostatic hyperplasia (BPH) (TZ volume > 30 g, negative prostate biopsy), histologically confirmed prostatitis or age matched tmaffected healthy men (PSA _<1.25 ng/ml) were used to train and develop a decision tree classification algorithm. RESULTS: Of the 435 men evaluated 56 had histologically confined prostate cancer, 51 had BPH, 56 prostatitis and 272 were unaffected, age matched men. With the above mentioned new protein profiling classification approach an overall sensitivity of 93% and a specificity of 100% was obtained in differentiating patients with prostate cancer from patients and volunteers without prostate cancer. CONCLUSIONS: The high sensitivity and -more important- the high specificity obtained by the serum protein profiling approach presented in this study demonstrate that this approach conthined with an artificial intelligence classification algorithm can facilitate the discovery of better biomarkers for prostate diseases and can be used for the early detection of prostate cancer.