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Supplementary Data from The MicroRNA Profile of Prostate Carcinoma Obtained by Deep Sequencing

Prostate cancer is a leading cause of tumor mortality. To characterize the underlying molecular mechanisms, we have compared the microRNA (miRNA) profile of primary prostate cancers and noncancer prostate tissues using deep sequencing. MiRNAs are small noncoding RNAs of 21 to 25 nucleotides that regulate gene expression through the inhibition of protein synthesis. We find that 33 miRNAs were upregulated or downregulated >1.5-fold. The deregulation of selected miRNAs was confirmed by both Northern blotting and quantitative reverse transcription-PCR in established prostate cancer cell lines and clinical tissue samples. A computational search indicated the 3′-untranslated region (UTR) of the mRNA for myosin VI (MYO6) as a potential target for both miR-143 and miR-145, the expression of which was reduced in the tumor tissues. Upregulation of myosin VI in prostate cancer was previously shown by immunohistochemistry. The level of MYO6 mRNA was significantly induced in all primary tumor tissues compared with the nontumor tissue from the same patient. This finding was matched to the upregulation of myosin VI in established prostate cancer cell lines. In luciferase reporter analysis, we find a significant negative regulatory effect on the MYO6 3′UTR by both miR-143 and miR-145. Mutation of the potential binding sites for miR-143 and miR-145 in the MYO6 3′UTR resulted in a loss of responsiveness to the corresponding miRNA. Our data indicate that miR-143 and miR-145 are involved in the regulation of MYO6 expression and possibly in the development of prostate cancer. Mol Cancer Res; 8(4); 529–38. ©2010 AACR.

Cancers 14(12), 2943 (2022). doi:10.3390/cancers14122943 special issue: "Special Issue "Bio-Pathological Markers for the Diagnosis and Therapy of Cancers, Volume II" / Special Issue Editors: Dr. Lucia Salvatorelli, Guest Editor; Dr. Giuseppe Broggi, Guest Editor", Published by MDPI, Basel

Zusammenfassung Das Reformationsjubiläum 2017 war durch eine vielfältige Erinnerungs- und Eventkultur geprägt. Die Konzeption stand nicht vorab fest, sie wurde in der Gestaltung des Reformations­jubiläums als Reformationsdekade entwickelt. Ergebnisse der kirchenhistorischen Forschung wurden einbezogen und zugleich nach der Relevanz für Gegenwart und Zukunft gefragt. Das Reformationsjubiläum war historisch-diskursiv, international und ökumenisch ausgerichtet und wurde zu einem Beteiligungsjubiläum, Bildungsereignis und Motivationsschub.

Impaired Ca2+ signaling in prostate cancer contributes to several cancer hallmarks, such as enhanced proliferation and migration and a decreased ability to induce apoptosis. Na+ influx via transient receptor potential melastatin 4 channel (TRPM4) can reduce store-operated Ca2+ entry (SOCE) by decreasing the driving force for Ca2+. In patients with prostate cancer, gene expression of TRPM4 is elevated. Recently, TRPM4 was identified as a cancer driver gene in androgen-insensitive prostate cancer. We investigated TRPM4 protein expression in cancer tissue samples from 20 patients with prostate cancer. We found elevated TRPM4 protein levels in prostatic intraepithelial neoplasia (PIN) and prostate cancer tissue compared to healthy tissue. In primary human prostate epithelial cells (hPEC) from healthy tissue and in the androgen-insensitive prostate cancer cell lines DU145 and PC3, TRPM4 mediated large Na+ currents. We demonstrated significantly increased SOCE after siRNA targeting of TRPM4 in hPEC and DU145 cells. In addition, knockdown of TRPM4 reduced migration but not proliferation of DU145 and PC3 cells. Taken together, our data identify TRPM4 as a regulator of SOCE in hPEC and DU145 cells, demonstrate a role for TRPM4 in cancer cell migration and suggest that TRPM4 is a promising potential therapeutic target.

In prostate cancer, reactive oxygen species (ROS) are elevated and Ca(2+) signaling is impaired. Thus, several novel therapeutic strategies have been developed to target altered ROS and Ca(2+) signaling pathways in prostate cancer. Here, we investigate alterations of intracellular Ca(2+) and inhibition of cell viability caused by ROS in primary human prostate epithelial cells (hPECs) from healthy tissue and prostate cancer cell lines (LNCaP, DU145, and PC3). In hPECs, LNCaP and DU145 H2O2 induces an initial Ca(2+) increase, which in prostate cancer cells is blocked at high concentrations of H2O2. Upon depletion of intracellular Ca(2+) stores, store-operated Ca(2+) entry (SOCE) is activated. SOCE channels can be formed by hexameric Orai1 channels; however, Orai1 can form heteromultimers with its homolog, Orai3. Since the redox sensor of Orai1 (Cys-195) is absent in Orai3, the Orai1/Orai3 ratio in T cells determines the redox sensitivity of SOCE and cell viability. In prostate cancer cells, SOCE is blocked at lower concentrations of H2O2 compared with hPECs. An analysis of data from hPECs, LNCaP, DU145, and PC3, as well as previously published data from naive and effector TH cells, demonstrates a strong correlation between the Orai1/Orai3 ratio and the SOCE redox sensitivity and cell viability. Therefore, our data support the concept that store-operated Ca(2+) channels in hPECs and prostate cancer cells are heteromeric Orai1/Orai3 channels with an increased Orai1/Orai3 ratio in cells derived from prostate cancer tumors. In addition, ROS-induced alterations in Ca(2+) signaling in prostate cancer cells may contribute to the higher sensitivity of these cells to ROS.

BACKGROUND In vivo model systems in prostate cancer research that authentically reproduce tumor growth are still sparse. While orthotopic implantation is technically difficult, particularly in the mouse, most models favor subcutaneous tumor growth. This however provides little information about natural tumor growth behavior and tumor stroma interaction. Furthermore, established prostate cancer cell lines grown as in vivo xenografts are not able to reflect the variety of tumor specific growth patterns and growth behavior in men. Primary cell cultures are difficult to handle and an induction of orthotopic tumors has not been successful yet. Therefore, a tumorgraft model using tumor tissue from prostatectomy specimens was developed. METHODS Balb/c nude mice were used to graft fresh prostate tumor tissue by renal subcapsular and orthotopic implantation. Testosterone propionate was supplemented. Animals were tracked by means of 30 MHz ultrasound to monitor tumor engraftment and growth. Autopsy, histology, PSA measurements as well as immunostaining and PCR for human tissue were performed to confirm orthotopic tumor growth. RESULTS Renal subcapsular engraftment was seen in 2 of 3 mice. Orthotopic engraftment was observed in 7 of 11 animals (63.6%) with an overall engraftment of 5 out of 9 patient specimens (55.6%). Ultrasound confirmed the tumor growth over time. Of interest, the tumorgrafts not only retained essential features of the parental tumors, but also stained positive for tumor specific markers such as AR, PSA, and AMACR. Tumor positive animals showed highly elevated serum PSA levels with confirmation of a human specific PCR sequence and a human endothelial cell lining in the tumor vessels. CONCLUSIONS Standardized implantation of fresh tumor tissue in nude mice prostates generates tumorgrafts with histological properties of organ-confined prostate cancer. These tumorgrafts display a new approach for an optimized in vivo model of prostate cancer and will allow further investigations on specific pathways of tumor initiation and progression as well as therapeutic response. Prostate 75:1526–1537, 2015. © 2015 Wiley Periodicals, Inc.

Posttranscriptional gene regulation by microRNAs (miRNAs) contributes to the induction and maintenance of prostate carcinoma (PCa). To identify mRNAs enriched or removed from Ago2-containing RISC complexes, these complexes were immunoprecipitated from normal prostate fibroblasts (PNFs) and the PCa line DU145 and the bound mRNAs were quantified by microarray. The analysis of Ago complexes derived from PNFs or DU145 confirmed the enrichment or depletion of a variety of mRNAs already known from the literature to be deregulated. Novel potential targets were analyzed by luciferase assays with miRNAs known to be deregulated in PCa. We demonstrate that the mRNAs of the death effector domain-containing protein (DEDD), the tumor necrosis factor receptor superfamily, member 10b protein (TNFRSF10B), the tumor protein p53 inducible nuclear protein 1 (TP53INP1), and the secreted protein, acidic, cysteine-rich (SPARC; osteonectin) are regulated by miRNAs miR-148a, miR-20a, miR-24, and miR-29a/b, respectively. Therefore, these miRNAs represent potential targets for therapy. Surprisingly, overexpression of miR-24 induced focus formation and proliferation of DU145 cells, while miR-29b reduced proliferation. The study confirms genes deregulated in PCa by virtue of their presence/absence in the Ago2-complex. In conjunction with the already published miRNA profiles of PCa, the data can be used to identify miRNA-regulated mRNAs.

Labelled 5α-dihydrotestosterone (DHT) binding experiments have shown that expression levels of (yet unidentified) membrane androgen receptors (mAR) are elevated in prostate cancer and correlate with a negative prognosis. However, activation of these receptors which mediate a rapid androgen response can counteract several cancer hallmark functions such as unlimited proliferation, enhanced migration, adhesion and invasion and the inability to induce apoptosis. Here, we investigate the downstream signaling pathways of mAR and identify rapid DHT induced activation of store-operated Ca2+ entry (SOCE) in primary cultures of human prostate epithelial cells (hPEC) from non-tumorous tissue. Consequently, down-regulation of Orai1, the main molecular component of Ca2+ release-activated Ca2+ (CRAC) channels results in an almost complete loss of DHT induced SOCE. We demonstrate that this DHT induced Ca2+ influx via Orai1 is important for rapid androgen triggered prostate specific antigen (PSA) release. We furthermore identified alterations of the molecular components of CRAC channels in prostate cancer. Three lines of evidence indicate that prostate cancer cells down-regulate expression of the Orai1 homolog Orai3: First, Orai3 mRNA expression levels are significantly reduced in tumorous tissue when compared to non-tumorous tissue from prostate cancer patients. Second, mRNA expression levels of Orai3 are decreased in prostate cancer cell lines LNCaP and DU145 when compared to hPEC from healthy tissue. Third, the pharmacological profile of CRAC channels in prostate cancer cell lines and hPEC differ and siRNA based knock-down experiments indicate changed Orai3 levels are underlying the altered pharmacological profile. The cancer-specific composition and pharmacology of CRAC channels identifies CRAC channels as putative targets in prostate cancer therapy.

Introduction Emerging evidence suggest that microRNAs could serve as non-invasive biomarker for cancer patients. Our study was designed to analyze circulating serum microRNAs in patients with renal cell carcinoma (RCC). Materials and methods Serum RNA was isolated from patients with clear cell RCC (ccRCC) and non-malignant disease; an artificial microRNA (cel-miR-39) was spiked-in prior the isolation procedure to control isolation efficiency. The levels of miR-26a-2*, miR-191, miR-337-3p and miR-378 in serum were determined using quantitative real-time PCR; the microRNA levels were normalized to cel-miR-39. Results First, miR-26a-2*, miR-191, miR-337-3p and miR-378 were quantified in serum of each 25 patients with ccRCC and non-malignant disease. The level of miR-378 was significantly increased in ccRCC patients, and thus chosen for validation. The analysis of miR-378 in the validation cohort with 117 RCC patients and 123 control subjects did not confirm a different level of miR-378. Also, miR-378 was not correlated to pT-stage, lymph node/distant metastasis, vascular invasion and Fuhrman grade. Conclusions The analysis of circulating serum levels of miR-26a-2*, miR-191, miR-337-3p and miR-378 is unlikely to provide helpful diagnostic/prognostic information in RCC patients.

The molecular carcinogenesis of lung cancer has yet to be clearly elucidated. We investigated the possible oncogenic function of SEC62 in lung cancer, which was predicted based on our previous findings that lung and thyroid cancer tissue samples exhibited increased Sec62 protein levels. The SEC62 gene locus is at 3q26.2, and 3q amplification is reportedly the most common genomic alteration in non–small cell lung cancer. We analyzed SEC62 mRNA and protein levels in tissue samples from lung cancer patients by real-time quantitative PCR, Western blot, and IHC and found significantly increased SEC62 mRNA and protein levels in tumors compared with tumor-free tissue samples from the same patients. Correlation analyses revealed significantly higher Sec62 levels in tumors with lymph node metastases compared with nonmetastatic tumors, as well as in poorly compared with moderately differentiated tumors. On the basis of these promising results, we examined the role of Sec62 in cancer cell biology in vitro . Cell migration assays with lung and thyroid cancer cells showed distinct stimulation of migration in SEC62 -overexpressing cells and inhibition of migration in Sec62-depleted cells. Moreover, we found that SEC62 silencing sensitized the cells to thapsigargin-induced endoplasmic reticulum stress. Thus, our results indicate that SEC62 represents a potential candidate oncogene in the amplified 3q region in cases of non–small cell lung cancer and harbors various functions in cancer cell biology.

The clinical course of prostate cancer, the most common cancer in men, is very variable. Despite intense research activities over the years and besides histopathological criteria, prognostic markers that reliably predict tumor behavior and the necessity for treatment are still missing. A likely explanation for this fact is the lack of good tumor models, mimicking the in vivo situation. These models are not only essential for a better understanding of the pathogenesis of prostate cancer but also play an important role in the development of new therapeutic strategies. Since results of permanent cell culture experiments reflect only in part real tumor behavior and primary cultures from patient material cannot be grown indefinitely, novel approaches need to be developed to achieve reliable and clinically relevant prostate cancer research.In this work the development of several approaches for culturing primary prostate cancer tissue is illustrated and a forecast of future research plans utilizing xenograft models in mice is made.

BACKGROUND We previously reported that over-expression of the SEC62 gene is a widespread phenomenon in prostate cancer. Since the use of endoplasmic reticulum (ER) stress-inducing substances such as thapsigargin in prostate cancer therapy is widely discussed in the literature, we investigated the influence of Sec62 protein content on the cellular response to these drugs. METHODS Growth effects were analyzed by real-time cell analysis and viability tests in DU145-cells representing an increased SEC62 expression or PC3- and LNCaP-cells representing a similar SEC62 expression compared to non-tumor cells. Ca2+-imaging in an established HeLa-system with fluorescent dye was used to study molecular effects of Sec62 depletion. RESULTS We found a lower propensity toward apoptotic cell death after thapsigargin treatment for DU145 cells compared to PC3 or LNCaP and siRNA-mediated silencing of SEC62 resulted in a reduced viability of thapsigargin-treated PC3 cells, indicating that Sec62 functions in cellular stress response. Measurement of cytosolic [Ca2+] demonstrated the influence of Sec62 on the cellular response to thapsigargin on a molecular level. Using real-time cell analysis, we observed the loss of androgen stimulation of LNCaP cells in the presence of thapsigargin, and an additional negative effect on cell growth of Sec62 depletion. Also, for PC3- and DU145-cells Sec62 depletion inhibited growth after thapsigargin treatment. CONCLUSIONS Our data indicate a crucial function of Sec62 in the response to thapsigargin-induced ER stress. This will be of great significance on the background of elevated Sec62 protein levels in prostate cancer cells when treatment with thapsigargin analogs is considered. Prostate 71:1074–1083, 2011. © 2011 Wiley-Liss, Inc.

Sec62 is part of the protein translocation apparatus in the membrane of the endoplasmic reticulum (ER). In yeast, Sec62 participates in the post-translational translocation of proteins into the ER, but its function in mammals remains elusive. Previously we described the amplification and over-expression of the SEC62 gene in prostate cancer cell lines and the protein has been described as a potential target gene in prostate cancer. In the current study we show that in the tumor tissue of prostate cancer patients Sec62 protein levels are elevated compared with tumor-free tissue derived from the same patients or from prostates of control group patients and that the higher Sec62 protein content correlates with an increasing de-differentiation of the cells. Therefore, up-regulation of Sec62 protein content indeed is a phenomenon associated with prostate cancer progression. Analysis of a multi-tissue tumor array showed that in addition to prostate cancer, overproduction of Sec62 is observed in various other tumors, most significantly in tumors of the lung and the thyroid. To examine the tumor-related functions of Sec62, we silenced the SEC62 gene in the prostate cancer cell-line PC3 as well as in a set of other tumor cell-lines with two different siRNAs. In general, after silencing of SEC62 the cell migration and the invasive potential of the cells was blocked or at least dramatically reduced while cell viability was hardly affected. Thus, the SEC62 gene may indeed be considered as a target gene in the therapy of various tumors.

Prostate cancer is a leading cause of tumor mortality. To characterize the underlying molecular mechanisms, we have compared the microRNA (miRNA) profile of primary prostate cancers and noncancer prostate tissues using deep sequencing. MiRNAs are small noncoding RNAs of 21 to 25 nucleotides that regulate gene expression through the inhibition of protein synthesis. We find that 33 miRNAs were upregulated or downregulated >1.5-fold. The deregulation of selected miRNAs was confirmed by both Northern blotting and quantitative reverse transcription-PCR in established prostate cancer cell lines and clinical tissue samples. A computational search indicated the 3′-untranslated region (UTR) of the mRNA for myosin VI (MYO6) as a potential target for both miR-143 and miR-145, the expression of which was reduced in the tumor tissues. Upregulation of myosin VI in prostate cancer was previously shown by immunohistochemistry. The level of MYO6 mRNA was significantly induced in all primary tumor tissues compared with the nontumor tissue from the same patient. This finding was matched to the upregulation of myosin VI in established prostate cancer cell lines. In luciferase reporter analysis, we find a significant negative regulatory effect on the MYO6 3′UTR by both miR-143 and miR-145. Mutation of the potential binding sites for miR-143 and miR-145 in the MYO6 3′UTR resulted in a loss of responsiveness to the corresponding miRNA. Our data indicate that miR-143 and miR-145 are involved in the regulation of MYO6 expression and possibly in the development of prostate cancer. Mol Cancer Res; 8(4); 529–38. ©2010 AACR.

Zahlreiche Erkenntnisse in der Prostatakarzinomforschung basieren auf Zellkulturergebnissen. Neuere genomische Untersuchungen zeigen jedoch, dass verwendete permanente Prostatakarzinomzelllinien sich deutlich von dem klinisch relevanten, primaren Prostatakarzinom unterscheiden und damit die Ubertragbarkeit dieser Zellkulturergebnisse auf die Klinik nur bedingt gegeben ist. Das Arbeiten mit Primarzellkulturen aus Gewebestuckchen von Prostatektomiepraparaten bietet eine sehr gute Alternative, doch die Etablierung von Primarkulturen gestaltet sich schwierig und recht aufwandig. In dieser Arbeit wurde ein Primarzellkulturmodell mit einem Invasionssystem kombiniert. Hiermit gelang es nicht nur invasiv wachsende Zellpopulationen aus Primarkulturen zu selektionieren, sondern auch diese Zellen in einem 3D-Modell unter der Ausbildung von Spharoiden weiter zu kultivieren. Zur Charakterisierung dieser Zellpopulation haben wir vergleichende genomische Hybridisierungen durchgefuhrt, die zahlreiche genetische Alterationen aufzeigen. Das hier dargestellte Modell ermoglicht es erstmalig, invasive Zellklone aus primarem Prostatakarzinomgewebe zu gewinnen und durch Kultivierung fur weitere Untersuchungen zu verwenden.

Satraplatin is an orally available platinum analog. The purpose of this study was to better characterize satraplatin's preclinical antitumor efficacy in a variety of sensitive and resistant human tumor cell lines and in a prostate cancer xenograft model and to evaluate the effect of satraplatin on PSA expression and/or secretion in a prostate cancer cell line.Satraplatin and its primary metabolite JM-118 were preclinically tested for their cytotoxic activity in a range of cancer cells including: human prostate, those forming the NCI drug screening panel, and those resistant to anti-cancer drugs. Also, the antiproliferative efficacy of satraplatin was tested in vivo in a human prostate cancer model. The effect of satraplatin and JM-118 on PSA transcription was measured by quantitative real time PCR.Satraplatin and JM-118 inhibited in vitro and in vivo the growth of prostate cancer cells in a dose-dependent fashion. The IC50 cytotoxicity values for satraplatin ranged from 1 to 3 microM for androgen-insensitive cells and was 11 microM for the androgen-sensitive cell line. Interestingly, JM-118 was up to 16-fold more potent than satraplatin. Oral administration of satraplatin to nude mouse PC-3 xenograft models inhibited the growth of these human tumors. Satraplatin had no direct effect on PSA transcription and the observed decrease in secreted PSA correlated with a decrease in cell number. When evaluated in the NCI drug-screening panel, satraplatin was most active in leukemia and small cell lung cancer cell lines. Both satraplatin and JM-118 were tested on cells resistant to chemotherapeutic agents. Satraplatin and JM-118 were equally active in the cisplatin-resistant A129cp80 ovarian carcinoma cell line, with activity comparable to that observed in the parent line. Neither expression of MDR1, BCRP, MRP1, nor altered tubulin or topoisomerase I were found to mediate resistance to satraplatin or JM-118. Although these resistance mechanisms contribute to drug resistance for a number of chemotherapeutics, they do not appear to play a role in satraplatin resistance.These results demonstrate that satraplatin and JM-118 have preclinical antitumor activity in human prostate cancer and other tumor types as well, including several cell lines displaying drug resistance to cisplatin, docetaxel and mitoxantrone. In addition, the results suggest that PSA should be further evaluated as a relevant marker of clinical response in patients with prostate cancer treated with satraplatin.

BACKGROUND Fibulins, encoded by FBLN genes, are extracellular matrix proteins influencing cell adhesion and migration. Altered expression of fibulins is associated with progression of several cancer types, but has not been studied in prostate cancer. METHODS Expression of FBLN1 (major splice forms C and D), FBLN4, FBLN5, SPOCK1, and TENC was compared between 47 prostate cancer samples and 13 benign prostatic tissues by quantitative RT-PCR. Fibulin-1 and fibulin-5 expression was studied by immunohistochemistry. Effects of androgens and the DNA methylation inhibitor 5-aza-2′-deoxycytidine on fibulin expression were investigated in different prostate cancer cell lines. RESULTS Our recent microarray analysis suggested downregulation of three fibulins, FBLN1, FBLN4, and FBLN5, in prostate cancer, while two further ECM genes, SPOCK1 (testican) and TENC (tenascin C), appeared upregulated or unchanged. These observations were corroborated by quantitative RT-PCR. Accordingly, FBLN1 and FBLN4 were weakly expressed in carcinoma lines compared to normal prostate epithelial cells (PrECs). Only FBLN4 was induced by 5-aza-2′-deoxycytidine, but its promoter was unmethylated. Androgen did not affect expression of FBLN genes. The FBLN1C and FBLN1D splice forms were coordinately expressed. Fibulin-1 protein was weakly detectable in benign PrECs, but tended to accumulate in cancer cells. Fibulin-5 was predominantly located in the stroma with a strong gradient from the periurethral to the peripheral zone, and lost in cancers. CONCLUSIONS Three FBLN genes are significantly downregulated in prostate cancer, whereas SPOCK1 is often upregulated. FBLN5 downregulation fits its postulated anticancerous function, whereas FBLN1 and FBLN4 behave different than in certain other cancers. Prostate 67: 1770–1780, 2007. © 2007 Wiley-Liss, Inc.

In vivo model systems in prostate cancer research that authentically reproduce tumor growth are still sparse. While orthotopic implantation is technically difficult, particularly in the mouse, most models favor subcutaneous tumor growth. This however provides little information about natural tumor growth behavior and tumor stroma interaction. Furthermore, established prostate cancer cell lines grown as in vivo xenografts are not able to reflect the variety of tumor specific growth patterns and growth behavior in men. Primary cell cultures are difficult to handle and an induction of orthotopic tumors has not been successful yet. Therefore, a tumorgraft model using tumor tissue from prostatectomy specimens was developed.Balb/c nude mice were used to graft fresh prostate tumor tissue by renal subcapsular and orthotopic implantation. Testosterone propionate was supplemented. Animals were tracked by means of 30 MHz ultrasound to monitor tumor engraftment and growth. Autopsy, histology, PSA measurements as well as immunostaining and PCR for human tissue were performed to confirm orthotopic tumor growth.Renal subcapsular engraftment was seen in 2 of 3 mice. Orthotopic engraftment was observed in 7 of 11 animals (63.6%) with an overall engraftment of 5 out of 9 patient specimens (55.6%). Ultrasound confirmed the tumor growth over time. Of interest, the tumorgrafts not only retained essential features of the parental tumors, but also stained positive for tumor specific markers such as AR, PSA, and AMACR. Tumor positive animals showed highly elevated serum PSA levels with confirmation of a human specific PCR sequence and a human endothelial cell lining in the tumor vessels.Standardized implantation of fresh tumor tissue in nude mice prostates generates tumorgrafts with histological properties of organ-confined prostate cancer. These tumorgrafts display a new approach for an optimized in vivo model of prostate cancer and will allow further investigations on specific pathways of tumor initiation and progression as well as therapeutic response.

Gain at chromosome 3q25-q26 has been reported to commonly occur in prostate cancer. To map the 3q25-q26 amplification unit and to identify the candidate genes of amplification, we did fluorescence in situ hybridization and quantitative real-time PCR for gene copy number and mRNA expression measurements in prostate cancer cell lines and prostate cancer samples from radical prostatectomy specimens. The minimal overlapping region of DNA copy number gains in the cell lines could be narrowed down to 700 kb at 3q26.2. Of all positional and functional candidates in this region, the gene TLOC1/SEC62 revealed the highest frequency (50%) of copy number gains in the prostate cancer samples and was found to be up-regulated at the mRNA level in all samples analyzed. TLOC1/Sec62 protein was also shown to be overexpressed by Western blot analysis. Intriguingly, the TLOC1/SEC62 gene copy number was increased in prostate tumors from patients who had a lower risk of and a longer time to progression following radical prostatectomy. These findings make TLOC1/SEC62 the best candidate within the 3q amplification unit in prostate cancer. TLOC1/Sec62 protein is a component of the endoplasmic reticulum protein translocation machinery, whose function during prostate carcinogenesis remains to be determined. (Mol Cancer Res 2006;4(3):169–76)

Gene amplifications and deletions play an important role in the pathogenesis of solid tumors, including prostate cancer. Real-time PCR is a powerful tool for quantitative DNA analysis, particularly when starting quantities of tumor tissue are minimal (1)(2)(3)(4)(5). In the present study, we describe a modification of the 2−ΔΔCT method, which recently was shown to be suitable for relative gene expression analyses (6). We used our technique to analyze prostate cancer cell lines and tissue samples to determine the relationship between homeodomain-containing transcription factor 3.1 (NKX3.1) and MYC gene copy number alterations in this tumor type. The specimens analyzed included (a) blood samples from healthy donors; (b) the prostate cancer cell lines DU145 (ATCC no. HTB-81) and PC3 (ATCC no. CRL-1435) and their derived sublines DU145MN1, PC3-N, and PC3-125-1L (7); (c) the colorectal cell line COLO320DM (ATCC no. CCL-220), which harbors a high-level MYC amplification; and (d) primary prostate adenocarcinoma samples obtained after radical prostatectomy from previously untreated patients. The specimens were histologically verified, and samples were taken as described previously (8). We extracted DNA from the snap-frozen prostate carcinoma samples, blood samples, and cell lines, using the Blood and Cell Culture DNA Midi Kit (Qiagen). We performed quantitative real-time PCR using the LightCycler™ system (Roche Diagnostics) with the FastStart DNA Master SYBR Green I LightCycler Kit (Roche Diagnostics). PCRs were run in duplicate with 20-μL reaction volumes containing 1× SYBR Green I PCR Buffer Mix, 5 mM MgCl2, 0.5 μM each primer, 1× FastStart Taq DNA Polymerase (Roche Diagnostics), and 50 ng of genomic DNA. The cycling conditions are given in Table 1⇓ . The hot start PCR method was applied to prevent incomplete DNA denaturation as discussed by Wilhelm et al. (9). Melting curve analysis was performed …

Vasodilator-stimulated phosphoprotein (VASP) and mammalian-enabled protein (MENA) share similar cellular localisation and functions (signal transduction pathways, regulation of actin cytoskeleton dynamics). Functional substitution and compensation among Ena/ VASP proteins have been proposed as the reason for the absence of major morphological and functional deficits in VASP�/� mice. The aim of this study was to investigate VASP expression in the mouse cochlea, to analyse cochlear function in VASP�/� mice compared with wildtype mice, and to analyse cochlear MENA distribution taking into account that MENA protein might compensate VASP loss in the cochlea of VASP�/� mice. We confirmed specific VASP expression in the pillar cells of the mice organ of Corti as previously reported for rat cochlea. By analysing the hearing function in VASP�/� mice, we found no differences in auditory brainstem responses and distortion product otoacoustic emissions from those of wildtype mice but evidence for an increased noise sensitivity at lower frequencies. When MENA protein levels in cochlea tissue were tested in mutant and wildtype mice by Western blot analysis, no significant differences were found, as was also seen with regard to MENA mRNA levels in laser-microdissected single pillar cells. Most surprisingly, however, MENA protein was absent in pillar cells of VASP�/� mice, whereas it was detected in other cochlear cells. The finding of a cell-specific, and not organ-specific, redundancy of MENA protein expression noted for the first time in VASP�/� mice is proposed as the reason for the observed distinct cochlear phenotype.

Juvenile Angiofibrome erscheinen histologisch gutartig, wachsen aber lokal aggressiv. Sie treten uberwiegend bei mannlichen Adoleszenten auf. Als einzige genetische Veranderung sind bisher β-Catenin-Mutationen bei diesem Tumor bekannt. Angiofibromgewebe von 7 Patienten wurde mittels der vergleichenden genomischen Hybridisierung (CGH) auf quantitative Genomveranderungen hin analysiert. In 6 von 7 untersuchten Angiofibromen konnten mit der CGH auf 18 verschiedenen Chromsomen Alterationen nachgewiesen werden. Zugewinne von chromosomalen Abschnitten zeigten sich gehauft auf den Chromosomen 4q, 6q und 8q. In 4 von 7 Fallen zeigte sich ein vollstandiger Verlust des Y-Chromosoms. Die CGH ist zur Analyse chromosomaler Veranderungen bei Angiofibromen geeignet. Mogliche Zielsequenzen fur weitere Genomuntersuchungen, v. a. zum Verlust des Y-Chromosoms, konnten lokalisiert werden.