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urologie.uniklinikum-dresden.de, *e-mail: doreen.kunze@uniklinikum-dresden.de

urologie.uniklinikum-dresden.de, *e-mail: doreen.kunze@uniklinikum-dresden.de

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  1. (a) siRNA-D5 ns-siRNA untreated PSA/TBP p38MAPK C13orf19/TBP β-Actin phospho-p38MAPK (b) 3.6 % 3.9 % 3.5 % time (h) time (h) β-Actin b) 72 h a) 1.3 % 1.6 % 0.8 % • Material and Mathods II: • hormonsensitive LNCaP cells were incubated overnight in RPMI medium with 10% charcoal-stripped FCS in the absence of androgen • cell culture was continued for additional 48 h in this medium in the presence of 1 or 10 nM of the synthetic androgen methyltrienolone (R1881, PerkinElmer Life Sciences) • for antiandrogen treatment, cells were incubated with 1 or 10 nM R1881 and 10µM bicalutamide (AstraZeneca) untreated ns-siRNA siRNA-D5 FIGURE 3 C13orf19 mRNA expression in LNCaP cells after treatment with the synthetic androgen R1881 or rather combined with R1881 and the antiandrogen bicalutamide. In contrast the androgen dependence of the prostate specific antigen (PSA) mRNA expression measured in the same experiment. Both transcript numbers were normalized to the reference gene TBP. • Material and Mathods III: • p38MAPK inhibitor treatment: BPH-1 and PC-3 cells were incubated in DMEM without FCS for 24‑72 h before treatment with the inhibitor SB203580 (0.5‑8.0 µM) for 1 h • → qPCR analysis of the C13orf19 mRNA expression • inhibition of the C13orf19 mRNA expression with siRNA-D5 • → Western blotting with primary phospho‑p38 MAP kinase and p38 MAP kinase (detects total p38MAPK protein) antibodies (1:1000; Cell Signaling Technology), secondary antibody: anti- rabbit IgG HRP-linked (1:1000, Amersham Biosciences), -actin served as a loading control • a yeast two-hybrid screen was performed by U. Stelzl3to detect putative physical interactions between C13orf19 and other proteins [5] FIGURE 4 Examination of C13orf19 interaction with p38MAPK. (a) C13orf19 mRNA expression in BPH‑1 and PC‑3 cells after treatment with the p38MAPK inhibitor SB203580 relative to the reference gene TBP normalized to DMSO-treated cells. (b) Western blotting of PC-3 cells 72 h and 96 h after transfection with siRNA‑D5. The antibodies recognized the phosphorylated (upper) or total (lower) p38MAPK protein respectively. -actin served as a loading control. • Results I:The effects of the siRNA‑D5-mediated C13orf19 mRNA inhibition in the prostate cell line BPH‑1 and the prostate cancer cell line PC‑3 were examined (all results shown for PC-3 cells – similar results with BPH-1): • remarkably reduction of the C13orf19 mRNA expression (Tab. 1) • no significant alterations in cell viability 24 h, 48 h and 72 h after transfection (Fig. 1) • no adjustments in cell cycle distribution and rate of apoptosis (Fig. 2) • low apoptosis rate in PC‑3 cells → combined treatment of siRNA-D5 and chemotherapy (etoposide, docetaxel) • → no synergistic effects • Results III: The putative interaction between C13orf19 and p38MAPK was investigated: • C13orf19 mRNA expression after treatment of BPH‑1 and PC‑3 cells with the p38MAPK inhibitor SB203580: • → no alterations in the gene expression (Fig. 4a) • p38MAPK expression and phosphorylation status after siRNA‑D5 transfection in PC‑3 cells (Western Blot): • → not changes in expression and phosphorylation of p38MAPK • yeast two-hybrid screen: • → no interactions between C13orf19 and p38MAPK • Functional analyzes of C13orf19 in prostate cell lines • Doreen Kunze1*, Uta Schmidt2, Susanne Fuessel1, Ulrich Stelzl3, Axel Meye1, Manfred P. Wirth1 • 1Department of Urology, Technical University Dresden; • 2 Department of Physiological Chemistry, University Ulm; • 3Max-Delbrueck-Centrum for Molecular Medicine, Berlin, Germany http://urologie.uniklinikum-dresden.de, *e-mail: doreen.kunze@uniklinikum-dresden.de TABLE I Inhibition of the C13orf19 mRNA expression after transfection with 250 nM siRNA‑D5 in the cell line PC‑3. The results are indicated relative to the reference gene PBGD and normalized to the non-silencing‑siRNA control. This study was partially supported by a grant from the Wilhelm-Sander-Stiftung (to A.M. & M.P.W.). Introduction:Chromosome 13q is often affected by genetic aberrations in prostate cancer (PCa), above all loss of heterozygosity [1]. These changes have led to the hypothesis that at least one prostate-specific tumor suppressor gene (TSG) resides on chromosome 13q. C13orf19 (NM 017569), also known as P38IP or FAM48A, was previously identified to be down-regulated in PCa [2]. It is localized on chromosome 13q13 between the TSGs BRCA-2 and RB-1. LOH in the chromosomal region of the C13orf19 gene in 47% of the patients for seven C13orf19-associated markers being higher than for the four markers of the RB1 locus (39%) and for the three BRCA2-related markers (25%) on 13q [3]. The presumed corresponding protein of C13orf19 with 733 amino acids resembles a transcription factor because of its putative nuclear localization signal and the glutamine clusters at the C terminus. The mRNA was expressed exclusively in the epithelia as shown by in situ hybridization [2]. Recently, we were able to validate our initial data on the PCa-associated downregulation of C13orf19 in a cohort of 61 patients; an at least 1.5-fold lower mRNA level was observed in samples from 46% of the patients [4]. The first database entry described C13orf19 as P38IP (Acc. No. AF093250), a protein which interacts with the p38 map kinase (MAPK). This description was given by J. Han (Scripps Research Institute, La Jolla, USA) due to the identification of P38IP in a yeast two-hybrid screen with p38MAPK (personal communication). The aim of this study was to show whether a C13orf19 knock-down by means of siRNA will have an effect on PCa cell growth, apoptosis or cell cycle distribution (I). Also, a possible regulation by androgens was investigated (II). Furthermore, the described interaction with p38MAPK should be verified pharmacologically and additional interaction partners should be identified in an independent yeast two-hybrid screen (III). FIGURE 1 Relative cell viability of PC-3 cells 48 h after transfection with 125 nM siRNA‑D5 normalised to cells treated with ns‑siRNA (100%). • Material and Methods I: • BPH-1 and PC-3 cells were transfected DOTAP-mediated with siRNA-D5 (nt 1288‑1306 [C13orf19 mRNA: NM_017569]; 125 or 250 nM) • mRNA expression of C13orf19 and TBP (reference gene) were measured by quantitative PCR (relative expression values were normalized to the non-silencing (ns) siRNA-control) • cellular viability (WST‑1), apoptosis (annexin V-staining), cell cycle distribution and clonogenic survival were examined • chemotherapeutical treatment: PC-3 cells were incubated 24 h after siRNA transfection with the chemotherapeutic agents docetaxel (0,5-40 nM) or etoposide (20 µM) for 24 h • Results II: The dependence of the C13orf19 mRNA expression on the androgen • R1881 was investigated in hormonsensitive LNCaP cells: • treatment with R1881 (1 and 10 nM): • → no changes of the C13orf19 transcript expression(Fig. 3) • combination of R1881 with the antiandrogen bicalutamide: • → no changes of the C13orf19 transcript expression (Fig. 3) DISCUSSION: The reduced expression of C13orf19 in PCa does not seem to play a key role in PCa formation. Probably the previously [4] detected decrease in expression represents an accompanying effect of LOH on chromosome 13q. Furthermore, the C13orf19 mRNA reduction could be associated with the progression of PCa. All three experiments to examine the postulated interaction between C13orf19 and p38MAPK did not confirm it. Therefore, the designation of the gene as P38IP is misleading and the gene should forthwith be named C13orf19 or FAM48A. REFERENCES 1 von Knobloch R, Konrad L, Barth PJ, Brandt H, Wille S, Heidenreich A, Moll R and Hofmann R: Genetic pathways and new progression markers for prostate cancer suggested by microsatellite allelotyping. Clin Cancer Res 10: 1064-1073, 2004. 2 Schmidt U, Fiedler U, Pilarsky CP, Ehlers W, Fuessel S, Haase M, Faller G, Sauter G and Wirth MP: Identification of a novel gene on chromosome 13 between BRCA‑2 and RB‑1. Prostate 47: 91-101, 2001. 3 Fiedler U, Ehlers W, Meye A, Fuessel S, Schmidt U and Wirth MP: LOH analyses in the region of the putative tumor suppressor gene C13 on chromosome 13q13. Anticancer Res 27: 2341‑2350, 2001. 4 Schmidt U, Fuessel S, Haase M, Kraemer K, Meye A and Wirth MP: Quantification of C13orf19/P38IP mRNA expression by quantitative real-time PCR in patients with urological malignancies. Cancer Lett 225: 253‑260, 2005. 5 Stelzl U, Worm U, Lalowski M, Haenig C, Brembeck FH, Goehler H, Stroedicke M, Zenkner M, Schoenherr A, Koeppen S, Timm J, Mintzlaff S, Abraham C, Bock N, Kietzmann S, Goedde A, Toksoz E, Droege A, Krobitsch S, Korn B, Birchmeier W, Lehrach H, Wanker EE: A human protein-protein interaction network: a resource for annotating the proteome. Cell 122: 957-968, 2005. FIGURE 2 Cell cycle distribution (a) and rate of apoptosis (b) of PC-3 cells 48 h after transfection with 125 nM siRNA‑D5. 1.5x104 cells were used for each assay. The proportions (%) of early (annexin V-positive, PI-negative; lower right) and late apoptotic cells (annexin V-positive, PI-positive; upper right) are shown.