Thus, Sirt-1 participates in the regulation of genome architecture and gene expression

l analysis Data were expressed as mean +/- SD, and analyzed 870281-82-6 price statistically by one way analysis of variance followed by post hoc t-tests or student 1692608 t-test to assess the differences between groups for in vitro and in vivo studies. Statistical significance was taken as p<0.05. Results Expression of pro-osteoblastic factors by PC3c cells From human androgen-resistant prostate cancer cell line PC3, we obtained after single cell population culture in vitro a new subculture cell line named PC3c cells that was chosen based on its epithelial phenotype. As expected and similarly to the parental PC3 cells, AR could not be detected by real-time PCR in PC3c while it was expressed in the hormone dependant cell line VCaP used as a positive control. On the other hand, the prostate markers P504S ) and the prostatic acid phosphatase were expressed in both cell lines confirming the prostate origin of the cells . Characterization by real-time PCR of PC3c cells indicated that ET1 and OPG, two factors 6099352 that have been implicated in the pathogenesis of osteosclerotic bone metastases from prostate cancer are overexpressed compared to the parental cell line PC3 while other factors such as fibroblast growth 4 New Androgen-Resistant Bone Metastasis Model factor 9 and TGF are similarly expressed in both cell lines. On the other hand, the expression of DKK1 and Noggin, two osteoblast inhibitors inhibitors), is decreased in PC3c versus PC3 . Moreover, PC3c similarly to PC3 cells expressed factors known to be implicated in prostate cancer osteomimicry such as OPN and Runx2. All together, these results suggest that PC3c cells may potentially induce osteoblastic lesions when compared with PC3 cells that are known to predominantly exhibit osteolytic lesions in bone. BV/TV Sham PC3 PC3c 22,4 +/- 2,9 3,6 +/- 4,1 39,5 +/- 3,0$ TB/STV 0 72,6 +/- 6,6 36,6 +/- 4,5$ BV/TV: bone volume/ total volume. TB/STV: tumor burden/soft tissue volume. Sham were performed as control. n is the number of legs with bone metastases. P<0,05; P<0,001 compared with Sham; $P<0,001 compared with PC3. doi: 10.1371/journal.pone.0075092.t001 PC3c cells induce mixed osteoblastic/osteolytic bone lesions In order to test the property of PC3c to induce bone lesions, intra-tibial injections were performed into male SCID mice. Ten weeks after tumor cell inoculation, radiographic analysis revealed that animals bearing PC3c tumors had bone lesions that included osteolytic and osteoblastic components while pure osteolytic lesions were observed in animal bearing PC3 tumors after 6 weeks. The capacity of PC3 and PC3c to induce pure osteolytic and mixed lesions, respectively, was confirmed using 3D micro-CT reconstruction , histology and histomorphometric analyses of tibiae. As expected no skeletal lesions were observed after PBS injection . By immunohistochemistry, we confirmed, in vivo, that ET-1 and OPG were highly expressed in PC3c tumors when compared with PC3. and Dentin matrix acidic phosphoprotein 1 expression was stimulated after 24h of treatment with PC3 and PC3c conditioned medium respectively while OPG and RANKL expression was not affected. PC3c cells induce robust osteoblastic reactions upon osteogenic conditions Because PC3c cells induced new bone formation in vivo, we next tested whether they could produce OBs markers. After immunostaining of bone metastatic tissue sections, OPN and BSP were found expressed in PC3c cells in situ. Moreover after 3 weeks of culture, in vitro, upon osteogenic conditions l analysis Data were expressed as mean +/- SD, and analyzed statistically by one way analysis of variance followed by post hoc t-tests or student t-test to assess the differences between groups for in vitro and in vivo studies. Statistical significance was taken as p<0.05. Results Expression of pro-osteoblastic factors by PC3c cells From human androgen-resistant prostate cancer cell line PC3, we obtained after single cell population culture in vitro a new subculture cell line named PC3c cells that was chosen based on its epithelial phenotype. As expected and similarly to the parental PC3 cells, AR could not be detected by real-time PCR in PC3c while it was expressed in the hormone dependant cell line VCaP used as a positive control. On the other hand, the prostate markers P504S ) and the prostatic acid phosphatase were expressed in both cell lines confirming the prostate origin of the cells . Characterization by real-time PCR of PC3c cells indicated that ET1 and OPG, two factors that have been implicated in the pathogenesis of osteosclerotic bone metastases from prostate cancer are overexpressed compared to the parental cell line PC3 while other factors such as fibroblast growth 4 New Androgen-Resistant Bone Metastasis Model factor 9 and TGF are similarly expressed in both cell lines. On the other hand, the expression of DKK1 and Noggin, two osteoblast inhibitors inhibitors), is decreased in PC3c versus PC3 . Moreover, PC3c similarly to PC3 cells expressed factors known to be implicated in prostate cancer osteomimicry such as OPN and Runx2. All together, these results suggest that PC3c cells may potentially induce osteoblastic lesions when compared with PC3 cells that are known to predominantly exhibit osteolytic lesions in bone. BV/TV Sham PC3 PC3c 22,4 +/- 2,9 3,6 +/- 4,1 39,5 +/- 3,0$ TB/STV 0 72,6 +/- 6,6 36,6 +/- 4,5$ BV/TV: bone volume/ total volume. TB/STV: tumor burden/soft tissue volume. Sham were performed as control. n is the number of legs with bone metastases. P<0,05; P<0,001 compared with Sham; $P<0,001 compared with PC3. doi: 10.1371/journal.pone.0075092.t001 PC3c cells induce mixed osteoblastic/osteolytic bone lesions In order to test the property of PC3c to induce bone lesions, intra-tibial injections were performed 12931192 into male SCID mice. Ten weeks after tumor cell inoculation, radiographic analysis revealed that animals bearing PC3c tumors had bone lesions that included osteolytic and osteoblastic components while pure osteolytic lesions were observed in animal bearing PC3 tumors after 6 weeks. The capacity of PC3 and PC3c to induce pure osteolytic and mixed lesions, respectively, was confirmed using 3D micro-CT reconstruction , histology and histomorphometric analyses of tibiae. As expected no skeletal lesions were observed after PBS injection . By immunohistochemistry, we confirmed, in vivo, that ET-1 and OPG were highly expressed in PC3c tumors when compared with PC3. and Dentin matrix acidic phosphoprotein 1 expression was stimulated after 24h of treatment with PC3 and PC3c conditioned medium respectively while OPG and RANKL expression was not affected. PC3c cells induce robust osteoblastic reactions upon osteogenic 18753409 conditions Because PC3c cells induced new bone formation in vivo, we next tested whether they could produce OBs markers. After immunostaining of bone metastatic tissue sections, OPN and BSP were found expressed in PC3c cells in situ. Moreover after 3 weeks of culture, in vitro, upon osteogenic conditions