Ibody (Fig. 1Bi). Smooth muscle cells have much stronger staining (3) than

Ibody (Fig. 1Bi). Smooth muscle cells have much stronger GBT-440 web staining (3) than fibroblast cells (0?+). The staining pattern of BMI1 protein was compared in stage II V CaP Galanthamine specimens (Fig. 1Bi). These data showed increased expression levels of BMI1 protein in high grade tumor in human CaP (Fig. 1Bi). The box plots of the data for BMI1 protein expression in stroma exhibited a wide interspecimen variation in cancer specimens, compared with normal tissues and revealed a significant difference in the level of protein between normal and CaP tissues (p,0.05, Fig. 1Bii). The average score for the staining intensity of BMI1 in stroma of normal tissues was 0.8160.07 (n = 70), and was significantly lower than highgrade stage II (1.860.08; n = 36), stage III (2.2660.10 n = 28) and stage IV (2.860.11; n = 6) cancer specimens (Fig. 1Bii; p,0.05). A similar pattern of staining in pair-matched CaP specimens was observed in the epithelial of the prostatic specimens. Taken together, these data show that expression of BMI1 increases with increasing stage of CaP.Androgen treatment of cellsFor this reason, Caucasian and African American prostate epithelial cells were treated with androgen analogue (R1881) for 12 h. After 12 h, media was discarded and cells were grown in further 12 h. After 24 h, cells were harvested to be evaluated for intracellular BMI1 expression by western blot analysis.Statistical analysesGraphical summaries of the distribution of staining intensity were made using scatter plots and box plots. Simple linear egression and correlation methods were use to evaluate associations between BMI1, PSA and CaP rank (1 = normal, 2 = Stage II, 3 = Stage III, 4 = Stage IV). To correct for skewness, BMI1 and PSA were analyzed on a log (base2) scale. A p-value of ,0.05 was considered to be statistically significant.BMI1 expression in normal and neoplastic prostatic cells representing CaP 16574785 disease in Caucasian menAs an attempt towards identifying the expression of BMI1 in CaP progression, we first measured protein expression levels by immunoblot analysis in several human Caucasian CaP cell lines, LNCaP, Du145 and PC3 and compared them to NHPE (normal primary prostate epithelial cell) and RWPE1 (representing normal immortalized prostatic epithelial cells), respectively. Among the CaP cell lines used, LNCaP is androgen-dependent whereas Du145 and PC3 are androgen-independent. The choice of these cells was based on the fact that 80 CaP patients present with androgen-dependent disease at the time of diagnosis which later transforms into more aggressive, androgen-independent disease [19]. As shown in Figure 2(Ai i), all CaP cell lines exhibited a higher expression of BMI1 protein than in normal prostate epithelial cells. When the protein expression of BMI1 was compared, based on the densitometric analysis of the immunoblots, highly aggressive PC3 cells and Du145 exhibited higher expression than in LNCaP cells (Fig. 2Aii). Interestingly, we also detected BMI1 expression in the prostate stromal cells (WPMY1) (Fig. 2Ai i). Interestingly, BMI1 expression was found to be very low in prostate epithelial cells representing benign prostatic hyperplasia (BPH) condition (data not shown).Results Bmi1 protein levels in prostatic tissues increases with progressive stages of disease in transgenic TRAMP mouse modelsGlinsky et al. [18] previously showed that Bmi1 protein is elevated in the prostatic tissues of TRAMP mice, an autochthonous mouse model of CaP development, we inves.Ibody (Fig. 1Bi). Smooth muscle cells have much stronger staining (3) than fibroblast cells (0?+). The staining pattern of BMI1 protein was compared in stage II V CaP specimens (Fig. 1Bi). These data showed increased expression levels of BMI1 protein in high grade tumor in human CaP (Fig. 1Bi). The box plots of the data for BMI1 protein expression in stroma exhibited a wide interspecimen variation in cancer specimens, compared with normal tissues and revealed a significant difference in the level of protein between normal and CaP tissues (p,0.05, Fig. 1Bii). The average score for the staining intensity of BMI1 in stroma of normal tissues was 0.8160.07 (n = 70), and was significantly lower than highgrade stage II (1.860.08; n = 36), stage III (2.2660.10 n = 28) and stage IV (2.860.11; n = 6) cancer specimens (Fig. 1Bii; p,0.05). A similar pattern of staining in pair-matched CaP specimens was observed in the epithelial of the prostatic specimens. Taken together, these data show that expression of BMI1 increases with increasing stage of CaP.Androgen treatment of cellsFor this reason, Caucasian and African American prostate epithelial cells were treated with androgen analogue (R1881) for 12 h. After 12 h, media was discarded and cells were grown in further 12 h. After 24 h, cells were harvested to be evaluated for intracellular BMI1 expression by western blot analysis.Statistical analysesGraphical summaries of the distribution of staining intensity were made using scatter plots and box plots. Simple linear egression and correlation methods were use to evaluate associations between BMI1, PSA and CaP rank (1 = normal, 2 = Stage II, 3 = Stage III, 4 = Stage IV). To correct for skewness, BMI1 and PSA were analyzed on a log (base2) scale. A p-value of ,0.05 was considered to be statistically significant.BMI1 expression in normal and neoplastic prostatic cells representing CaP 16574785 disease in Caucasian menAs an attempt towards identifying the expression of BMI1 in CaP progression, we first measured protein expression levels by immunoblot analysis in several human Caucasian CaP cell lines, LNCaP, Du145 and PC3 and compared them to NHPE (normal primary prostate epithelial cell) and RWPE1 (representing normal immortalized prostatic epithelial cells), respectively. Among the CaP cell lines used, LNCaP is androgen-dependent whereas Du145 and PC3 are androgen-independent. The choice of these cells was based on the fact that 80 CaP patients present with androgen-dependent disease at the time of diagnosis which later transforms into more aggressive, androgen-independent disease [19]. As shown in Figure 2(Ai i), all CaP cell lines exhibited a higher expression of BMI1 protein than in normal prostate epithelial cells. When the protein expression of BMI1 was compared, based on the densitometric analysis of the immunoblots, highly aggressive PC3 cells and Du145 exhibited higher expression than in LNCaP cells (Fig. 2Aii). Interestingly, we also detected BMI1 expression in the prostate stromal cells (WPMY1) (Fig. 2Ai i). Interestingly, BMI1 expression was found to be very low in prostate epithelial cells representing benign prostatic hyperplasia (BPH) condition (data not shown).Results Bmi1 protein levels in prostatic tissues increases with progressive stages of disease in transgenic TRAMP mouse modelsGlinsky et al. [18] previously showed that Bmi1 protein is elevated in the prostatic tissues of TRAMP mice, an autochthonous mouse model of CaP development, we inves.