Downmodulation of Bcl-2 sensitizes metastatic LNCaP-LN3 cells to undergo apoptosis via the intrinsic pathway.

BACKGROUND: We explored the mechanisms of apoptosis after Bcl-2 protein downmodulation in metastatic LNCaP-LN3 cells (LN3). METHODS: LNCaP, LNCaP-Pro5 (Pro5) and LN3 cells were cultured in 5% charcoal-stripped serum (CSS) or in R1881 (synthetic androgen) and bicalutamide (synthetic anti-androgen) and growth inhibition was assessed. Expression levels of androgen receptor (AR) and Bcl-2 were determined. LN3 cells were transfected with small interfering RNA Bcl-2 (siRNA Bcl-2) or control siRNA oligonucleotides. Rates of apoptosis and proliferation were obtained. Cytochrome c localization in treated and control cells was assessed +/- cyclosporine A (CsA). Caspases 9, 3, and poly (ADP-ribose) polymerase cleavage (PARP) were measured upon downmodulation of Bcl-2; and cell growth inhibition in vitro after Bcl-2 modulation combined with docetaxel chemotherapy was determined. RESULTS: LN3 cells maintained growth under castrate conditions in vitro. AR protein amplification did not explain castrate-resistant LN3 cell growth. Bcl-2 protein levels in LN3 cells were significantly higher than in Pro5 cells, and were effectively downmodulated by siRNA Bcl-2. Subsequently increased apoptosis and decreased proliferation mediated by cytochrome c was noted and this was reversed by CsA. siRNA Bcl-2-transfected LN3 cells exhibited elevated levels of caspases 9, 3, and PARP cleavage. Exposure of LN3 cells to docetaxel led to increased apoptosis, and simultaneous downmodulation of Bcl-2 substantially enhanced this effect. CONCLUSIONS: Downmodulation of Bcl-2 in metastatic castrate-resistant LNCaP-LN3 cells led to apoptosis via a cytochrome c-dependent pathway that was enhanced with docetaxel treatment.

The use of short tandem repeat profiling to characterize human bladder cancer cell lines.

PURPOSE: Cross-contamination of cell lines is a serious but often unrecognized problem. We describe the authentication of a panel of transitional cell carcinoma cell lines using the short tandem repeat profiling technique to detect cross-contamination. MATERIALS AND METHODS: Genomic DNA was isolated from UM-UC-1, UM-UC-2, UM-UC-3 (ATCC), UM-UC-6, UM-UC-9, UM-UC-10, UM-UC-11, UM-UC-13, UM-UC-14, UM-UC-16, T24 and KU7 cell lines. Short tandem repeat loci (D3S1358, D16S539, vWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317 and D7S820) and a segment of the X-Y homologous gene amelogenin were co-amplified by polymerase chain reaction. Profiling was done using POP-4TM performance optimized polymer 4 (Applied Biosystems) with an ABI Prism 310 genetic analyzer. DNA sequencing of TP53 and immunohistochemistry for p53 were performed in UM-UC-3 and UM-UC-3-GFP. RESULTS: All cell lines had a unique short tandem repeat profile except UM-UC-2 and T24, which were virtually identical. T24 short tandem repeat profiles matched those of early passage number UM-UC-2. Stable transfection of the green fluorescence protein marker gene did not alter UM-UC-6, UM-UC-14 or KU7 profiles. However, the short tandem repeat profile for UM-UC-3-GFP was different from that of UM-UC-3. DNA sequencing showed a difference in TP53 between UM-UC-3 and UM-UC-3-GFP, confirming that UM-UC-3-GFP is not derived from UM-UC-3. CONCLUSIONS: Short tandem repeat profiling provides a unique genetic signature of human cell lines that does not significantly change with passage or green fluorescence protein transduction. Using short tandem repeat profiling we noted that the cell line UM-UC-2 is T24. DNA fingerprinting using short tandem repeat profiling is an easy and reliable tool that can be used to verify cell lines.

Analysis of the expression of biomarkers in urinary bladder cancer using a tissue microarray.

Dysregulation of Akt, PTEN, Drg-1, Cx-26, and L-plastin expression appear to be important in the progression of various cancers. Their expression in bladder cancer has not been well characterized. To assess the expression of these genes and their relationship to the outcome of bladder cancer, we used a bladder cancer tissue microarray (TMA) of 251 transitional cell carcinomas. We quantitated immunohistochemical staining of each protein using both automated and manual methods and correlated the expression levels with the clinicopathologic characteristics of the tumor and patient survival. Overall, the results from both automated and manual analyses were similar. We found a significant correlation between the expression of PTEN, Cx-26 and L-plastin with known clinically important pathologic features of bladder cancer (tumor grade, stage, and growth pattern). Aberrant localization patterns of Cx-26 and Drg-1 were observed in bladder tumors. There was also a significant correlation in expression among pAkt, PTEN, and L-plastin. Although the expression of these genes correlated with factors known to be associated with patient outcome, none of them was an independent predictor of progression-free or overall survival.

Spindle checkpoint function requires Mad2-dependent Cdc20 binding to the Mad3 homology domain of BubR1.

The mitotic spindle assembly checkpoint delays anaphase until all chromosomes achieve bipolar attachment to the spindle microtubules. The spindle assembly checkpoint protein BubR1 is thought to act by forming an inhibitory complex with Cdc20. We here identify two Cdc20 binding sites on BubR1. A strong Cdc20 binding site is located between residues 490 and 560, but mutations that disrupt Cdc20 binding to this region have no effect upon checkpoint function. A second Cdc20 binding site present between residues 1 and 477 is highly specific for Cdc20 already bound to Mad2. Mutation of a conserved lysine in this region weakened Cdc20 binding and correspondingly reduced checkpoint function. Our results indicate that there may be more than one checkpoint complex containing BubR1, Mad2, and Cdc20. They also lead us to propose that in vivo checkpoint inhibition of Cdc20 is a two-step process in which prior binding of Mad2 to Cdc20 is required to make Cdc20 sensitive to inhibition by BubR1. Thus, Mad2 and BubR1 must cooperate to inhibit Cdc20 activity.