Antisense RNA Down-Regulation of bcl-2 Expression in DU145 Prostate Cancer Cells Does Not Diminish the Cytostatic Effects of G3139 (Oblimersen)

A, representative reverse transcription-PCR (RT-PCR) analysis demonstrating the production of the antisense bcl-2 mRNA in infected DU145 prostate cancer cells. In the bcl-2 AS1 clone, a 113 bp RT-PCR product was observed, which was not seen in the AS2 clone, or in the mock-infected or wild-type cells. Conversely, in the AS2 clone, a 413 bp product was observed, which was not seen in any other cell line. When a combination of the 5′ AS1 and 3′ AS2 primers were used, no products were observed in any cell line. A control G3PDH RT-PCR is also shown. In the absence of RTase, no PCR products were seen. B, representative Western blot analysis demonstrating down-regulation of bcl-2 expression in AS1- and AS2-infected DU145 prostate cancer cells by an antisense RNA strategy. Greatest down-regulation was observed in the AS1 clone 15 (94%) and AS2 clone 1 (83%). Protein samples (30–40 μg of protein/lane) were analyzed as described in “Materials and Methods,” with tubulin used as a control protein species. Percentage inhibition versus mock-transfected cells was determined by laser-scanning densitometry. D, parental DU145 cells; M, mock-infected (with empty viral vector) cells.

Representative Western blot analysis demonstrating that down-regulation of bcl-2 expression in AS1 and AS2 by antisense RNA strategy leads to the up-regulation of protein kinase C (PKC)-α, catalase, and cIAP-1 protein expression, and down-regulation of cIAP-2. In contrast, the expression of bcl-xL and bax proteins were only slightly changed, whereas no change in the levels of expression of nuclear factor κB (p50), relA (p65), XIAP, and SOD were observed. Protein samples (30–40 μg of protein/lane) were analyzed as described in “Materials and Methods,” with tubulin used as a control protein species. The percentage of inhibition versus control, untreated cells was determined by laser-scanning densitometry.

Flow cytometric analysis of oligonucleotide-treated mock-transfected (A) or AS1 DU145 cells (B) analyzed after double staining with fluoresceinated anti-Annexin V monoclonal antibody staining (X axis) and propidium iodide (Y axis). 10⁴ cells/sample were analyzed at a rate of 100–200 cells/s. Cells were treated with complexes of G3139, G4126, G4232, or 2009 (400 nm) and Lipofectin (15 μg/ml) for 5 h in Opti-MEM, and then for an additional 67 h in complete medium without complexes. Data points in the top right quadrant represent individual cells in late apoptosis/early necrosis. Early apoptosis is represented in the bottom right quadrant, and necrosis in the top left quadrant. Treatment with G3139 and related oligonucleotides did not cause significant apoptosis. a, untreated cells; b, G4126; c, G3139; d, G4232; e, 2009.

Flow cytometric analysis of bromodeoxyuridine incorporation (Y axis; FL1 channel) and DNA content as determined by propidium iodide staining (X axis; FL3 channel) in oligonucleotide-treated mock (A) and AS1 transfected (B) DU145 cells. Cells were either untreated (a); or treated for 5 h with complexes of G4126 (b), G3139 (c), G4232 (d), or 2009 (e; 400 nm) and Lipofectin (15 μg/ml) for 5 h in Opti-MEM, and then for an additional 67 h in complete medium without complexes. Nineteen h later, as described in “Materials and Methods,” cells were pulsed with 10 μm bromodeoxyuridine for 2 h, harvested, and analyzed (A and B; and region gates for the quantitative analysis are indicated). Integrated data are presented in Tables 2<$REFLINK> and 3<$REFLINK> .

Representative histograms of the mean fluorescence channels of yields 2′-7′ dichlorofluorescein (DCF) and ethidium (E) fluorescence from the flow cytometric analysis of reactive oxygen species (ROS) production in oligonucleotide-treated mock, AS1- and AS2-infected DU145 prostate cancer cells, as demonstrated by the oxidation of H₂DCF to DCF and hydroethidium (HE) to E. The production of ROS in oligonucleotide-treated AS1and AS2 cells was identical to the ROS production in mock-transfected cells. G3139 and 2009 caused a significant shift in fluorescence of both markers of ROS production, whereas G4126 and G4232 produced only a small increase. Cells were treated with oligomer (400 nm)/lipofectin (15 μg/ml) complexes for 5 h, and assayed after 3 days, as described. Fold increases in mean fluorescence channel were normalized against untreated cells. Experiments were done in triplicate and data are presented as mean; bars, ±SD.

A, representative Western blot analysis demonstrating that small interfering RNA bcl-2 (D6 and D1) down-regulates bcl-2 protein expression. In contrast, essentially no change in the levels of expression of protein kinase C (PKC)-α or bax proteins were observed. DU145 cells were treated either with complexes of D6, D1, or D3 (25 nm) and Lipofectamine 2000 (1.33 μg/ml) for 24 h in Opti-MEM and then for an additional 48 h in complete medium without complexes, and harvested. Protein samples (30–40 μg of protein/lane) were analyzed by Western blotting as described in “Materials and Methods,” with tubulin used as a control protein species. The percentage of inhibition versus control, untreated cells was determined by laser-scanning densitometry. B, time dependence of the effects of siRNA bcl-2 (D6 and D1) on bcl-2 protein expression. DU145 cells were treated either with complexes of D6, D1, or D3 (25 nm) and Lipofectamine 2000 (1.33 μg/ml) for 24 h in Opti-MEM and then for an additional 2–9 days in complete medium without complexes. Cells were harvested, and protein samples (30–40 μg of protein/lane) were analyzed by Western blotting 3–10 days after the treatment as described in “Materials and Methods,” with tubulin used as a control protein species. The percentage of inhibition versus control, untreated cells was determined by laser-scanning densitometry.

DU145 cells in which bcl-2 protein expression has been down-regulated previously are not sensitized to treatment with cytotoxic agents. Mock-transfected and the AS1 or AS2 clones were treated for 72 h with Taxol (A) or mitoxantrone (B and C) at the indicated concentrations. Cellular viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as described in “Materials and Methods,” and absorbance was measured at 570 nm. Cellular viability was determined as a ratio of the absorbance of the treated cells to that of the corresponding untreated cells. Data are presented as the average; bars, ±SD; n = 4. Wild-type DU145 cells were also not sensitized to cytotoxic agents after small interfering RNA D6 treatment (D–F). 25 × 10⁴ cells were seeded in six-well plates, treated either with complexes of D6 or control D3 small interfering RNAs (25 nm) and Lipofectamine 2000 (1.33 μg/ml) for 24 h in Opti-MEM, and then for an additional 2 days in complete medium without complexes. The cells were then reseeded in 96-well plates (0.4 × 10⁴ cells/well) at the same density, and next day treated for 72 h with Taxol (D), mitoxantrone (E), or thapsigargin (F) at the indicated concentrations. Cellular viability was determined as a ratio of the absorbance of the treated cells to that of the corresponding untreated cells. Data are presented as the average; bars, ±SD; n = 4.