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The role of IKK in constitutive activation of NF-B transcription factor in prostate carcinoma cells

¹ AMC Cancer Research Center, Denver, CO 80214, USA
² Regulation of Cell Growth Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, PO Box B, Frederick, MD 21702, USA
³ N. N. Blokhin Cancer Research Center, Moscow, 115478, Russia

View larger version (23K): [in a new window]   Fig. 1. Analysis of constitutive B DNA-binding in human primary prostate cells and PC cell lines. (A) EMSA analysis of B-binding. PrEC, normal epithelial prostate primary cultures; LNCaP and MDA PC 2b are androgen-dependent cell lines; CL2 (derived from LNCaP cells), JCA1, PC3 and DU145 are androgen-independent PC cell lines. Nuclear proteins (10 µg/reaction) from untreated cells and LNCaP cells treated with TNF- were used for electrophoretic mobility shift assay (EMSA). Composition of dimers is indicated. Data are shown for one representative experiment. (B) Identification of nuclear B-binding complexes by EMSSA. Nuclear proteins from PC3 cells were incubated with a labeled B oligonucleotide and antibodies against p50 and p65 proteins. DNA binding activity was analyzed by EMSA. Composition of dimers is indicated. (C) EMSA analysis of Sp1-binding. Nuclear proteins (10 µg/reaction) from the same cells as in Fig. 1A, were used for EMSA with Sp1 oligonucleotide. Composition of dimers is indicated. Data are shown for one representative experiment.

View larger version (22K): [in a new window]   Fig. 2. Analysis of constitutive NF-B transcriptional activity in human primary prostate cells and PC cell lines. (A) Constitutive activity of B-luciferase reporter. Prostate cells were cotransfected with B-luciferase reporter and pRL-null construct. Luciferase activity was measured 24 hours after transfection in untreated prostate cells by dual luciferase assay. Data are shown as FL/RL ratio for one representative experiment. (B) Northern blot analysis of constitutive IB and IL-6 genes expression. Northern blots containing total RNA (20 µg/lane) from untreated normal prostate and PC cells were probed for expression of IB and IL-6 genes. The membranes were also hybridized with a 7S RNA probe as a control for equal RNA loading.

View larger version (46K): [in a new window]   Fig. 3. Western blot analysis of NF-B and IB protein expression in human primary prostate cells and PC cell lines. Western blots containing whole cell protein extracts (10 µg/lane) from untreated cells were probed for expression of p50, p65, IB, IBß and IB.

View larger version (40K): [in a new window]   Fig. 4. Proteasomal inhibitor MG132 decreased B DNA binding in androgen-independent PC cells. Androgen-independent cell lines were treated with proteasomal inhibitor MG132 (7.5 µg/ml) for 30-60 minutes. Nuclear proteins (5 µg/reaction) were used for EMSA.

View larger version (34K): [in a new window]   Fig. 5. Analysis of IB phosphorylation in androgen-independent PC cells. Western blots containing whole cell protein extracts (10 µg/lane) from untreated cells were probed for expression of IB and IB-P. Data are shown for the one representative experiment.

View larger version (13K): [in a new window]   Fig. 6. The rate of IB phosphorylation in androgen-dependent and androgen-independent PC cell lines. Indicated cell lines were treated with proteasomal inhibitor MG132 (7.5 µg/ml) for 0.5-6 hours. (A) Western blots containing cytosol proteins (10 µg/lane) were probed for expression of IB-P. (B) Western blots shown in A are plotted as a percentage of the maximum IB-P expression level. Abscissa: time after MG132 treatment (hours). Ordinate: relative amount of IB-P. Data are shown for the one representative experiment.

View larger version (14K): [in a new window]   Fig. 7. The rate of IB-P degradation is higher in androgen-independent PC cells. Indicated cell lines were treated with cycloheximide (CHX, 10 µg/ml) in combination with 15-deoxy-12,14-prostaglandin J2 (15dPGJ2) for 0.5-6 hours. (A) Western blots containing cytosol protein extracts (10 µg/lane) were probed for expression of IB-P. (B) Western blots shown in A are plotted as a percentage of the initial IB-P expression level. Abscissa: time after treatment (hours). Ordinate: relative amount of IB-P. Data are shown for the one representative experiment.

View larger version (24K): [in a new window]   Fig. 8. Effect of cycloheximide on NF-B binding in PC cells. Indicated cell lines were treated with CHX (10 µg/ml) for 0.5-3 hours. Nuclear proteins (5 µg/reaction) from cells were used for electrophoretic mobility shift assay (EMSA). (A) NF-B binding activity. (B) NF-B binding activity shown in A is plotted as a percentage of the maximum (at 3 hours of CHX treatment) p65/p50 binding level. Abscissa: time after treatment (minutes). Ordinate: relative amount of NF-B binding. Data are shown for the one representative experiment.

View larger version (21K): [in a new window]   Fig. 9. Pulse-chase analysis of IB degradation in PC cell lines. LNCaP and DU145 cells were metabolically labeled with 35S-Met-Cys and harvested at indicated time points. IB was immunoprecipitated, resolved on 12.5% denaturating PAAG and transferred to membrane. Dried membrane was subjected to radiography.

View larger version (38K): [in a new window]   Fig. 10. Analysis of IKK expression and IKK activity in normal prostate cells and PC cell lines. (A) Western blot analysis of IKK and IKKß expression. Western blots containing whole cell protein extracts from untreated cells (10 µg/lane) were probed for expression of IKK and IKKß. (B) Analysis of IKK activity. Protein extracts from untreated cells were immunoprecipitated with a combination of IKK and IKKß antisera, and used for in vitro kinase reaction. Protein extract from LNCaP cells stimulated by TNF- (7.5 ng/ml, 10 minutes) was used as a positive control.

View larger version (14K): [in a new window]   Fig. 11. Effect of d.n. IKK mutants on NF-B constitutive activity in normal prostate cells and the PC3 cell line. (A) Prostate cells were co-transfected with B-luciferase, pRL-null and (1) control vector; (2) IKK d.n. mutant; (3) IKKß d.n. mutant; and (4) IB d.n. mutant. (B) Prostate cells were co-transfected with MMTV-luciferase, pRL-null and (1) control vector; (2) IKK d.n. mutant; (3) IKKß d.n. mutant; and (4) IB d.n. mutant. Luciferase activity was measured 24 hours after transfection in untreated prostate cells by dual luciferase assay. Data are shown as FL/RL luciferase activities ratio (% to control) for one representative experiment. PrEC, normal epithelial prostate primary cultures.

View larger version (36K): [in a new window]   Fig. 12. Effect of IB d.n. mutant on apoptosis in prostate cells. (A) Western blot detection of PARP cleavage. PC3 and LNCaP prostate cells were infected with adenovirus expressing GFP and IB mutant lacking Ser32 and Ser36 (AdV-d.n.IB) or adenovirus expressing only GFP (AdV-control). 24 hours later cell cultures were left untreated or treated with TNF- (7.5 ng) for 10 hours. PARP cleavage was detected by western blotting with antibody that detects the full length PARP (116 kDa) and PARP cleavage product (85 kDa). Adherent cells and detached floaters were combined for whole-cell lysate preparations. (1) Untreated cells; (2) AdV-d.n.IB-infected cells; (3) AdV-d.n.IB-infected cells treated with TNF-; (4) AdV-control-infected cells; (5) AdV-control-infected cells treated with TNF-. (B) Effect of IB d.n mutant on morphology of PC3 cells. Micrographs (x300) depicting representative morphological response of PC3 cells 48 hours after infection: (1) with AdV-control; (2) with AdV-d.n.IB; and (3) with AdV-d.n.IB and treated with TNF-. Note numerous blebbing cells in cell cultures treated with TNF-.