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First published online 15 July 2008
doi: 10.1242/jcs.030742

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An AR-Skp2 pathway for proliferation of androgen-dependent prostate-cancer cells

¹ Department of Developmental and Molecular Biology, The Albert Einstein Comprehensive Cancer Center and Liver Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA
² Department of Urologic Oncology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA

View larger version (30K): [in this window] [in a new window]   Fig. 1. Effects of Skp2 knockdown on proliferation of various cancer cell lines. LNCaP is the only cell line whose proliferation was near completely inhibited by Skp2 knockdown. (A) Various cell lines, as indicated, were plated at 50% confluency and transduced with lentiviruses expressing control miRNA (C) or Skp2 miRNA (S) for 2 days before western blots of Skp2 and Cdk2 were performed. (B) Relative S-phase population as determined by BrdU incorporation during a 2-hour pulse for each cell line marked in A.

View larger version (58K): [in this window] [in a new window]   Fig. 2. Effects of knockdown of AR or Skp2 on the proliferation and differentiation of LNCaP cells in FBS media. (A) LNCaP cells were transduced with the indicated lentiviral constructs for 2 days and used for the cell-proliferation assay (see Materials and Methods). Relative cell number at the end of a 12-day culture in FBS-media are presented. (B) Western blots of LNCaP cells transduced with the indicated lentiviral constructs (top) for 3 days. (C) Phase-contrast photographs of cells shown in A before harvesting for the cell-proliferation assay. Results shown are representative of at least three independent experiments with LNCaP cells at various passages.

View larger version (35K): [in this window] [in a new window]   Fig. 3. Status of the AR-Skp2 pathway in various prostate-cancer cells as indicated. (A,C,D) Western blots and relative cell proliferation of LAPC4, C4-2 and 22Rv1 cells, respectively, after transduction with the indicated lentiviral constructs and culturing in 10% FBS medium. These experiments were performed twice with similar results. (B) Western blots of CWR22 xenograft tumors harvested at the indicated times.

View larger version (62K): [in this window] [in a new window]   Fig. 4. Regulation of Skp2 protein levels in LNCaP cells by androgen. Androgen regulates Skp2 protein levels in LNCaP cells. (A) Proliferation of LNCaP cells, measured 12 days after culture in the indicated media. (B) Western blots of LNCaP cells cultured in the indicated media for 2 days at about 70% confluency. (C) Time-course experiments with LNCaP cells. Cells were incubated in CDT media for 2 days before the start of this experiment. Cells were harvested at the indicated time points for western blot analyses.

View larger version (56K): [in this window] [in a new window]   Fig. 5. Regulation of Skp2 mRNA and protein stability of androgen. Androgen regulates Skp2 mRNA levels and protein stability. (A) Western blots of LNCaP cells cultured in the indicated conditions. (B) Semi-quantitative RT-PCR of LNCaP cells cultured in the indicated conditions. (C) Western blots of LNCaP cells incubated in the indicated conditions for 3 days [treatment of MG-132 (50 µM) was for the last 16 hours]. (D) CHX treatment and chase to determine the degradation of Skp2 and Cdk2 after LNCaP cells were transferred from FBS media to either CDT or CDT+R1881 media. Western blot films were scanned, quantified and plotted. All results are representative of three independent experiments.

View larger version (39K): [in this window] [in a new window]   Fig. 6. Stabilization of CMV-expressed FLAG-Skp2 by androgen. Androgen can stabilize exogenous Skp2 that is overexpressed from a CMV promoter. (A) Skp2 protein levels in LNCaP, LN-empty and LN-FLAG-Skp2 cells. (B) Relative proliferation of LN-FLAG-Skp2 cells incubated in the four media conditions, as marked, for 12 days. (C) Western blot of LN-FLAG-Skp2 cells after culture in the indicated conditions for 2 days. FLAG-Skp2 was detected with an anti-FLAG antibody. (D) CHX experiment as in Fig. 5D. All results are representative of three independent experiments.

View larger version (56K): [in this window] [in a new window]   Fig. 7. Inhibition of cell proliferation and induction of CMV-expressed FLAG-Skp2 protein accumulation by high concentrations of androgen. High concentrations of androgen inhibit cell proliferation and induce accumulation of CMV-expressed FLAG-Skp2. (A) BrdU labeling of LN-FLAG-Skp2 cells cultured in various concentrations of R1881 as indicated (20p, 20 pM; 2n, 2 nM). (B) Western blots of LN-FLAG-Skp2 cells cultured at the indicated conditions for 3 days. (C) CHX experiment as in Fig. 5D. (D) LN-FLAG-Skp2 cells were transduced with lentiviruses expressing control or AR miRNA and treated with various concentrations of R1881 to determine their effects on FLAG-Skp2 protein levels as in B. These results are representative of three independent experiments. (E) LAPC4 and DU145 prostate-cancer cell lines were similarly treated with increasing concentrations of R1881 for 3 days and subjected to western blot analysis. These experiments were performed twice with identical results.

View larger version (37K): [in this window] [in a new window]   Fig. 8. Presence of a transferable, D-box-dependent and androgen-responsive degron in the Skp2 N-terminus. The Skp2 N-terminus contains a transferable, D-box-dependent and androgen-responsive degron. (A,B) Cartoon presentation of various GFP fusion proteins. LRR, leucine-rich repeats; CRS, cytoplasm-retention signals. (C) LNCaP cells were transduced with lentiviruses expressing GFP, NGFP, 20NGFP or BGFP, incubated in CDT media with or without MG-132 (50 µM) for 24 hours, then harvested for western blotting. (D) The same transduced LNCaP cells were incubated in the indicated conditions (R1881 at 2 nM) for 3 days and then harvested for western blotting. All results are representative of three independent experiments.

View larger version (30K): [in this window] [in a new window]   Fig. 9. Green-fluorescence flow-cytometry analysis of the effects of androgen. (A) LNCaP cells transduced with lentiviruses expressing the indicated proteins (top) were incubated in CDT or CDT+R1881 (100 pM) for 3 days. Cells were trypsinized, resuspended in PBS and used immediately for flow-cytometry analysis. Twenty thousand cells were counted. The position of uninfected (GFP negative) cells in the same cell cultures was representatively indicated by a filled arrow in the top-left FACS plot. (B) LNCaP cells expressing NGFP were subjected to the same analysis, except that a serum-free-medium condition was included. All results are representative of three independent experiments.

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