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. 2005 Apr 6;5(1):8.
doi: 10.1186/1475-2867-5-8.

Androgen receptor signaling is required for androgen-sensitive human prostate cancer cell proliferation and survival

Qing Yang  1 , Kar-Ming Fung, Wanda V Day, Bradley P Kropp, Hsueh-Kung Lin
Affiliations

Androgen receptor signaling is required for androgen-sensitive human prostate cancer cell proliferation and survival

Qing Yang et al. Cancer Cell Int. .

Abstract

BACKGROUND: Androgens and androgen receptors (AR) regulate normal prostate development and growth. They also are involved in pathological development of prostatic diseases, including benign prostatic hyperplasia (BPH) and prostate cancer (PCa). Antiandrogen therapy for PCa, in conjunction with chemical or surgical castration, offers initial positive responses and leads to massive prostate cell death. However, cancer cells later appear as androgen-independent PCa. To investigate the role of AR in prostate cell proliferation and survival, we introduced a vector-based small interfering RNA (siRNA). This siRNA targeted 5'-untranslated region of AR mRNA for extended suppression of AR expression in androgen-sensitive human prostate LNCaP cells. RESULTS: The siRNA design successfully suppressed endogenous AR expression, as revealed by western blotting and immunofluorescence staining in LNCaP cells. LNCaP cells did not proliferate in the absence of AR and underwent apoptosis, based on elevated phospho-Histone H2B expression and higher number of apoptotic body as compared to control cells. CONCLUSION: We demonstrated that AR is vital for prostate cell proliferation and survival in this androgen-sensitive prostate cell line. These results further strengthen the hypothesis that AR can be a therapeutic target for treating androgen-sensitive stages of PCa. Unlike antiandorgens, however, siRNA targeting AR provides a direct inactivation of AR function through the suppression of AR protein expression.

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Figures

Figure 1
Figure 1
A vector-based plasmid construct for suppression of AR expression in LNCaP cells. (A) Two single-stranded oligonucleotides were synthesized consisting of a 19 nucleotide of -25 to -7 bp 5'-UTR of the AR mRNA separated by a short loop sequence from the reverse complement of the same sequence. The sense strand of synthesized oligonucleatides was ended with five thymidines as termination signal. The annealed ds DNA contained Bgl II and Sfi I restriction at its 5' and 3' end of the oligonucleotides, respectively. (B) Schematic drawing of the pSiAR-EGFP vector. The expression construct was designed to expression AR siRNA driven by U6 promoter for suppression of endogenous AR expression in mammalian cells. The expression construct also contained EGFP driven by the CMV promoter constitutive expression of GFP in target cells.
Figure 2
Figure 2
Suppression of endogenous AR expression in LNCaP cells using the pSiAR-EGFP expression construct. LNCaP cells were transfected with either the pSiAR-EGFP or the pSiAR-EGFP construct using the Lipofectemine 2000 protocol. (A) Immunocytochemical detection of AR expression in transfected cells. To determine AR expression in LNCaP cells, following transfection, cells were stained with mouse anti-human AR monoclonal antibody (1:100 dilution) followed by AlexaR FluorR 594 conjugated goat anti-mouse IgG secondary antibody (2 mg/ml) incubation. The AR staining was detected by fluorescent microscopy (BX51, Olympus). The images were composite between the red with the positive in AR staining with the phase-contract from the same field. Suppression of endogenous AR expression was demonstrated by the absence of red fluorescence staining as indicated by arrows. DAPI staining showed the number of cells in the same field. (B) Western blot analysis of AR expression in pSiAR-EGFP and control transfected cells. At 7 days following transfection, both GFP-positive and GFP-negative cells were collected through cell sorter; and cells were lysed to prepare cellular protein extracts. Aliquots of 20 μg total cellular protein were loaded into Tris-HCl gels and transferred to PDVF membranes. The AR expression was determined by incubating with mouse anti-human AR monoclonal antibody (1:500) followed by the HRP-conjugated anti-mouse IgG (1:125,000) secondary antibody incubation. Immunoreactive signals were detected using ECL. Levels of β-actin expression was also determined in each sample and used as protein loading control.
Figure 3
Figure 3
Suppression of LNCaP cell proliferation in the absence of endogenous AR. LNCaP cells were seeded in tissue culture plates and transfected with a mixture of either pSiAR-EGFP or pSiCon-EGFP plasmid construct with Lipofecamine 2000 in OPTI-MEM. (A) Cell proliferation without separation of GFP-positive and negative cells. At 24 hours following transfection, cells were trypsinized and distributed into each well (1,000 cells/well) of 96-well tissue culture plates in the presence the complete medium. Cell proliferation was determined using the XTT assay kit for a period of 9 days; data from days 11 and 14 were not included since parental and pSiCon-EGFP transfected LNCaP cells reached confluence after day9. (B) LNCaP cell proliferation following enrichment of GFP-positive cells. At 24 hours after transfection, cells were trypsinized and EGFP-positive cells were collected through the MoStar cell sorting system. GFP-positive cells were seeded into each well (1,000 cells/well) of 96-well plates for cell viability assay. Cell proliferation was determined for a period of 14 days. Data were calculated as absorbance at days following transfection normalized to the absorbance at the day of cell sorting, and presented as fold induction in absorbance. LNCaP cells transfected with pSiCon-EGFP plasmid construct were used as the AR-positive control. * represents significant statistical difference between LNCaP cells with and without AR (P < 0.001). Each time point represents the mean ± SEM from 3 independent experiments.
Figure 4
Figure 4
Elevated expression of phospho-histone H2B S(14) in AR-knockdown LNCaP cells. LNCaP cells were either transfected with pSiAR-EGFP or pSiCon-EGFP plasmid construct. On day 6 after transfection, cells were harvested. Cellular proteins were extracted using acid extraction method, electrophoresized through gradient Tris-HCl gels, and electroblotted onto PVDF membranes. Levels of phospho-histone H2B expression was detected by an immunoassay procedure.
Figure 5
Figure 5
Elevated number of apoptotic LNCaP cells transfected pSiAR-EGFP construct. Apoptotic bodies in LNCaP cells transfected with control vector pSiCon-EGFP (A) and pSiAR-EGFP (B). Following transfection with these plasmid constructs, LNCaP cells were collected through centrifugation, fixed in neutralized formalin, and encased in agrose blocks. Cell blocks were paraffin-embedded, sectioned, deparaffinized, rehydrated, and stained with hematoxylin. Apoptotic bodies which showed condensed and/or cleaved nucleus were counted from random fields and numerical data are shown in Table 1.
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References

    1. Heinlein CA, Chang C. Androgen receptor in prostate cancer. Endocr Rev. 2004;25:276–308. doi: 10.1210/er.2002-0032. - DOI - PubMed
    1. Roche PJ, Hoare SA, Parker MG. A consensus DNA-binding site for the androgen receptor. Mol Endocrinol. 1992;6:2229–2235. doi: 10.1210/me.6.12.2229. - DOI - PubMed
    1. Nelson PS, Clegg N, Arnold H, Ferguson C, Bonham M, White J, Hood L, Lin B. The program of androgen-responsive genes in neoplastic prostate epithelium. Proc Natl Acad Sci USA. 2002;99:11890–11895. doi: 10.1073/pnas.182376299. - DOI - PMC - PubMed
    1. Culig Z, Hobisch A, Cronauer MV, Cato ACB, Hittmair A, Radmayr C, Eberle J, Bartsch G, Klocker H. Mutant androgen receptor detected in an advanced-stage prostatic carcinoma is activated by adrenal androgens and progesterone. Mol Endocrinol. 1993;7:1541–1550. doi: 10.1210/me.7.12.1541. - DOI - PubMed
    1. Buchanan G, Yang M, Harris JM, Nahm HS, Han G, Moore N, Bentel JM, Matusik RJ, Horsfall DJ, Marshall VR, Greenberg NM, Tilley WD. Mutations at the boundary of the hinge and ligand binding domain of the androgen receptor confer increased transactivation function. Mol Endocrinol. 2001;15:46–56. doi: 10.1210/me.15.1.46. - DOI - PubMed