doi:10.1016/j.cellsig.2006.07.011 
Copyright © 2006 Elsevier Inc. All rights reserved.
Androgen dependent regulation of protein kinase A subunits in prostate cancer cells
Anne-Katrine Kvissela, b, Håkon Rambergc, Turid Eidec, Aud Svindlandd, Bjørn Steen Skålhegga and Kristin Austlid Taskénc, 
, 
aDepartment of Nutrition, University of Oslo, N-0317 Oslo, Norway
bDepartment of Biochemistry, University of Oslo, N-0317 Oslo, Norway
cFaculty Division Aker University Hospital, University of Oslo, Oslo Urological University Clinic, Urological Research Institute, building 23, Aker University Hospital, Trondheimsveien 235, N-0514 Oslo, Norway
dFaculty Division Aker University Hospital, University of Oslo, Department of Pathology, Aker University Hospital, N-0514 Oslo, Norway
Received 28 April 2006;
revised 11 July 2006;
accepted 18 July 2006.
Available online 25 July 2006.
References and further reading may be available for this article. To view references and further reading you must
purchase this article.
Abstract
Neuroendocrine (NE) cells may play a role in prostate cancer progression. Both androgen deprivation and cAMP are well known inducers of NE differentiation (NED) in the prostate. Gene-expression profiling of LNCaP cells, incubated in androgen stripped medium, showed that the Cβ isoform of PKA is up-regulated during NE differentiation. Furthermore, by using semi-quantitative RT-PCR and immunoblotting analysis, we observed that the Cβ splice variants are differentially regulated during this process. Whereas the Cβ2 splice variant is down-regulated in growth arrested LNCaP cells, the Cβ1, Cβ3 and Cβ4 variants, as well as the RIIβ subunit of PKA, are induced in NE-like LNCaP cells. The opposite effect of Cβ expression could be mimicked by androgen stimulation, implying the Cβ gene of PKA as a putative new target gene for the androgen receptor in prostate cancer. Moreover, to investigate expression of PKA subunits during prostate cancer progression, we did immunoblotting of several prostatic cell lines and normal and tumor tissue from prostate cancer patients. Interestingly, multiple Cβ subunits were also observed in human prostate specimens, and the Cβ2 variant was up-regulated in tumor cells. In conclusion, it seems that the Cβ isoforms play different roles in proliferation and differentiation and could therefore be potential markers for prostate cancer progression.
Keywords: Prostate cancer; Protein kinase A (PKA); Catalytic subunit; Neuroendocrine differentiation; Androgen
 |
Fig. 1. Messenger RNA for Cβ is up-regulated during NE differentiation of LNCaP cells and the Cβ splice variants are differentially regulated. A) Invert phase microscopy pictures of LNCaP cells cultured in FCS and NE-like LNCaP cells cultured in CSS for 3 days (20× objective). B) RNA isolated from LNCaP cells incubated with fetal calf serum (FCS) or charcoal-stripped FCS (CSS) for 4 days were subjected to microarray analysis, revealing up-regulation of the Cβ isoform of PKA in CSS treated cells. The results were verified by semi-quantitative RT-PCR (sqRT-PCR) using primers recognizing all Cβ splice variants. The data was normalized against the Glucose-6-phosphate dehydrogenase (G6PD) mRNA level. The level of Cβ mRNA normalized to G6PD mRNA in cells maintained in CSS is represented as fold change over normalized Cβ levels in cells maintained in FCS (mean +/− SEM; n = 3). C) RNA isolated from LNCaP cells incubated with fetal calf serum (FCS) or charcoal-stripped FCS (CSS) for 4 days were subjected to semi-quantitative RT-PCR using Cβ1, Cβ2, Cβ3 and Cβ4-specific primers. (mean +/− SEM; n = 3). The data was normalized against G6PD and is presented as in B).
Fig. 2. PKA subunits are differentially regulated at the protein level during NE differentiation of LNCaP cells. Protein extracts were prepared from LNCaP cells incubated with FCS for 0 and 14 days, or incubated with CSS for 4, 6, 8 and 14 days and analyzed by western blot analysis using antisera to pan-C, RIα, RIIα and RIIβ. The PKA subunit antibody is indicated to the left and the different C subunit splice variants indicated to the right, as determined by apparent molecular weight. Whereas one representative immunoblot for each PKA subunit is presented in the upper left panel, the results from densitometric scanning of at least three independent experiments are shown for Cβ2 (upper right panel), Cα1/Cβ1 (lower left panel) and Cβ3/Cβ4 (lower right panel).
 |
Fig. 3. Androgen dependent regulation of PKA-Cβ splice variants in LNCaP cells. A) LNCaP cells were grown in CSS medium for two days prior to stimulation with increasing concentrations of R1881 (0.001–10 nM) or 5-α-androstan-3-α, 17-β-diol (3-α-diol) (1 nM and 10 nM) for 65 h. Cells grown in FCS or CSS for the total time period without androgen stimulation were used as controls. RNA was isolated and subjected to semi-quantitative RT-PCR using Cβ1 (upper, left panel), Cβ2 (upper, right panel), Cβ3 (lower, left panel) and Cβ4 (lower, right panel) specific primers. The data was normalized against the TATA binding protein (TBP) mRNA level. The level of Cβ mRNA normalized to TBP mRNA in the various samples is represented as fold change over levels in cells maintained in FCS (mean +/− SEM; n = 3). B) Protein extracts were prepared from LNCaP-cells treated as described in A) as above, and western blot analysis performed using equal protein loading of lanes and antisera to pan-C. Anti-RIα was used as loading control. The antibody used is indicated to the left and the different C subunit splice variants indicated to the right (upper left panel) as determined by apparent molecular weight. One representative experiment is presented and the densitometric scanning intensity shown for three independent experiments: Cβ2 (upper right panel), Cα1/Cβ1 (lower left panel) and Cβ3/Cβ4 (lower right panel).
Fig. 4. Expression of PKA subunits in different prostatic cancer cell lines. Protein extracts were prepared from the hormone sensitive LNCaP cell line, the hormone refractory LNCaP sublines; Rf, C4 and C4-2B and the hormone independent cell lines; DU 145 and PC3 and western blot analysis performed using antisera to pan-C, RIα, RIIα and RIIβ. The PKA subunit antibody is indicated to the left and the different C subunit splice variants indicated to the right (upper left panel) as determined by apparent molecular weight. One representative immunoblot is presented for each PKA subunit (upper left panel) whereas densitometric scanning results from replicate experiments are shown for Cβ2 (upper right panel), Cα1/Cβ1 (lower left panel) and Cβ3/Cβ4 (lower right panel).
Fig. 5. Expression of PKA subunits in normal and tumor tissue from prostate cancer patients. A small slice of normal (N) and tumor (T) tissue was obtained from radical prostatectomies of three different patients, protein extracts were prepared and western blot analysis performed using antisera to pan-C, RIα, RIIα and RIIβ. The PKA subunit antibody is indicated to the left and the different C subunit splice variants indicated to the right as determined by apparent molecular weight (upper panel). The intensities of the bands representing Cα1/Cβ1, Cβ2 and Cβ3/Cβ4 in normal and tumor tissue from eight different patients were measured densitometrically. The results for Cβ2 and Cβ3/Cβ4 were normalized against Cα1/Cβ1, a Mann–Whitney test performed and the results presented as box-plot (lower panel).
Abstract
Purchase PDF (713 K)
E-mail Article
Add to my Quick Links
Cited By in Scopus (3)
Purchase PDF (368 K)
