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Cyclin-dependent kinase 5 acts as a critical determinant of AKT-dependent proliferation and regulates differential gene expression by the androgen receptor in prostate cancer cells.

Lindqvist J, Imanishi SY, Torvaldson E, Malinen M, Remes M, Örn F, Palvimo JJ, Eriksson JE - Mol. Biol. Cell (2015)

Bottom Line: Contrary to cell cycle-associated cyclin-dependent kinases, CDK5 is best known for its regulation of signaling processes in differentiated cells and its destructive activation in Alzheimer's disease.However, the amplified cell growth was found to be separated from AR signaling, further corroborated by CDK5-dependent proliferation of AR cells.Instead, we found that the key growth-promoting effect was due to specific CDK5-mediated AKT activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences, Faculty of Science and Engineering, Åbo Akademi University, FI-20520 Turku, Finland Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, FI-20520 Turku, Finland.

No MeSH data available.


Related in: MedlinePlus

Down-regulation of CDK5 protein disturbs prostate cancer cell proliferation independently of androgen signaling. (A) Androgen-dependent LNCaP cells were transfected with indicated siRNA overnight, and three positions from a transfected well were imaged with Cell-IQ (CM Technologies) live-cell imaging platform (Supplemental Videos S1 and S2). From the video material, the cell confluency in each position was automatically quantified with Cell IQ Analyser software (CM Technologies) at fixed settings, and the relative cell confluency (relative area growth) is plotted in the graph over time. Images represent the endpoint of the experiment. A prominent change in cell morphology accompanied the loss of CDK5. Transfection efficiency was in the end validated with Western blotting. (B) LNCaP cells were transfected with indicated siRNA, and the cell population size was calculated manually at each time point. Results are plotted as relative proliferation and compared with the 0-h time point, confirming that the absence of CDK5 impairs proliferation of LNCaP cells. (C) LNCaP cells were transfected with empty plasmid (mock) or WT-CDK5 overnight, imaged with Cell-IQ (Supplemental Videos S3 and S4), and analyzed with similar settings as before. Expression of WT-CDK5 is detected as a double band in Western blots. (D) LNCaP cells were transfected as described and counted after 48 h. Cell counts were normalized to the starting time point. Indeed, cell population size was promoted after CDK5 overexpression (WT-CDK5) compared with empty vector control (mock). (E) CDK5 knockdown in androgen-independent (AR deficient) PC-3 cells causes rounding up of cells similarly to LNCaP cells. Cell counting experiments reveal that CDK5 siRNA keeps the PC-3 cell population distinctly smaller than with negative control cells (Scr). Results are plotted as mean ± SEM, *p < 0.05, Student's t test, n ≥ 3).
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Figure 1: Down-regulation of CDK5 protein disturbs prostate cancer cell proliferation independently of androgen signaling. (A) Androgen-dependent LNCaP cells were transfected with indicated siRNA overnight, and three positions from a transfected well were imaged with Cell-IQ (CM Technologies) live-cell imaging platform (Supplemental Videos S1 and S2). From the video material, the cell confluency in each position was automatically quantified with Cell IQ Analyser software (CM Technologies) at fixed settings, and the relative cell confluency (relative area growth) is plotted in the graph over time. Images represent the endpoint of the experiment. A prominent change in cell morphology accompanied the loss of CDK5. Transfection efficiency was in the end validated with Western blotting. (B) LNCaP cells were transfected with indicated siRNA, and the cell population size was calculated manually at each time point. Results are plotted as relative proliferation and compared with the 0-h time point, confirming that the absence of CDK5 impairs proliferation of LNCaP cells. (C) LNCaP cells were transfected with empty plasmid (mock) or WT-CDK5 overnight, imaged with Cell-IQ (Supplemental Videos S3 and S4), and analyzed with similar settings as before. Expression of WT-CDK5 is detected as a double band in Western blots. (D) LNCaP cells were transfected as described and counted after 48 h. Cell counts were normalized to the starting time point. Indeed, cell population size was promoted after CDK5 overexpression (WT-CDK5) compared with empty vector control (mock). (E) CDK5 knockdown in androgen-independent (AR deficient) PC-3 cells causes rounding up of cells similarly to LNCaP cells. Cell counting experiments reveal that CDK5 siRNA keeps the PC-3 cell population distinctly smaller than with negative control cells (Scr). Results are plotted as mean ± SEM, *p < 0.05, Student's t test, n ≥ 3).

Mentions: First we wanted to establish the extent to which CDK5 itself regulates the proliferation of different prostate cancer cells that are distinguished by distinct molecular features. To investigate how the absence of CDK5 affects prostate cancer cell behavior, we transfected androgen-dependent LNCaP cells with negative control scrambled small interfering RNA (siRNA; Scr) or a pool of CDK5-specific siRNA, yielding efficient down-regulation of CDK5 (Figure 1A). In live-cell imaging, cell populations with down-regulated CDK5 displayed a striking reduction in cell growth as compared with control cells (Figure 1A and Supplemental Videos S1 and S2). Quantification of cell confluence further highlighted the obvious cell growth retardation (Figure 1A), which was additionally confirmed with cell counting of Scr and CDK5 siRNA–transfected cells (Figure 1B). Next, we sought to investigate whether LNCaP proliferation could be stimulated by expression of exogenous WT-CDK5, the transfection success being confirmed by Western blotting (Figure 1C). Measured as before, CDK5 transfection induced an increase in cell proliferation (Figure 1C and Supplemental Videos S3 and S4), which was confirmed by overexpression of CDK5 and manual counting of the cells 48 h after transfection (Figure 1D). We confirmed our observations with another prostate cancer cell line, 22Rv1, which is androgen responsive rather than strictly androgen dependent. We obtained an equally prominent response with this cell line (Supplemental Figure S1 and Supplemental Videos S5–S8).


Cyclin-dependent kinase 5 acts as a critical determinant of AKT-dependent proliferation and regulates differential gene expression by the androgen receptor in prostate cancer cells.

Lindqvist J, Imanishi SY, Torvaldson E, Malinen M, Remes M, Örn F, Palvimo JJ, Eriksson JE - Mol. Biol. Cell (2015)

Down-regulation of CDK5 protein disturbs prostate cancer cell proliferation independently of androgen signaling. (A) Androgen-dependent LNCaP cells were transfected with indicated siRNA overnight, and three positions from a transfected well were imaged with Cell-IQ (CM Technologies) live-cell imaging platform (Supplemental Videos S1 and S2). From the video material, the cell confluency in each position was automatically quantified with Cell IQ Analyser software (CM Technologies) at fixed settings, and the relative cell confluency (relative area growth) is plotted in the graph over time. Images represent the endpoint of the experiment. A prominent change in cell morphology accompanied the loss of CDK5. Transfection efficiency was in the end validated with Western blotting. (B) LNCaP cells were transfected with indicated siRNA, and the cell population size was calculated manually at each time point. Results are plotted as relative proliferation and compared with the 0-h time point, confirming that the absence of CDK5 impairs proliferation of LNCaP cells. (C) LNCaP cells were transfected with empty plasmid (mock) or WT-CDK5 overnight, imaged with Cell-IQ (Supplemental Videos S3 and S4), and analyzed with similar settings as before. Expression of WT-CDK5 is detected as a double band in Western blots. (D) LNCaP cells were transfected as described and counted after 48 h. Cell counts were normalized to the starting time point. Indeed, cell population size was promoted after CDK5 overexpression (WT-CDK5) compared with empty vector control (mock). (E) CDK5 knockdown in androgen-independent (AR deficient) PC-3 cells causes rounding up of cells similarly to LNCaP cells. Cell counting experiments reveal that CDK5 siRNA keeps the PC-3 cell population distinctly smaller than with negative control cells (Scr). Results are plotted as mean ± SEM, *p < 0.05, Student's t test, n ≥ 3).
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Figure 1: Down-regulation of CDK5 protein disturbs prostate cancer cell proliferation independently of androgen signaling. (A) Androgen-dependent LNCaP cells were transfected with indicated siRNA overnight, and three positions from a transfected well were imaged with Cell-IQ (CM Technologies) live-cell imaging platform (Supplemental Videos S1 and S2). From the video material, the cell confluency in each position was automatically quantified with Cell IQ Analyser software (CM Technologies) at fixed settings, and the relative cell confluency (relative area growth) is plotted in the graph over time. Images represent the endpoint of the experiment. A prominent change in cell morphology accompanied the loss of CDK5. Transfection efficiency was in the end validated with Western blotting. (B) LNCaP cells were transfected with indicated siRNA, and the cell population size was calculated manually at each time point. Results are plotted as relative proliferation and compared with the 0-h time point, confirming that the absence of CDK5 impairs proliferation of LNCaP cells. (C) LNCaP cells were transfected with empty plasmid (mock) or WT-CDK5 overnight, imaged with Cell-IQ (Supplemental Videos S3 and S4), and analyzed with similar settings as before. Expression of WT-CDK5 is detected as a double band in Western blots. (D) LNCaP cells were transfected as described and counted after 48 h. Cell counts were normalized to the starting time point. Indeed, cell population size was promoted after CDK5 overexpression (WT-CDK5) compared with empty vector control (mock). (E) CDK5 knockdown in androgen-independent (AR deficient) PC-3 cells causes rounding up of cells similarly to LNCaP cells. Cell counting experiments reveal that CDK5 siRNA keeps the PC-3 cell population distinctly smaller than with negative control cells (Scr). Results are plotted as mean ± SEM, *p < 0.05, Student's t test, n ≥ 3).
Mentions: First we wanted to establish the extent to which CDK5 itself regulates the proliferation of different prostate cancer cells that are distinguished by distinct molecular features. To investigate how the absence of CDK5 affects prostate cancer cell behavior, we transfected androgen-dependent LNCaP cells with negative control scrambled small interfering RNA (siRNA; Scr) or a pool of CDK5-specific siRNA, yielding efficient down-regulation of CDK5 (Figure 1A). In live-cell imaging, cell populations with down-regulated CDK5 displayed a striking reduction in cell growth as compared with control cells (Figure 1A and Supplemental Videos S1 and S2). Quantification of cell confluence further highlighted the obvious cell growth retardation (Figure 1A), which was additionally confirmed with cell counting of Scr and CDK5 siRNA–transfected cells (Figure 1B). Next, we sought to investigate whether LNCaP proliferation could be stimulated by expression of exogenous WT-CDK5, the transfection success being confirmed by Western blotting (Figure 1C). Measured as before, CDK5 transfection induced an increase in cell proliferation (Figure 1C and Supplemental Videos S3 and S4), which was confirmed by overexpression of CDK5 and manual counting of the cells 48 h after transfection (Figure 1D). We confirmed our observations with another prostate cancer cell line, 22Rv1, which is androgen responsive rather than strictly androgen dependent. We obtained an equally prominent response with this cell line (Supplemental Figure S1 and Supplemental Videos S5–S8).

Bottom Line: Contrary to cell cycle-associated cyclin-dependent kinases, CDK5 is best known for its regulation of signaling processes in differentiated cells and its destructive activation in Alzheimer's disease.However, the amplified cell growth was found to be separated from AR signaling, further corroborated by CDK5-dependent proliferation of AR cells.Instead, we found that the key growth-promoting effect was due to specific CDK5-mediated AKT activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences, Faculty of Science and Engineering, Åbo Akademi University, FI-20520 Turku, Finland Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, FI-20520 Turku, Finland.

No MeSH data available.


Related in: MedlinePlus