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Selective CREB-dependent cyclin expression mediated by the PI3K and MAPK pathways supports glioma cell proliferation.

Daniel P, Filiz G, Brown DV, Hollande F, Gonzales M, D'Abaco G, Papalexis N, Phillips WA, Malaterre J, Ramsay RG, Mantamadiotis T - Oncogenesis (2014)

Bottom Line: CREB overexpression in transgenic animals imparts oncogenic properties on cells in various tissues, and aberrant CREB expression is associated with tumours.Cyclin D1 is highly CREB-dependent, whereas cyclin B1 and PCNA are co-regulated by both CREB-dependent and -independent mechanisms.The precise regulatory network involved appears to differ depending on the tumour-suppressor phosphatase and tensin homolog status of the GBM cells, which in turn allows CREB to regulate the activity of the PI3K itself.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The University of Melbourne, Parkville, Victoria, Australia.

ABSTRACT
The cyclic-AMP response element binding (CREB) protein has been shown to have a pivotal role in cell survival and cell proliferation. Transgenic rodent models have revealed a role for CREB in higher-order brain functions, such as memory and drug addiction behaviors. CREB overexpression in transgenic animals imparts oncogenic properties on cells in various tissues, and aberrant CREB expression is associated with tumours. It is the central position of CREB, downstream from key developmental and growth signalling pathways, which gives CREB this ability to influence a spectrum of cellular activities, such as cell survival, growth and differentiation, in both normal and cancer cells. We show that CREB is highly expressed and constitutively activated in patient glioma tissue and that this activation closely correlates with tumour grade. The mechanism by which CREB regulates glioblastoma (GBM) tumour cell proliferation involves activities downstream from both the mitogen-activated protein kinase and phosphoinositide 3-kinase (PI3K) pathways that then modulate the expression of three key cell cycle factors, cyclin B, D and proliferating cell nuclear antigen (PCNA). Cyclin D1 is highly CREB-dependent, whereas cyclin B1 and PCNA are co-regulated by both CREB-dependent and -independent mechanisms. The precise regulatory network involved appears to differ depending on the tumour-suppressor phosphatase and tensin homolog status of the GBM cells, which in turn allows CREB to regulate the activity of the PI3K itself. Given that CREB sits at the hub of key cancer cell signalling pathways, understanding the role of glioma-specific CREB function may lead to improved novel combinatorial anti-tumour therapies, which can complement existing PI3K-specific drugs undergoing early phase clinical trials.

No MeSH data available.


Related in: MedlinePlus

Knockdown of CREB alters human GBM cell cycle kinetics through regulation of cyclin B1, cyclin D1 and PCNA. (a, b) Synchronized GBM cell lines T98G and U118 were treated with either siCREB or scramble control siRNA and then stimulated with serum for 24 h before being analysed for cell cycle proportions using DNA-content analysis. (c, d) Synchronized GBM cell lines T98G and U118 were treated with either siCREB or scramble control siRNA, stimulated with serum and harvested every 12 h for analysis for cyclin B1, cyclin D1 and PCNA expression. (e, f) Synchronized GBM cell lines T98G and U118 were treated with either siCREB or scrambled control siRNA and then stimulated with serum for 24 h before harvesting and analysis using qRT–PCR for CCNB1, CCND1 and PCNA mRNA expression. ***P<0.0005, **P<0.005, *P<0.05. All western blottings were performed at least three times, and blottings for total CREB assay were imaged, then membranes were stripped and reprobed for pCREB detection.
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fig5: Knockdown of CREB alters human GBM cell cycle kinetics through regulation of cyclin B1, cyclin D1 and PCNA. (a, b) Synchronized GBM cell lines T98G and U118 were treated with either siCREB or scramble control siRNA and then stimulated with serum for 24 h before being analysed for cell cycle proportions using DNA-content analysis. (c, d) Synchronized GBM cell lines T98G and U118 were treated with either siCREB or scramble control siRNA, stimulated with serum and harvested every 12 h for analysis for cyclin B1, cyclin D1 and PCNA expression. (e, f) Synchronized GBM cell lines T98G and U118 were treated with either siCREB or scrambled control siRNA and then stimulated with serum for 24 h before harvesting and analysis using qRT–PCR for CCNB1, CCND1 and PCNA mRNA expression. ***P<0.0005, **P<0.005, *P<0.05. All western blottings were performed at least three times, and blottings for total CREB assay were imaged, then membranes were stripped and reprobed for pCREB detection.

Mentions: To determine the mechanism by which CREB regulates GBM cell proliferation, cell cycle parameters were measured using DNA content by flow cytometry in T98G and U118 cells, which were the cell lines showing the greatest CREB-dependent decrease in proliferation. CREB knockdown increased the proportion of cells in G0/G1 phase in both cell lines tested (Figures 5a and b). siCREB T98G cells also showed significantly reduced proportions in cell cycle phase distribution in S- and G2/M phases compared with control cells, whereas siCREB U118 cells exhibited increased G0/G1 and fewer G2/M phase cells only. To explore the molecular basis underlying disruption of the cell cycle, we measured the expression of cell cycle/proliferation factors previously reported to be transcriptionally regulated by CREB, including cyclins B1, D128 and PCNA.14, 29 Protein expression of cyclins B1, D1 and PCNA was assessed in T98G and U118 cells in CREB knockdown or scrambled control siRNA-treated GBM cell lines (Figures 5c and d). Cells were serum deprived for 24 h before the addition of serum to synchronize cell cycle activation, and cyclin and PCNA protein levels were determined every 12 h, over 48 h. The dynamics of expression seen was consistent with the reported cyclin and PCNA expression profiles for mammalian cells, where PCNA and cyclin D1 are consistently expressed throughout the cell cycle while cyclin B1 expression peaks at G2/M.30 Upon treatment with serum (triggering cell cycle entry), we observed inhibition of protein expression of cyclin B1, cyclin D1 and PCNA in both T98G and U118 cell lines as early as 12 h (not shown) with maximal inhibition reached at 24 h, compared with no-serum cells (Figures 5c and d). T98G cells showed a consistent and almost complete block in cyclin D1, cyclin B1 and PCNA expression over 48 h in CREB knockdown cells, in contrast to U118 cells, which exhibited a more modest inhibition of cyclin D1, over 48 h. In U118 cells, cylin B1 protein expression was maximally inhibited at 24 h, but little effect was seen in PCNA expression (Figure 5d). Moreover, in U118 cells cyclin B1 inhibition was not sustained, with expression approaching control levels beyond 24 h. Given that cyclin B1, D1 and PCNA harbour cAMP-resonsive elements (CREB-binding sequences) in their promoters (see Supplementary Data), we tested whether CREB exerted its influence on these target genes directly; we performed reverse transcriptase–PCR (RT–PCR) at 24 h following siCREB treatment to measure mRNA levels of cyclin B1, D1 and PCNA. CREB knockdown robustly inhibited cyclin D1 mRNA expression in both T98G and U118 cells, while cyclin B1 mRNA expression was significantly reduced in T98G cells only. Surprisingly, PCNA mRNA expression was unaffected by CREB knockdown in both cell lines (Figures 5e and f), implying that CREB exerts its affect on PCNA protein expression indirectly. The transcriptional influence CREB exerts in the expression of these genes reflects the context of the cAMP-resonsive elements in their respective promoters (see Supplementary Data), with cyclin D1 showing the best context with a full (8-base pair consensus) cAMP-resonsive element positioned closest to the transcription start site.


Selective CREB-dependent cyclin expression mediated by the PI3K and MAPK pathways supports glioma cell proliferation.

Daniel P, Filiz G, Brown DV, Hollande F, Gonzales M, D'Abaco G, Papalexis N, Phillips WA, Malaterre J, Ramsay RG, Mantamadiotis T - Oncogenesis (2014)

Knockdown of CREB alters human GBM cell cycle kinetics through regulation of cyclin B1, cyclin D1 and PCNA. (a, b) Synchronized GBM cell lines T98G and U118 were treated with either siCREB or scramble control siRNA and then stimulated with serum for 24 h before being analysed for cell cycle proportions using DNA-content analysis. (c, d) Synchronized GBM cell lines T98G and U118 were treated with either siCREB or scramble control siRNA, stimulated with serum and harvested every 12 h for analysis for cyclin B1, cyclin D1 and PCNA expression. (e, f) Synchronized GBM cell lines T98G and U118 were treated with either siCREB or scrambled control siRNA and then stimulated with serum for 24 h before harvesting and analysis using qRT–PCR for CCNB1, CCND1 and PCNA mRNA expression. ***P<0.0005, **P<0.005, *P<0.05. All western blottings were performed at least three times, and blottings for total CREB assay were imaged, then membranes were stripped and reprobed for pCREB detection.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig5: Knockdown of CREB alters human GBM cell cycle kinetics through regulation of cyclin B1, cyclin D1 and PCNA. (a, b) Synchronized GBM cell lines T98G and U118 were treated with either siCREB or scramble control siRNA and then stimulated with serum for 24 h before being analysed for cell cycle proportions using DNA-content analysis. (c, d) Synchronized GBM cell lines T98G and U118 were treated with either siCREB or scramble control siRNA, stimulated with serum and harvested every 12 h for analysis for cyclin B1, cyclin D1 and PCNA expression. (e, f) Synchronized GBM cell lines T98G and U118 were treated with either siCREB or scrambled control siRNA and then stimulated with serum for 24 h before harvesting and analysis using qRT–PCR for CCNB1, CCND1 and PCNA mRNA expression. ***P<0.0005, **P<0.005, *P<0.05. All western blottings were performed at least three times, and blottings for total CREB assay were imaged, then membranes were stripped and reprobed for pCREB detection.
Mentions: To determine the mechanism by which CREB regulates GBM cell proliferation, cell cycle parameters were measured using DNA content by flow cytometry in T98G and U118 cells, which were the cell lines showing the greatest CREB-dependent decrease in proliferation. CREB knockdown increased the proportion of cells in G0/G1 phase in both cell lines tested (Figures 5a and b). siCREB T98G cells also showed significantly reduced proportions in cell cycle phase distribution in S- and G2/M phases compared with control cells, whereas siCREB U118 cells exhibited increased G0/G1 and fewer G2/M phase cells only. To explore the molecular basis underlying disruption of the cell cycle, we measured the expression of cell cycle/proliferation factors previously reported to be transcriptionally regulated by CREB, including cyclins B1, D128 and PCNA.14, 29 Protein expression of cyclins B1, D1 and PCNA was assessed in T98G and U118 cells in CREB knockdown or scrambled control siRNA-treated GBM cell lines (Figures 5c and d). Cells were serum deprived for 24 h before the addition of serum to synchronize cell cycle activation, and cyclin and PCNA protein levels were determined every 12 h, over 48 h. The dynamics of expression seen was consistent with the reported cyclin and PCNA expression profiles for mammalian cells, where PCNA and cyclin D1 are consistently expressed throughout the cell cycle while cyclin B1 expression peaks at G2/M.30 Upon treatment with serum (triggering cell cycle entry), we observed inhibition of protein expression of cyclin B1, cyclin D1 and PCNA in both T98G and U118 cell lines as early as 12 h (not shown) with maximal inhibition reached at 24 h, compared with no-serum cells (Figures 5c and d). T98G cells showed a consistent and almost complete block in cyclin D1, cyclin B1 and PCNA expression over 48 h in CREB knockdown cells, in contrast to U118 cells, which exhibited a more modest inhibition of cyclin D1, over 48 h. In U118 cells, cylin B1 protein expression was maximally inhibited at 24 h, but little effect was seen in PCNA expression (Figure 5d). Moreover, in U118 cells cyclin B1 inhibition was not sustained, with expression approaching control levels beyond 24 h. Given that cyclin B1, D1 and PCNA harbour cAMP-resonsive elements (CREB-binding sequences) in their promoters (see Supplementary Data), we tested whether CREB exerted its influence on these target genes directly; we performed reverse transcriptase–PCR (RT–PCR) at 24 h following siCREB treatment to measure mRNA levels of cyclin B1, D1 and PCNA. CREB knockdown robustly inhibited cyclin D1 mRNA expression in both T98G and U118 cells, while cyclin B1 mRNA expression was significantly reduced in T98G cells only. Surprisingly, PCNA mRNA expression was unaffected by CREB knockdown in both cell lines (Figures 5e and f), implying that CREB exerts its affect on PCNA protein expression indirectly. The transcriptional influence CREB exerts in the expression of these genes reflects the context of the cAMP-resonsive elements in their respective promoters (see Supplementary Data), with cyclin D1 showing the best context with a full (8-base pair consensus) cAMP-resonsive element positioned closest to the transcription start site.

Bottom Line: CREB overexpression in transgenic animals imparts oncogenic properties on cells in various tissues, and aberrant CREB expression is associated with tumours.Cyclin D1 is highly CREB-dependent, whereas cyclin B1 and PCNA are co-regulated by both CREB-dependent and -independent mechanisms.The precise regulatory network involved appears to differ depending on the tumour-suppressor phosphatase and tensin homolog status of the GBM cells, which in turn allows CREB to regulate the activity of the PI3K itself.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The University of Melbourne, Parkville, Victoria, Australia.

ABSTRACT
The cyclic-AMP response element binding (CREB) protein has been shown to have a pivotal role in cell survival and cell proliferation. Transgenic rodent models have revealed a role for CREB in higher-order brain functions, such as memory and drug addiction behaviors. CREB overexpression in transgenic animals imparts oncogenic properties on cells in various tissues, and aberrant CREB expression is associated with tumours. It is the central position of CREB, downstream from key developmental and growth signalling pathways, which gives CREB this ability to influence a spectrum of cellular activities, such as cell survival, growth and differentiation, in both normal and cancer cells. We show that CREB is highly expressed and constitutively activated in patient glioma tissue and that this activation closely correlates with tumour grade. The mechanism by which CREB regulates glioblastoma (GBM) tumour cell proliferation involves activities downstream from both the mitogen-activated protein kinase and phosphoinositide 3-kinase (PI3K) pathways that then modulate the expression of three key cell cycle factors, cyclin B, D and proliferating cell nuclear antigen (PCNA). Cyclin D1 is highly CREB-dependent, whereas cyclin B1 and PCNA are co-regulated by both CREB-dependent and -independent mechanisms. The precise regulatory network involved appears to differ depending on the tumour-suppressor phosphatase and tensin homolog status of the GBM cells, which in turn allows CREB to regulate the activity of the PI3K itself. Given that CREB sits at the hub of key cancer cell signalling pathways, understanding the role of glioma-specific CREB function may lead to improved novel combinatorial anti-tumour therapies, which can complement existing PI3K-specific drugs undergoing early phase clinical trials.

No MeSH data available.


Related in: MedlinePlus