Limits...
The functional role of Notch signaling in HPV-mediated transformation is dose-dependent and linked to AP-1 alterations.

Henken FE, De-Castro Arce J, Rösl F, Bosch L, Meijer CJ, Snijders PJ, Steenbergen RD - Cell Oncol (Dordr) (2012)

Bottom Line: We examined whether the dual findings in literature may be explained by gene dosage effects and determined the relation with AP-1, a downstream target of Notch.Moderate Notch activation contributed to increased viability and anchorage independent growth, whereas high level Notch activation decreased anchorage independent growth.Moderate Notch expression led to an increased AP-1 transcriptional activity and DNA binding activity, but did not affect complex composition.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Unit of Molecular Pathology, VU University Medical Center, Amsterdam, The Netherlands.

ABSTRACT

Background: The role of Notch signaling in HPV-mediated transformation has been a long standing debate, as both tumor suppressive and oncogenic properties have been described. We examined whether the dual findings in literature may be explained by gene dosage effects and determined the relation with AP-1, a downstream target of Notch.

Methods: SiHa cervical cancer cells were transfected with two doses of intracellular active Notch. Non-tumorigenic HPV16-immortalized keratinocytes (FK16A) were transfected with Fra1 specific siRNAs and non-targeting controls. Transfectants were analysed for Notch, Hes, cJun, cFos and Fra1 mRNA expression, Notch pathway activation using luciferase assays, cell viability using MTT assays, anchorage independent growth, AP-1 activity and/or AP-1 complex composition using EMSA.

Results: In SiHa cells two activation states of Notch signaling pathway were obtained. Moderate Notch activation contributed to increased viability and anchorage independent growth, whereas high level Notch activation decreased anchorage independent growth. The shift in phenotypical outcome was correlated to altered AP-1 activity and complex composition. Moderate Notch expression led to an increased AP-1 transcriptional activity and DNA binding activity, but did not affect complex composition. High levels of Notch additionally led to a change in AP-1 complex composition, from cJun/cFos to cJun/Fra1 dimers, which is exemplary for non-tumorigenic HPV-immortalized cell lines. Conversely, silencing of Fra1 in non-tumorigenic HPV16-immortalized keratinocytes, leading to an enrichment of cJun/cFos dimers, was accompanied with increased colony formation.

Conclusion: The functional role of Notch in HPV-mediated transformation is dosage dependent and correlated to a change in AP-1.

Show MeSH

Related in: MedlinePlus

The effect of Fra1 silencing in HPV16-immortalized keratinocytes. a mRNA expression of AP-1 family members Fra1, cFos and cJun upon transfection with siRNAs targeting Fra1 or a non-targeting siRNA control. b EMSA for AP-1 composition using cells transfected with siRNAs targeting Fra1 compared to non-targeting control. The lower band is total AP-1 bound to the consensus oligo. Arrows indicate specific antibody supershifts. c Colony formation in soft agarose. Transfection with siRNAs targeting PLK1 which induces cell death was included as a control for transfection efficiency
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3306567&req=5

Fig4: The effect of Fra1 silencing in HPV16-immortalized keratinocytes. a mRNA expression of AP-1 family members Fra1, cFos and cJun upon transfection with siRNAs targeting Fra1 or a non-targeting siRNA control. b EMSA for AP-1 composition using cells transfected with siRNAs targeting Fra1 compared to non-targeting control. The lower band is total AP-1 bound to the consensus oligo. Arrows indicate specific antibody supershifts. c Colony formation in soft agarose. Transfection with siRNAs targeting PLK1 which induces cell death was included as a control for transfection efficiency

Mentions: Fra1 silencing resulted in an upregulation of cFos mRNA and had little effect on cJun mRNA expression (Fig. 4a). Examination of AP-1 complex composition in these cells showed that silencing of Fra1 resulted in a reduced Fra1 incorporation into the complex, with more cJun/cFos dimers being the consequence (Fig. 4b). HPV16-immortalized keratinocytes silenced for Fra1 displayed increased colony forming capacity in soft agarose (Fig. 4c) without an increase in proliferative potential (not shown). No effects were seen using non-targeting siRNAs on complex composition, proliferation or anchorage independent growth. These results further strengthen the notion that changes in AP-1 complex composition are important in HPV-mediated transformation.Fig. 4


The functional role of Notch signaling in HPV-mediated transformation is dose-dependent and linked to AP-1 alterations.

Henken FE, De-Castro Arce J, Rösl F, Bosch L, Meijer CJ, Snijders PJ, Steenbergen RD - Cell Oncol (Dordr) (2012)

The effect of Fra1 silencing in HPV16-immortalized keratinocytes. a mRNA expression of AP-1 family members Fra1, cFos and cJun upon transfection with siRNAs targeting Fra1 or a non-targeting siRNA control. b EMSA for AP-1 composition using cells transfected with siRNAs targeting Fra1 compared to non-targeting control. The lower band is total AP-1 bound to the consensus oligo. Arrows indicate specific antibody supershifts. c Colony formation in soft agarose. Transfection with siRNAs targeting PLK1 which induces cell death was included as a control for transfection efficiency
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3306567&req=5

Fig4: The effect of Fra1 silencing in HPV16-immortalized keratinocytes. a mRNA expression of AP-1 family members Fra1, cFos and cJun upon transfection with siRNAs targeting Fra1 or a non-targeting siRNA control. b EMSA for AP-1 composition using cells transfected with siRNAs targeting Fra1 compared to non-targeting control. The lower band is total AP-1 bound to the consensus oligo. Arrows indicate specific antibody supershifts. c Colony formation in soft agarose. Transfection with siRNAs targeting PLK1 which induces cell death was included as a control for transfection efficiency
Mentions: Fra1 silencing resulted in an upregulation of cFos mRNA and had little effect on cJun mRNA expression (Fig. 4a). Examination of AP-1 complex composition in these cells showed that silencing of Fra1 resulted in a reduced Fra1 incorporation into the complex, with more cJun/cFos dimers being the consequence (Fig. 4b). HPV16-immortalized keratinocytes silenced for Fra1 displayed increased colony forming capacity in soft agarose (Fig. 4c) without an increase in proliferative potential (not shown). No effects were seen using non-targeting siRNAs on complex composition, proliferation or anchorage independent growth. These results further strengthen the notion that changes in AP-1 complex composition are important in HPV-mediated transformation.Fig. 4

Bottom Line: We examined whether the dual findings in literature may be explained by gene dosage effects and determined the relation with AP-1, a downstream target of Notch.Moderate Notch activation contributed to increased viability and anchorage independent growth, whereas high level Notch activation decreased anchorage independent growth.Moderate Notch expression led to an increased AP-1 transcriptional activity and DNA binding activity, but did not affect complex composition.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Unit of Molecular Pathology, VU University Medical Center, Amsterdam, The Netherlands.

ABSTRACT

Background: The role of Notch signaling in HPV-mediated transformation has been a long standing debate, as both tumor suppressive and oncogenic properties have been described. We examined whether the dual findings in literature may be explained by gene dosage effects and determined the relation with AP-1, a downstream target of Notch.

Methods: SiHa cervical cancer cells were transfected with two doses of intracellular active Notch. Non-tumorigenic HPV16-immortalized keratinocytes (FK16A) were transfected with Fra1 specific siRNAs and non-targeting controls. Transfectants were analysed for Notch, Hes, cJun, cFos and Fra1 mRNA expression, Notch pathway activation using luciferase assays, cell viability using MTT assays, anchorage independent growth, AP-1 activity and/or AP-1 complex composition using EMSA.

Results: In SiHa cells two activation states of Notch signaling pathway were obtained. Moderate Notch activation contributed to increased viability and anchorage independent growth, whereas high level Notch activation decreased anchorage independent growth. The shift in phenotypical outcome was correlated to altered AP-1 activity and complex composition. Moderate Notch expression led to an increased AP-1 transcriptional activity and DNA binding activity, but did not affect complex composition. High levels of Notch additionally led to a change in AP-1 complex composition, from cJun/cFos to cJun/Fra1 dimers, which is exemplary for non-tumorigenic HPV-immortalized cell lines. Conversely, silencing of Fra1 in non-tumorigenic HPV16-immortalized keratinocytes, leading to an enrichment of cJun/cFos dimers, was accompanied with increased colony formation.

Conclusion: The functional role of Notch in HPV-mediated transformation is dosage dependent and correlated to a change in AP-1.

Show MeSH
Related in: MedlinePlus