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A role for the transcription factor HEY1 in glioblastoma.

Hulleman E, Quarto M, Vernell R, Masserdotti G, Colli E, Kros JM, Levi D, Gaetani P, Tunici P, Finocchiaro G, Baena RR, Capra M, Helin K - J. Cell. Mol. Med. (2008)

Bottom Line: In addition, we show by chromatin immunoprecipitations, luciferase assays and Northern blot experiments that HEY1 is a bona fide target of the E2F family of transcription factors, connecting the Ras and Notch signalling pathways.Finally, we show that ectopic expression of HEY1 induces cell proliferation in neural stem cells, while depletion of HEY1 by RNA interference reduces proliferation of glioblastoma cells in tissue culture.Moreover, HEY1 may represent a molecular marker to distinguish GBM patients with a longer survival prognosis from those at high risk.

View Article: PubMed Central - PubMed

Affiliation: European Institute of Oncology, Via Ripamonti, Milan, Italy.

ABSTRACT
Abstract Glioblastoma multiforme (GBM), the highest-grade glioma, is the most frequent tumour of the brain with a very poor prognosis and limited therapeutic options. Although little is known about the molecular mechanisms that underlie glioblastoma formation, a number of signal transduction routes, such as the Notch and Ras signalling pathways, seem to play an important role in the formation of GBM. In the present study, we show by in situ hybridization on primary tumour material that the transcription factor HEY1, a target of the Notch signalling pathway, is specifically up-regulated in glioma and that expression of HEY1 in GBM correlates with tumour-grade and survival. In addition, we show by chromatin immunoprecipitations, luciferase assays and Northern blot experiments that HEY1 is a bona fide target of the E2F family of transcription factors, connecting the Ras and Notch signalling pathways. Finally, we show that ectopic expression of HEY1 induces cell proliferation in neural stem cells, while depletion of HEY1 by RNA interference reduces proliferation of glioblastoma cells in tissue culture. Together, these data imply a role for HEY1 in the progression of GBM, and therefore we propose that HEY1 may be a therapeutic target for glioblastoma patients. Moreover, HEY1 may represent a molecular marker to distinguish GBM patients with a longer survival prognosis from those at high risk.

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Changes in HEY1 expression alter the rate of cell proliferation. (A) Neural stem cells (NSCs) infected with a retroviral vector expressing HEY1 form bigger neurospheres than NSCs infected with empty vector or E1A. (B) Immunofluorescence showing an induction of BrdU incorporation in non-differentiated NSCs. Non-infected (upper panels) or HEY1 infected (lower panels) NSCs were incubated for 24 hrs in the presence of BrdU and fixed on glass cover slips by cytospin-treatment. Cells were subsequently stained with DAPI or an antibody specific for BrdU. (C) FACS profiles of NSCs infected with a retroviral vector expressing HEY1 as compared to NSCs infected with empty vector.
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fig03: Changes in HEY1 expression alter the rate of cell proliferation. (A) Neural stem cells (NSCs) infected with a retroviral vector expressing HEY1 form bigger neurospheres than NSCs infected with empty vector or E1A. (B) Immunofluorescence showing an induction of BrdU incorporation in non-differentiated NSCs. Non-infected (upper panels) or HEY1 infected (lower panels) NSCs were incubated for 24 hrs in the presence of BrdU and fixed on glass cover slips by cytospin-treatment. Cells were subsequently stained with DAPI or an antibody specific for BrdU. (C) FACS profiles of NSCs infected with a retroviral vector expressing HEY1 as compared to NSCs infected with empty vector.

Mentions: To determine whether HEY1 might contribute to the development of GBM, full-length HEY1 was ectopically expressed in NSCs isolated from wild-type C57/Bl6 mice. As shown in Fig. 3A, overexpression of HEY1 led to the formation of very large neurospheres when compared to control cells. Overexpression of the adenovirus E1A protein also led to the formation of large colonies and was used as a positive control. Since the big neurospheres could have arisen by increased cell proliferation or by aggregation of smaller spheres, we determined whether HEY1 expression leads to an increase in DNA replication. NSCs were labelled with BrdU for 24 hrs and, as shown in Fig. 3B, NSCs infected with a plasmid expressing HEY1 show a clear increase in BrdU positive cells as compared to cells infected with an empty vector. The number of cells in G0/G1 phase decreased from about 76% in the control to 44% of HEY1-infected cells and, consistently with this, more HEY1-expressing cells were found in the S phase of the cell cycle when cells were infected with an HEY1-expressing vector (15%versus 42% respectively, Fig. 3C).


A role for the transcription factor HEY1 in glioblastoma.

Hulleman E, Quarto M, Vernell R, Masserdotti G, Colli E, Kros JM, Levi D, Gaetani P, Tunici P, Finocchiaro G, Baena RR, Capra M, Helin K - J. Cell. Mol. Med. (2008)

Changes in HEY1 expression alter the rate of cell proliferation. (A) Neural stem cells (NSCs) infected with a retroviral vector expressing HEY1 form bigger neurospheres than NSCs infected with empty vector or E1A. (B) Immunofluorescence showing an induction of BrdU incorporation in non-differentiated NSCs. Non-infected (upper panels) or HEY1 infected (lower panels) NSCs were incubated for 24 hrs in the presence of BrdU and fixed on glass cover slips by cytospin-treatment. Cells were subsequently stained with DAPI or an antibody specific for BrdU. (C) FACS profiles of NSCs infected with a retroviral vector expressing HEY1 as compared to NSCs infected with empty vector.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3823042&req=5

fig03: Changes in HEY1 expression alter the rate of cell proliferation. (A) Neural stem cells (NSCs) infected with a retroviral vector expressing HEY1 form bigger neurospheres than NSCs infected with empty vector or E1A. (B) Immunofluorescence showing an induction of BrdU incorporation in non-differentiated NSCs. Non-infected (upper panels) or HEY1 infected (lower panels) NSCs were incubated for 24 hrs in the presence of BrdU and fixed on glass cover slips by cytospin-treatment. Cells were subsequently stained with DAPI or an antibody specific for BrdU. (C) FACS profiles of NSCs infected with a retroviral vector expressing HEY1 as compared to NSCs infected with empty vector.
Mentions: To determine whether HEY1 might contribute to the development of GBM, full-length HEY1 was ectopically expressed in NSCs isolated from wild-type C57/Bl6 mice. As shown in Fig. 3A, overexpression of HEY1 led to the formation of very large neurospheres when compared to control cells. Overexpression of the adenovirus E1A protein also led to the formation of large colonies and was used as a positive control. Since the big neurospheres could have arisen by increased cell proliferation or by aggregation of smaller spheres, we determined whether HEY1 expression leads to an increase in DNA replication. NSCs were labelled with BrdU for 24 hrs and, as shown in Fig. 3B, NSCs infected with a plasmid expressing HEY1 show a clear increase in BrdU positive cells as compared to cells infected with an empty vector. The number of cells in G0/G1 phase decreased from about 76% in the control to 44% of HEY1-infected cells and, consistently with this, more HEY1-expressing cells were found in the S phase of the cell cycle when cells were infected with an HEY1-expressing vector (15%versus 42% respectively, Fig. 3C).

Bottom Line: In addition, we show by chromatin immunoprecipitations, luciferase assays and Northern blot experiments that HEY1 is a bona fide target of the E2F family of transcription factors, connecting the Ras and Notch signalling pathways.Finally, we show that ectopic expression of HEY1 induces cell proliferation in neural stem cells, while depletion of HEY1 by RNA interference reduces proliferation of glioblastoma cells in tissue culture.Moreover, HEY1 may represent a molecular marker to distinguish GBM patients with a longer survival prognosis from those at high risk.

View Article: PubMed Central - PubMed

Affiliation: European Institute of Oncology, Via Ripamonti, Milan, Italy.

ABSTRACT
Abstract Glioblastoma multiforme (GBM), the highest-grade glioma, is the most frequent tumour of the brain with a very poor prognosis and limited therapeutic options. Although little is known about the molecular mechanisms that underlie glioblastoma formation, a number of signal transduction routes, such as the Notch and Ras signalling pathways, seem to play an important role in the formation of GBM. In the present study, we show by in situ hybridization on primary tumour material that the transcription factor HEY1, a target of the Notch signalling pathway, is specifically up-regulated in glioma and that expression of HEY1 in GBM correlates with tumour-grade and survival. In addition, we show by chromatin immunoprecipitations, luciferase assays and Northern blot experiments that HEY1 is a bona fide target of the E2F family of transcription factors, connecting the Ras and Notch signalling pathways. Finally, we show that ectopic expression of HEY1 induces cell proliferation in neural stem cells, while depletion of HEY1 by RNA interference reduces proliferation of glioblastoma cells in tissue culture. Together, these data imply a role for HEY1 in the progression of GBM, and therefore we propose that HEY1 may be a therapeutic target for glioblastoma patients. Moreover, HEY1 may represent a molecular marker to distinguish GBM patients with a longer survival prognosis from those at high risk.

Show MeSH
Related in: MedlinePlus