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NBPF1, a tumor suppressor candidate in neuroblastoma, exerts growth inhibitory effects by inducing a G1 cell cycle arrest.

Andries V, Vandepoele K, Staes K, Berx G, Bogaert P, Van Isterdael G, Ginneberge D, Parthoens E, Vandenbussche J, Gevaert K, van Roy F - BMC Cancer (2015)

Bottom Line: Forced expression of NBPF1 in HEK293T cells resulted in a G1 cell cycle arrest that was accompanied by upregulation of the cyclin-dependent kinase inhibitor p21(CIP1/WAF1) in a p53-dependent manner.However, CDKN1A upregulation by NBPF1 was not observed in the DLD1 cells, which demonstrates that NBPF1 exerts cell-specific effects.In addition, proteome analysis of NBPF1-overexpressing DLD1 cells identified 32 differentially expressed proteins, of which several are implicated in carcinogenesis.

View Article: PubMed Central - PubMed

Affiliation: Inflammation Research Center, VIB, Ghent, Belgium. Vanessa.Andries@irc.vib-UGent.be.

ABSTRACT

Background: NBPF1 (Neuroblastoma Breakpoint Family, member 1) was originally identified in a neuroblastoma patient on the basis of its disruption by a chromosomal translocation t(1;17)(p36.2;q11.2). Considering this genetic defect and the frequent genomic alterations of the NBPF1 locus in several cancer types, we hypothesized that NBPF1 is a tumor suppressor. Decreased expression of NBPF1 in neuroblastoma cell lines with loss of 1p36 heterozygosity and the marked decrease of anchorage-independent clonal growth of DLD1 colorectal carcinoma cells with induced NBPF1 expression further suggest that NBPF1 functions as tumor suppressor. However, little is known about the mechanisms involved.

Methods: Expression of NBPF was analyzed in human skin and human cervix by immunohistochemistry. The effects of NBPF1 on the cell cycle were evaluated by flow cytometry. We investigated by real-time quantitative RT-PCR the expression profile of a panel of genes important in cell cycle regulation. Protein levels of CDKN1A-encoded p21(CIP1/WAF1) were determined by western blotting and the importance of p53 was shown by immunofluorescence and by a loss-of-function approach. LC-MS/MS analysis was used to investigate the proteome of DLD1 colon cancer cells with induced NBPF1 expression. Possible biological interactions between the differentially regulated proteins were investigated with the Ingenuity Pathway Analysis tool.

Results: We show that NBPF is expressed in the non-proliferative suprabasal layers of squamous stratified epithelia of human skin and cervix. Forced expression of NBPF1 in HEK293T cells resulted in a G1 cell cycle arrest that was accompanied by upregulation of the cyclin-dependent kinase inhibitor p21(CIP1/WAF1) in a p53-dependent manner. Additionally, forced expression of NBPF1 in two p53-mutant neuroblastoma cell lines also resulted in a G1 cell cycle arrest and CDKN1A upregulation. However, CDKN1A upregulation by NBPF1 was not observed in the DLD1 cells, which demonstrates that NBPF1 exerts cell-specific effects. In addition, proteome analysis of NBPF1-overexpressing DLD1 cells identified 32 differentially expressed proteins, of which several are implicated in carcinogenesis.

Conclusions: We demonstrated that NBPF1 exerts different tumor suppressive effects, depending on the cell line analyzed, and provide new clues into the molecular mechanism of the enigmatic NBPF proteins.

No MeSH data available.


Related in: MedlinePlus

Time-lapse microscopy of NBPF1 expression in transfected HEK293T cells stimulated with aphidicolin. EGFP-luciferase or EGFP-NBPF1 expression was followed for 15 h with images captured every 10 min. Individual frames are shown. Original movies are available as Additional files 3 and 4. Fluorescence levels are represented by pseudocolors, with blue indicating low EGFP levels and yellow indicating high EGFP levels (ImageJ LUT; color codes shown at the bottom). (A) Expression of EGFP-luciferase in cells stimulated with aphidicolin remained at a constant level at all the indicated time points. (B) Expression of EGFP-NBPF1 in cells stimulated with aphidicolin showed increased accumulation in the form of particulate or aggregated structures. In no case were cells with decreasing levels of EGFP-NBPF1 observed
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Fig3: Time-lapse microscopy of NBPF1 expression in transfected HEK293T cells stimulated with aphidicolin. EGFP-luciferase or EGFP-NBPF1 expression was followed for 15 h with images captured every 10 min. Individual frames are shown. Original movies are available as Additional files 3 and 4. Fluorescence levels are represented by pseudocolors, with blue indicating low EGFP levels and yellow indicating high EGFP levels (ImageJ LUT; color codes shown at the bottom). (A) Expression of EGFP-luciferase in cells stimulated with aphidicolin remained at a constant level at all the indicated time points. (B) Expression of EGFP-NBPF1 in cells stimulated with aphidicolin showed increased accumulation in the form of particulate or aggregated structures. In no case were cells with decreasing levels of EGFP-NBPF1 observed

Mentions: Therefore, we investigated the spatio-temporal dynamics of NBPF1 expression in the human embryonic kidney cell line, HEK293T. These cells were transfected with a control chimeric EGFP-luciferase expressing plasmid or with a plasmid expressing a chimeric EGFP-NBPF1 protein. EGFP fluorescence for both setups was followed in real-time. Interestingly, striking differences were seen between the control cells and the EGFP-NBPF1 positive cells. Whereas EGFP-luciferase expression remained at a constant level for at least 15 h (Fig. 2A and representative movie in Additional file 1: Movie 1), EGFP-NBPF1 expression often decreased rapidly (Fig. 2B and representative movie in Additional file 2: Movie 2). Also, we observed cells in the EGFP-NBPF1 setup that were EGFP negative at the onset of the experiment, but became EGFP positive later on. Importantly, we never observed any cell expressing EGFP-NBPF1 that completed a full cell cycle, whereas untransfected cells and EGFP-luciferase-transfected control cells divided frequently. Therefore, we hypothesized that the cell cycle was arrested in EGFP-NBPF1 positive cells, at least during the period of examination. Additionally, the disappearance of EGFP-NBPF1 positive cells suggested that the protein was degraded in proliferating cells. To further investigate this, we induced growth arrest by using a chemotherapeutic agent, aphidicolin, which is an inhibitor of eukaryotic nuclear DNA replication and blocks the cell cycle at the early S phase. HEK293T cells were transfected with an EGFP-luciferase control plasmid or with a plasmid expressing a chimeric EGFP-NBPF1 protein. After 24 h, the cells were treated with aphidicolin and EGFP fluorescence for both setups was followed in real-time 24 h later. Strikingly, EGFP-NBPF1 expression did not decrease but the protein accumulated in the form of particulate or aggregated structures during the time of imaging (Fig. 3B and representative movie in Additional file 3: Movie 3) whereas EGFP-luciferase expression remained at a constant level and no aggregates were seen (Fig. 3A and representative movie in Additional file 4: Movie 4). Therefore, we hypothesized that NBPF1 expression is indeed lost in proliferating cells, and that upon artificial induction of a cell cycle block, NBPF1 escapes degradation but loses its functionality due to its aggregation. Taken together, these data indicate that NBPF1 overexpression affects cell cycle and cell fate, and reveal that NBPF1 expression is cell cycle dependent: expression occurs mainly in G1 and is lost during mitosis.Fig. 2


NBPF1, a tumor suppressor candidate in neuroblastoma, exerts growth inhibitory effects by inducing a G1 cell cycle arrest.

Andries V, Vandepoele K, Staes K, Berx G, Bogaert P, Van Isterdael G, Ginneberge D, Parthoens E, Vandenbussche J, Gevaert K, van Roy F - BMC Cancer (2015)

Time-lapse microscopy of NBPF1 expression in transfected HEK293T cells stimulated with aphidicolin. EGFP-luciferase or EGFP-NBPF1 expression was followed for 15 h with images captured every 10 min. Individual frames are shown. Original movies are available as Additional files 3 and 4. Fluorescence levels are represented by pseudocolors, with blue indicating low EGFP levels and yellow indicating high EGFP levels (ImageJ LUT; color codes shown at the bottom). (A) Expression of EGFP-luciferase in cells stimulated with aphidicolin remained at a constant level at all the indicated time points. (B) Expression of EGFP-NBPF1 in cells stimulated with aphidicolin showed increased accumulation in the form of particulate or aggregated structures. In no case were cells with decreasing levels of EGFP-NBPF1 observed
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4440459&req=5

Fig3: Time-lapse microscopy of NBPF1 expression in transfected HEK293T cells stimulated with aphidicolin. EGFP-luciferase or EGFP-NBPF1 expression was followed for 15 h with images captured every 10 min. Individual frames are shown. Original movies are available as Additional files 3 and 4. Fluorescence levels are represented by pseudocolors, with blue indicating low EGFP levels and yellow indicating high EGFP levels (ImageJ LUT; color codes shown at the bottom). (A) Expression of EGFP-luciferase in cells stimulated with aphidicolin remained at a constant level at all the indicated time points. (B) Expression of EGFP-NBPF1 in cells stimulated with aphidicolin showed increased accumulation in the form of particulate or aggregated structures. In no case were cells with decreasing levels of EGFP-NBPF1 observed
Mentions: Therefore, we investigated the spatio-temporal dynamics of NBPF1 expression in the human embryonic kidney cell line, HEK293T. These cells were transfected with a control chimeric EGFP-luciferase expressing plasmid or with a plasmid expressing a chimeric EGFP-NBPF1 protein. EGFP fluorescence for both setups was followed in real-time. Interestingly, striking differences were seen between the control cells and the EGFP-NBPF1 positive cells. Whereas EGFP-luciferase expression remained at a constant level for at least 15 h (Fig. 2A and representative movie in Additional file 1: Movie 1), EGFP-NBPF1 expression often decreased rapidly (Fig. 2B and representative movie in Additional file 2: Movie 2). Also, we observed cells in the EGFP-NBPF1 setup that were EGFP negative at the onset of the experiment, but became EGFP positive later on. Importantly, we never observed any cell expressing EGFP-NBPF1 that completed a full cell cycle, whereas untransfected cells and EGFP-luciferase-transfected control cells divided frequently. Therefore, we hypothesized that the cell cycle was arrested in EGFP-NBPF1 positive cells, at least during the period of examination. Additionally, the disappearance of EGFP-NBPF1 positive cells suggested that the protein was degraded in proliferating cells. To further investigate this, we induced growth arrest by using a chemotherapeutic agent, aphidicolin, which is an inhibitor of eukaryotic nuclear DNA replication and blocks the cell cycle at the early S phase. HEK293T cells were transfected with an EGFP-luciferase control plasmid or with a plasmid expressing a chimeric EGFP-NBPF1 protein. After 24 h, the cells were treated with aphidicolin and EGFP fluorescence for both setups was followed in real-time 24 h later. Strikingly, EGFP-NBPF1 expression did not decrease but the protein accumulated in the form of particulate or aggregated structures during the time of imaging (Fig. 3B and representative movie in Additional file 3: Movie 3) whereas EGFP-luciferase expression remained at a constant level and no aggregates were seen (Fig. 3A and representative movie in Additional file 4: Movie 4). Therefore, we hypothesized that NBPF1 expression is indeed lost in proliferating cells, and that upon artificial induction of a cell cycle block, NBPF1 escapes degradation but loses its functionality due to its aggregation. Taken together, these data indicate that NBPF1 overexpression affects cell cycle and cell fate, and reveal that NBPF1 expression is cell cycle dependent: expression occurs mainly in G1 and is lost during mitosis.Fig. 2

Bottom Line: Forced expression of NBPF1 in HEK293T cells resulted in a G1 cell cycle arrest that was accompanied by upregulation of the cyclin-dependent kinase inhibitor p21(CIP1/WAF1) in a p53-dependent manner.However, CDKN1A upregulation by NBPF1 was not observed in the DLD1 cells, which demonstrates that NBPF1 exerts cell-specific effects.In addition, proteome analysis of NBPF1-overexpressing DLD1 cells identified 32 differentially expressed proteins, of which several are implicated in carcinogenesis.

View Article: PubMed Central - PubMed

Affiliation: Inflammation Research Center, VIB, Ghent, Belgium. Vanessa.Andries@irc.vib-UGent.be.

ABSTRACT

Background: NBPF1 (Neuroblastoma Breakpoint Family, member 1) was originally identified in a neuroblastoma patient on the basis of its disruption by a chromosomal translocation t(1;17)(p36.2;q11.2). Considering this genetic defect and the frequent genomic alterations of the NBPF1 locus in several cancer types, we hypothesized that NBPF1 is a tumor suppressor. Decreased expression of NBPF1 in neuroblastoma cell lines with loss of 1p36 heterozygosity and the marked decrease of anchorage-independent clonal growth of DLD1 colorectal carcinoma cells with induced NBPF1 expression further suggest that NBPF1 functions as tumor suppressor. However, little is known about the mechanisms involved.

Methods: Expression of NBPF was analyzed in human skin and human cervix by immunohistochemistry. The effects of NBPF1 on the cell cycle were evaluated by flow cytometry. We investigated by real-time quantitative RT-PCR the expression profile of a panel of genes important in cell cycle regulation. Protein levels of CDKN1A-encoded p21(CIP1/WAF1) were determined by western blotting and the importance of p53 was shown by immunofluorescence and by a loss-of-function approach. LC-MS/MS analysis was used to investigate the proteome of DLD1 colon cancer cells with induced NBPF1 expression. Possible biological interactions between the differentially regulated proteins were investigated with the Ingenuity Pathway Analysis tool.

Results: We show that NBPF is expressed in the non-proliferative suprabasal layers of squamous stratified epithelia of human skin and cervix. Forced expression of NBPF1 in HEK293T cells resulted in a G1 cell cycle arrest that was accompanied by upregulation of the cyclin-dependent kinase inhibitor p21(CIP1/WAF1) in a p53-dependent manner. Additionally, forced expression of NBPF1 in two p53-mutant neuroblastoma cell lines also resulted in a G1 cell cycle arrest and CDKN1A upregulation. However, CDKN1A upregulation by NBPF1 was not observed in the DLD1 cells, which demonstrates that NBPF1 exerts cell-specific effects. In addition, proteome analysis of NBPF1-overexpressing DLD1 cells identified 32 differentially expressed proteins, of which several are implicated in carcinogenesis.

Conclusions: We demonstrated that NBPF1 exerts different tumor suppressive effects, depending on the cell line analyzed, and provide new clues into the molecular mechanism of the enigmatic NBPF proteins.

No MeSH data available.


Related in: MedlinePlus