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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

Polyclonal antibody sc-82241 recognizes NBPF proteins specifically and detects NBPF expression in suprabasal layers of normal human skin and cervix. (A) Upon overexpression of NBPF1, fused to EGFP, in HEK293T cells, a clear overlap is detected between the EGFP signal and the signal of the anti-NBPF antibody sc-82241. Nuclei were stained with DAPI. Scale bar: 10 μm. (B) The anti-NBPF antibody yields a specific cytoplasmic staining in DLD1Tr21/NBPF1 cells when NBPF1 expression is induced in the presence of dox. Scale bar: 10 μm. (C) Paraffin sections from human (left panel) and mouse skin (right panel) were stained with anti-NBPF antibody sc-82241. In the human epidermis, staining for NBPF is observed only in the non-proliferating, differentiated cells of the suprabasal layer and not in the proliferating basal cell layer (arrows). The middle panel shows a magnification of the boxed area. Mouse skin served as a negative control: no staining is seen in any of the living layers of the epidermis. The cornified envelope of the epidermis shows aspecific staining, since the signal is obtained in both mouse and human samples. Scale bars: 25 μm. (D) Normal human cervix stained with anti-NBPF antibody sc-82241 shows strong immunopositivity of the suprabasal layer. The middle panel shows a magnification of the boxed area. In the negative control (right panel), the primary antibody was omitted. Arrows point to basal cell layers. Scale bars: 25 μm
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Fig1: Polyclonal antibody sc-82241 recognizes NBPF proteins specifically and detects NBPF expression in suprabasal layers of normal human skin and cervix. (A) Upon overexpression of NBPF1, fused to EGFP, in HEK293T cells, a clear overlap is detected between the EGFP signal and the signal of the anti-NBPF antibody sc-82241. Nuclei were stained with DAPI. Scale bar: 10 μm. (B) The anti-NBPF antibody yields a specific cytoplasmic staining in DLD1Tr21/NBPF1 cells when NBPF1 expression is induced in the presence of dox. Scale bar: 10 μm. (C) Paraffin sections from human (left panel) and mouse skin (right panel) were stained with anti-NBPF antibody sc-82241. In the human epidermis, staining for NBPF is observed only in the non-proliferating, differentiated cells of the suprabasal layer and not in the proliferating basal cell layer (arrows). The middle panel shows a magnification of the boxed area. Mouse skin served as a negative control: no staining is seen in any of the living layers of the epidermis. The cornified envelope of the epidermis shows aspecific staining, since the signal is obtained in both mouse and human samples. Scale bars: 25 μm. (D) Normal human cervix stained with anti-NBPF antibody sc-82241 shows strong immunopositivity of the suprabasal layer. The middle panel shows a magnification of the boxed area. In the negative control (right panel), the primary antibody was omitted. Arrows point to basal cell layers. Scale bars: 25 μm

Mentions: To explore the expression of the NBPF family members in more detail, we detected endogenous NBPF proteins in paraffin-embedded sections of human skin and cervix using a commercially available polyclonal anti-NBPF antibody (sc-82241). This antibody is raised against a peptide located in the protein domain encoded by exon types 5 and 1 (amino acids 240–290, UniProtKB Q3BBV1) and recognizes several NBPF proteins, including NBPF1. Because only few anti-NBPF antibodies are commercially available, it was important to confirm the specificity of this particular antibody. In immunofluorescence experiments, we used sc-82241 to stain HEK293T cells overexpressing an EGFP-fused NBPF1 protein: there was a clear overlap between the EGFP signal and sc-82241 reactivity (Fig. 1A). We then stained the inducible DLD1Tr21/NBPF1 stable cell line [3]: sc-82241 reactivity was observed only in the presence of doxycycline (dox), implying NBPF1-specific recognition (Fig. 1B). Taken together, these experiments clearly show that sc-82241 specifically detects human NBPF proteins.Fig. 1


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)

Polyclonal antibody sc-82241 recognizes NBPF proteins specifically and detects NBPF expression in suprabasal layers of normal human skin and cervix. (A) Upon overexpression of NBPF1, fused to EGFP, in HEK293T cells, a clear overlap is detected between the EGFP signal and the signal of the anti-NBPF antibody sc-82241. Nuclei were stained with DAPI. Scale bar: 10 μm. (B) The anti-NBPF antibody yields a specific cytoplasmic staining in DLD1Tr21/NBPF1 cells when NBPF1 expression is induced in the presence of dox. Scale bar: 10 μm. (C) Paraffin sections from human (left panel) and mouse skin (right panel) were stained with anti-NBPF antibody sc-82241. In the human epidermis, staining for NBPF is observed only in the non-proliferating, differentiated cells of the suprabasal layer and not in the proliferating basal cell layer (arrows). The middle panel shows a magnification of the boxed area. Mouse skin served as a negative control: no staining is seen in any of the living layers of the epidermis. The cornified envelope of the epidermis shows aspecific staining, since the signal is obtained in both mouse and human samples. Scale bars: 25 μm. (D) Normal human cervix stained with anti-NBPF antibody sc-82241 shows strong immunopositivity of the suprabasal layer. The middle panel shows a magnification of the boxed area. In the negative control (right panel), the primary antibody was omitted. Arrows point to basal cell layers. Scale bars: 25 μm
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4440459&req=5

Fig1: Polyclonal antibody sc-82241 recognizes NBPF proteins specifically and detects NBPF expression in suprabasal layers of normal human skin and cervix. (A) Upon overexpression of NBPF1, fused to EGFP, in HEK293T cells, a clear overlap is detected between the EGFP signal and the signal of the anti-NBPF antibody sc-82241. Nuclei were stained with DAPI. Scale bar: 10 μm. (B) The anti-NBPF antibody yields a specific cytoplasmic staining in DLD1Tr21/NBPF1 cells when NBPF1 expression is induced in the presence of dox. Scale bar: 10 μm. (C) Paraffin sections from human (left panel) and mouse skin (right panel) were stained with anti-NBPF antibody sc-82241. In the human epidermis, staining for NBPF is observed only in the non-proliferating, differentiated cells of the suprabasal layer and not in the proliferating basal cell layer (arrows). The middle panel shows a magnification of the boxed area. Mouse skin served as a negative control: no staining is seen in any of the living layers of the epidermis. The cornified envelope of the epidermis shows aspecific staining, since the signal is obtained in both mouse and human samples. Scale bars: 25 μm. (D) Normal human cervix stained with anti-NBPF antibody sc-82241 shows strong immunopositivity of the suprabasal layer. The middle panel shows a magnification of the boxed area. In the negative control (right panel), the primary antibody was omitted. Arrows point to basal cell layers. Scale bars: 25 μm
Mentions: To explore the expression of the NBPF family members in more detail, we detected endogenous NBPF proteins in paraffin-embedded sections of human skin and cervix using a commercially available polyclonal anti-NBPF antibody (sc-82241). This antibody is raised against a peptide located in the protein domain encoded by exon types 5 and 1 (amino acids 240–290, UniProtKB Q3BBV1) and recognizes several NBPF proteins, including NBPF1. Because only few anti-NBPF antibodies are commercially available, it was important to confirm the specificity of this particular antibody. In immunofluorescence experiments, we used sc-82241 to stain HEK293T cells overexpressing an EGFP-fused NBPF1 protein: there was a clear overlap between the EGFP signal and sc-82241 reactivity (Fig. 1A). We then stained the inducible DLD1Tr21/NBPF1 stable cell line [3]: sc-82241 reactivity was observed only in the presence of doxycycline (dox), implying NBPF1-specific recognition (Fig. 1B). Taken together, these experiments clearly show that sc-82241 specifically detects human NBPF proteins.Fig. 1

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