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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

Immunofluorescent analysis of the DLD1Tr21/Mock and DLD1Tr21/NBPF1 cell cultures used for proteomic analysis. Cell cultures were grown for 4 days in standard medium supplemented with dox. The induced DLD1Tr21/Mock cells were post-metabolically labeled with light 12C6-NHS-propionate, and the induced DLD1Tr21/NBPF1 cells were post-metabolically labeled with heavy 13C6-NHS-propionate, and then submitted to proteomic analysis as described in Methods. As a control for proper NBPF1 induction, replicate cell cultures were stained for induction of EGFP and NBPF1 expression by immunofluorescent microscopy. Expression of EGFP (shown in green) is induced in both cell lines upon the addition of dox (untreated cultures were fully negative). NBPF1 (antibody sc82241; red channel) was expressed only in DLD1Tr21/NBPF1 cells and only in the presence of dox. Nuclei were visualized with DAPI. Scale bars: 10 μm
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Fig15: Immunofluorescent analysis of the DLD1Tr21/Mock and DLD1Tr21/NBPF1 cell cultures used for proteomic analysis. Cell cultures were grown for 4 days in standard medium supplemented with dox. The induced DLD1Tr21/Mock cells were post-metabolically labeled with light 12C6-NHS-propionate, and the induced DLD1Tr21/NBPF1 cells were post-metabolically labeled with heavy 13C6-NHS-propionate, and then submitted to proteomic analysis as described in Methods. As a control for proper NBPF1 induction, replicate cell cultures were stained for induction of EGFP and NBPF1 expression by immunofluorescent microscopy. Expression of EGFP (shown in green) is induced in both cell lines upon the addition of dox (untreated cultures were fully negative). NBPF1 (antibody sc82241; red channel) was expressed only in DLD1Tr21/NBPF1 cells and only in the presence of dox. Nuclei were visualized with DAPI. Scale bars: 10 μm

Mentions: To obtain complementary information with respect to the role of NBPF1, we investigated the differences between the proteomes of DLD1Tr21/NBPF1 cells and DLD1Tr21/Mock cells. Both cell types were grown for 4 days in the presence of dox in standard medium. Lysates of the induced DLD1Tr21/Mock cells (Fig. 15; + dox, no NBPF1 expression, only EGFP expression) were digested with endoproteinase Lys-C and post-metabolically labeled with light NHS-12C3-propionate [17]. On the other hand, lysates of the induced DLD1Tr21/NBPF1 cells (Fig. 15; + dox, NBPF1 and EGFP expression) were labeled with heavy NHS-13C3-propionate. LC-MS/MS analysis led to the identification of 3613 proteins. A total of 32 proteins showing differential expression was identified at the 99% confidence level [18] (Additional file 5: Table S1). Of these 32 proteins, 19 proteins were upregulated, whereas 13 proteins were downregulated upon NBPF1 expression.Fig. 15


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)

Immunofluorescent analysis of the DLD1Tr21/Mock and DLD1Tr21/NBPF1 cell cultures used for proteomic analysis. Cell cultures were grown for 4 days in standard medium supplemented with dox. The induced DLD1Tr21/Mock cells were post-metabolically labeled with light 12C6-NHS-propionate, and the induced DLD1Tr21/NBPF1 cells were post-metabolically labeled with heavy 13C6-NHS-propionate, and then submitted to proteomic analysis as described in Methods. As a control for proper NBPF1 induction, replicate cell cultures were stained for induction of EGFP and NBPF1 expression by immunofluorescent microscopy. Expression of EGFP (shown in green) is induced in both cell lines upon the addition of dox (untreated cultures were fully negative). NBPF1 (antibody sc82241; red channel) was expressed only in DLD1Tr21/NBPF1 cells and only in the presence of dox. Nuclei were visualized with DAPI. Scale bars: 10 μm
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig15: Immunofluorescent analysis of the DLD1Tr21/Mock and DLD1Tr21/NBPF1 cell cultures used for proteomic analysis. Cell cultures were grown for 4 days in standard medium supplemented with dox. The induced DLD1Tr21/Mock cells were post-metabolically labeled with light 12C6-NHS-propionate, and the induced DLD1Tr21/NBPF1 cells were post-metabolically labeled with heavy 13C6-NHS-propionate, and then submitted to proteomic analysis as described in Methods. As a control for proper NBPF1 induction, replicate cell cultures were stained for induction of EGFP and NBPF1 expression by immunofluorescent microscopy. Expression of EGFP (shown in green) is induced in both cell lines upon the addition of dox (untreated cultures were fully negative). NBPF1 (antibody sc82241; red channel) was expressed only in DLD1Tr21/NBPF1 cells and only in the presence of dox. Nuclei were visualized with DAPI. Scale bars: 10 μm
Mentions: To obtain complementary information with respect to the role of NBPF1, we investigated the differences between the proteomes of DLD1Tr21/NBPF1 cells and DLD1Tr21/Mock cells. Both cell types were grown for 4 days in the presence of dox in standard medium. Lysates of the induced DLD1Tr21/Mock cells (Fig. 15; + dox, no NBPF1 expression, only EGFP expression) were digested with endoproteinase Lys-C and post-metabolically labeled with light NHS-12C3-propionate [17]. On the other hand, lysates of the induced DLD1Tr21/NBPF1 cells (Fig. 15; + dox, NBPF1 and EGFP expression) were labeled with heavy NHS-13C3-propionate. LC-MS/MS analysis led to the identification of 3613 proteins. A total of 32 proteins showing differential expression was identified at the 99% confidence level [18] (Additional file 5: Table S1). Of these 32 proteins, 19 proteins were upregulated, whereas 13 proteins were downregulated upon NBPF1 expression.Fig. 15

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