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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

CDKN1A expression is not induced upon NBPF1 expression in DLD1Tr21/NBPF1 cells. Expression analysis of selected genes and proteins was executed in DLD1Tr21/Mock and DLD1Tr21/NBPF1 cells, with or without dox treatment. Real-time quantitative RT-PCR measurements are shown for the expression levels of the IRES-driven marker gene EGFP (A), NBPF1 (B) and CDKN1A (C). Cells analyzed were kept untreated (− dox) or were dox treated for 8, 24 or 48 h in order to induce NBPF1 expression. CDKN1A mRNA induction upon NBPF1 expression was not observed in the dox-induced DLD1Tr21/NBPF1 cells. (D) Western blot was performed with lysates of dox-treated and non-treated DLD1Tr21/Mock and DLD1Tr21/NBPF1 cells. Cells were induced for 48 h and immunoblotted with an anti-p21 antibody. p21 protein induction was not observed in dox-induced DLD1Tr21/NBPF1 cells. EGFP detection showed efficient dox-dependent induction in both cell lines. Actin detection was used as a loading control
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Fig13: CDKN1A expression is not induced upon NBPF1 expression in DLD1Tr21/NBPF1 cells. Expression analysis of selected genes and proteins was executed in DLD1Tr21/Mock and DLD1Tr21/NBPF1 cells, with or without dox treatment. Real-time quantitative RT-PCR measurements are shown for the expression levels of the IRES-driven marker gene EGFP (A), NBPF1 (B) and CDKN1A (C). Cells analyzed were kept untreated (− dox) or were dox treated for 8, 24 or 48 h in order to induce NBPF1 expression. CDKN1A mRNA induction upon NBPF1 expression was not observed in the dox-induced DLD1Tr21/NBPF1 cells. (D) Western blot was performed with lysates of dox-treated and non-treated DLD1Tr21/Mock and DLD1Tr21/NBPF1 cells. Cells were induced for 48 h and immunoblotted with an anti-p21 antibody. p21 protein induction was not observed in dox-induced DLD1Tr21/NBPF1 cells. EGFP detection showed efficient dox-dependent induction in both cell lines. Actin detection was used as a loading control

Mentions: We then investigated whether the NBPF1-dependent CDKN1A induction could be confirmed in p53-mutant DLD1Tr21/NBPF1 cells, in which we expect that NBPF1 plays a tumor suppressor role [3]. DLD1Tr21/NBPF1 and DLD1Tr21/Mock cells were induced with dox for 8, 24 or 48 h and the expression levels of CDKN1A, EGFP and NBPF transcripts were determined by real-time quantitative RT-PCR. This expression analysis confirmed that DLD1Tr21/NBPF1 and DLD1Tr21/Mock cells expressed EGFP upon dox addition (Fig. 13A), and that NBPF1 expression was induced only in the DLD1Tr21/NBPF1 cell line in the presence of dox (Fig. 13B). However, in this cell line we did not detect any NBPF1-dependent changes in the mRNA level of CDKN1A (Fig. 13C). In addition, protein lysates of induced DLD1Tr21/NBPF1 cells showed that also the p21CIP1/WAF1 protein levels were not upregulated upon NBPF1 overexpression (Fig. 13D). Moreover, investigation of the different cell cycle phases upon induction of NBPF1 showed no changes in G1 or G2 phases (Fig. 14A), nor did we observe nuclear accumulation of p53 upon NBPF1 induction (Fig. 14B). These data indicate that the induction of CDKN1A upon NBPF1 overexpression is cell-type specific, and that the tumor suppressive effect of NBPF1 may act with or without cell cycle arrest.Fig. 13


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)

CDKN1A expression is not induced upon NBPF1 expression in DLD1Tr21/NBPF1 cells. Expression analysis of selected genes and proteins was executed in DLD1Tr21/Mock and DLD1Tr21/NBPF1 cells, with or without dox treatment. Real-time quantitative RT-PCR measurements are shown for the expression levels of the IRES-driven marker gene EGFP (A), NBPF1 (B) and CDKN1A (C). Cells analyzed were kept untreated (− dox) or were dox treated for 8, 24 or 48 h in order to induce NBPF1 expression. CDKN1A mRNA induction upon NBPF1 expression was not observed in the dox-induced DLD1Tr21/NBPF1 cells. (D) Western blot was performed with lysates of dox-treated and non-treated DLD1Tr21/Mock and DLD1Tr21/NBPF1 cells. Cells were induced for 48 h and immunoblotted with an anti-p21 antibody. p21 protein induction was not observed in dox-induced DLD1Tr21/NBPF1 cells. EGFP detection showed efficient dox-dependent induction in both cell lines. Actin detection was used as a loading control
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig13: CDKN1A expression is not induced upon NBPF1 expression in DLD1Tr21/NBPF1 cells. Expression analysis of selected genes and proteins was executed in DLD1Tr21/Mock and DLD1Tr21/NBPF1 cells, with or without dox treatment. Real-time quantitative RT-PCR measurements are shown for the expression levels of the IRES-driven marker gene EGFP (A), NBPF1 (B) and CDKN1A (C). Cells analyzed were kept untreated (− dox) or were dox treated for 8, 24 or 48 h in order to induce NBPF1 expression. CDKN1A mRNA induction upon NBPF1 expression was not observed in the dox-induced DLD1Tr21/NBPF1 cells. (D) Western blot was performed with lysates of dox-treated and non-treated DLD1Tr21/Mock and DLD1Tr21/NBPF1 cells. Cells were induced for 48 h and immunoblotted with an anti-p21 antibody. p21 protein induction was not observed in dox-induced DLD1Tr21/NBPF1 cells. EGFP detection showed efficient dox-dependent induction in both cell lines. Actin detection was used as a loading control
Mentions: We then investigated whether the NBPF1-dependent CDKN1A induction could be confirmed in p53-mutant DLD1Tr21/NBPF1 cells, in which we expect that NBPF1 plays a tumor suppressor role [3]. DLD1Tr21/NBPF1 and DLD1Tr21/Mock cells were induced with dox for 8, 24 or 48 h and the expression levels of CDKN1A, EGFP and NBPF transcripts were determined by real-time quantitative RT-PCR. This expression analysis confirmed that DLD1Tr21/NBPF1 and DLD1Tr21/Mock cells expressed EGFP upon dox addition (Fig. 13A), and that NBPF1 expression was induced only in the DLD1Tr21/NBPF1 cell line in the presence of dox (Fig. 13B). However, in this cell line we did not detect any NBPF1-dependent changes in the mRNA level of CDKN1A (Fig. 13C). In addition, protein lysates of induced DLD1Tr21/NBPF1 cells showed that also the p21CIP1/WAF1 protein levels were not upregulated upon NBPF1 overexpression (Fig. 13D). Moreover, investigation of the different cell cycle phases upon induction of NBPF1 showed no changes in G1 or G2 phases (Fig. 14A), nor did we observe nuclear accumulation of p53 upon NBPF1 induction (Fig. 14B). These data indicate that the induction of CDKN1A upon NBPF1 overexpression is cell-type specific, and that the tumor suppressive effect of NBPF1 may act with or without cell cycle arrest.Fig. 13

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