Limits...
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

NBPF1 overexpression increases p21CIP1/WAF1 protein levels. (A) HEK293T cells were transfected with plasmids encoding either EGFP-luciferase or EGFP-NBPF1. Both EGFP-positive (+) and EGFP-negative populations (−) were isolated by FACS, and the protein levels of p21CIP1/WAF1 were detected by western blotting (top row). Treatment of cells with doxorubicin (+ Doxo) served as a positive control for p21 induction. Detection with the anti-NBPF antibody (sc-82241) showed the successful isolation of EGFP-NBPF1 transfected cells (second row). Detection with an anti-EGFP antibody (third row) showed successful isolation of transfected cells that were EGFP-luciferase positive (indicated by *) or EGFP-NBPF1 positive (indicated by **). Vinculin expression acted as a loading control (bottom row). (B) Quantification of p21CIP1/WAF1 signals in the blot of (A), normalized against vinculin signals. In addition to the positive control (+ Doxo), only cells expressing EGFP-NBPF1 showed clear induction of p21. p-values were calculated with one-way ANOVA
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig7: NBPF1 overexpression increases p21CIP1/WAF1 protein levels. (A) HEK293T cells were transfected with plasmids encoding either EGFP-luciferase or EGFP-NBPF1. Both EGFP-positive (+) and EGFP-negative populations (−) were isolated by FACS, and the protein levels of p21CIP1/WAF1 were detected by western blotting (top row). Treatment of cells with doxorubicin (+ Doxo) served as a positive control for p21 induction. Detection with the anti-NBPF antibody (sc-82241) showed the successful isolation of EGFP-NBPF1 transfected cells (second row). Detection with an anti-EGFP antibody (third row) showed successful isolation of transfected cells that were EGFP-luciferase positive (indicated by *) or EGFP-NBPF1 positive (indicated by **). Vinculin expression acted as a loading control (bottom row). (B) Quantification of p21CIP1/WAF1 signals in the blot of (A), normalized against vinculin signals. In addition to the positive control (+ Doxo), only cells expressing EGFP-NBPF1 showed clear induction of p21. p-values were calculated with one-way ANOVA

Mentions: In addition, protein lysates of EGFP-positive and EGFP-negative populations from both settings showed that not only the CDKN1A mRNA levels, but also the p21CIP1/WAF1 protein levels were upregulated upon NBPF1 overexpression in HEK293T cells (Fig. 7). Doxorubicin (Doxo) treatment of mock transfected cells served as a positive control for p21CIP1/WAF1 induction. Taken together, these data indicated that NBPF1 overexpression in HEK293T cells induces p21CIP1/WAF1 expression at both the mRNA and protein levels.Fig. 7


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)

NBPF1 overexpression increases p21CIP1/WAF1 protein levels. (A) HEK293T cells were transfected with plasmids encoding either EGFP-luciferase or EGFP-NBPF1. Both EGFP-positive (+) and EGFP-negative populations (−) were isolated by FACS, and the protein levels of p21CIP1/WAF1 were detected by western blotting (top row). Treatment of cells with doxorubicin (+ Doxo) served as a positive control for p21 induction. Detection with the anti-NBPF antibody (sc-82241) showed the successful isolation of EGFP-NBPF1 transfected cells (second row). Detection with an anti-EGFP antibody (third row) showed successful isolation of transfected cells that were EGFP-luciferase positive (indicated by *) or EGFP-NBPF1 positive (indicated by **). Vinculin expression acted as a loading control (bottom row). (B) Quantification of p21CIP1/WAF1 signals in the blot of (A), normalized against vinculin signals. In addition to the positive control (+ Doxo), only cells expressing EGFP-NBPF1 showed clear induction of p21. p-values were calculated with one-way ANOVA
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig7: NBPF1 overexpression increases p21CIP1/WAF1 protein levels. (A) HEK293T cells were transfected with plasmids encoding either EGFP-luciferase or EGFP-NBPF1. Both EGFP-positive (+) and EGFP-negative populations (−) were isolated by FACS, and the protein levels of p21CIP1/WAF1 were detected by western blotting (top row). Treatment of cells with doxorubicin (+ Doxo) served as a positive control for p21 induction. Detection with the anti-NBPF antibody (sc-82241) showed the successful isolation of EGFP-NBPF1 transfected cells (second row). Detection with an anti-EGFP antibody (third row) showed successful isolation of transfected cells that were EGFP-luciferase positive (indicated by *) or EGFP-NBPF1 positive (indicated by **). Vinculin expression acted as a loading control (bottom row). (B) Quantification of p21CIP1/WAF1 signals in the blot of (A), normalized against vinculin signals. In addition to the positive control (+ Doxo), only cells expressing EGFP-NBPF1 showed clear induction of p21. p-values were calculated with one-way ANOVA
Mentions: In addition, protein lysates of EGFP-positive and EGFP-negative populations from both settings showed that not only the CDKN1A mRNA levels, but also the p21CIP1/WAF1 protein levels were upregulated upon NBPF1 overexpression in HEK293T cells (Fig. 7). Doxorubicin (Doxo) treatment of mock transfected cells served as a positive control for p21CIP1/WAF1 induction. Taken together, these data indicated that NBPF1 overexpression in HEK293T cells induces p21CIP1/WAF1 expression at both the mRNA and protein levels.Fig. 7

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