Limits...
Mutations in the PCNA-binding site of CDKN1C inhibit cell proliferation by impairing the entry into S phase.

Borges KS, Arboleda VA, Vilain E - Cell Div (2015)

Bottom Line: Mutations in the PCNA-binding site of CDKN1C significantly increase CDKN1C protein stability and prevent cell cycle progression into the S phase.However, the IMAGe-mutant CDKN1C protein decreased cell growth significantly more than both the wild-type or BWS protein.These findings bring new insights into the molecular events underlying IMAGe syndrome.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, David Geffen School of Medicine at UCLA, University of California, Los Angeles, 695 Charles E. Young Drive, Los Angeles, CA 90095 USA ; Department of Genetics, Ribeirão Preto Medical School, University of São, Ribeirão Preto, Av. Bandeirantes 3900, CEP 14049-900 Ribeirão Preto, SP Brazil.

ABSTRACT
CDKN1C (also known as P57 (kip2) ) is a cyclin-dependent kinase inhibitor that functions as a negative regulator of cell proliferation through G1 phase cell cycle arrest. Recently, our group described gain-of-function mutations in the PCNA-binding site of CDKN1C that result in an undergrowth syndrome called IMAGe Syndrome (Intrauterine Growth Restriction, Metaphyseal dysplasia, Adrenal hypoplasia, and Genital anomalies), with life-threatening consequences. Loss-of-function mutations in CDKN1C have been identified in 5-10% of individuals with Beckwith-Wiedemann syndrome (BWS), an overgrowth disorder with features that are the opposite of IMAGe syndrome. Here, we investigate the effects of IMAGe-associated mutations on protein stability, cell cycle progression and cell proliferation. Mutations in the PCNA-binding site of CDKN1C significantly increase CDKN1C protein stability and prevent cell cycle progression into the S phase. Overexpression of either wild-type or BWS-mutant CDKN1C inhibited cell proliferation. However, the IMAGe-mutant CDKN1C protein decreased cell growth significantly more than both the wild-type or BWS protein. These findings bring new insights into the molecular events underlying IMAGe syndrome.

No MeSH data available.


Related in: MedlinePlus

Clonogenic assay of the HEK293T and SW13 cells transfected with IMAGe-mutant show decreased clonogenic capacity compared to BWS or wild-type CDKN1C. After transfection of the wild-type CDKN1C, BWS-mutants (p.L42P), IMAGE-mutant (p.K278E) and an empty vector, single cell suspensions were split into six-well in triplicate to assay for clonogenic capacity. (A) SW13 and (B) HEK293T cells results. # indicates a p < 0.05 when compared to the empty-vector condition. *p = <0.05. (C) Representative dishes of SW13 cells transfected as described. Cells were fixed and stained with Crystal violet solution; violet staining represents a higher number of colonies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4389716&req=5

Fig4: Clonogenic assay of the HEK293T and SW13 cells transfected with IMAGe-mutant show decreased clonogenic capacity compared to BWS or wild-type CDKN1C. After transfection of the wild-type CDKN1C, BWS-mutants (p.L42P), IMAGE-mutant (p.K278E) and an empty vector, single cell suspensions were split into six-well in triplicate to assay for clonogenic capacity. (A) SW13 and (B) HEK293T cells results. # indicates a p < 0.05 when compared to the empty-vector condition. *p = <0.05. (C) Representative dishes of SW13 cells transfected as described. Cells were fixed and stained with Crystal violet solution; violet staining represents a higher number of colonies.

Mentions: All CDKN1C expressing plasmids caused a reduction in the clonogenic capacity (fewer and smaller colonies) when compared to the empty vector in both cell lines (Figure 4A and B). IMAGe-mutant transfected cells displayed even fewer colonies than cells transfected with wild-type CDKN1C in the two cell lines studied (Figure 4A and B).Figure 4


Mutations in the PCNA-binding site of CDKN1C inhibit cell proliferation by impairing the entry into S phase.

Borges KS, Arboleda VA, Vilain E - Cell Div (2015)

Clonogenic assay of the HEK293T and SW13 cells transfected with IMAGe-mutant show decreased clonogenic capacity compared to BWS or wild-type CDKN1C. After transfection of the wild-type CDKN1C, BWS-mutants (p.L42P), IMAGE-mutant (p.K278E) and an empty vector, single cell suspensions were split into six-well in triplicate to assay for clonogenic capacity. (A) SW13 and (B) HEK293T cells results. # indicates a p < 0.05 when compared to the empty-vector condition. *p = <0.05. (C) Representative dishes of SW13 cells transfected as described. Cells were fixed and stained with Crystal violet solution; violet staining represents a higher number of colonies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4389716&req=5

Fig4: Clonogenic assay of the HEK293T and SW13 cells transfected with IMAGe-mutant show decreased clonogenic capacity compared to BWS or wild-type CDKN1C. After transfection of the wild-type CDKN1C, BWS-mutants (p.L42P), IMAGE-mutant (p.K278E) and an empty vector, single cell suspensions were split into six-well in triplicate to assay for clonogenic capacity. (A) SW13 and (B) HEK293T cells results. # indicates a p < 0.05 when compared to the empty-vector condition. *p = <0.05. (C) Representative dishes of SW13 cells transfected as described. Cells were fixed and stained with Crystal violet solution; violet staining represents a higher number of colonies.
Mentions: All CDKN1C expressing plasmids caused a reduction in the clonogenic capacity (fewer and smaller colonies) when compared to the empty vector in both cell lines (Figure 4A and B). IMAGe-mutant transfected cells displayed even fewer colonies than cells transfected with wild-type CDKN1C in the two cell lines studied (Figure 4A and B).Figure 4

Bottom Line: Mutations in the PCNA-binding site of CDKN1C significantly increase CDKN1C protein stability and prevent cell cycle progression into the S phase.However, the IMAGe-mutant CDKN1C protein decreased cell growth significantly more than both the wild-type or BWS protein.These findings bring new insights into the molecular events underlying IMAGe syndrome.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, David Geffen School of Medicine at UCLA, University of California, Los Angeles, 695 Charles E. Young Drive, Los Angeles, CA 90095 USA ; Department of Genetics, Ribeirão Preto Medical School, University of São, Ribeirão Preto, Av. Bandeirantes 3900, CEP 14049-900 Ribeirão Preto, SP Brazil.

ABSTRACT
CDKN1C (also known as P57 (kip2) ) is a cyclin-dependent kinase inhibitor that functions as a negative regulator of cell proliferation through G1 phase cell cycle arrest. Recently, our group described gain-of-function mutations in the PCNA-binding site of CDKN1C that result in an undergrowth syndrome called IMAGe Syndrome (Intrauterine Growth Restriction, Metaphyseal dysplasia, Adrenal hypoplasia, and Genital anomalies), with life-threatening consequences. Loss-of-function mutations in CDKN1C have been identified in 5-10% of individuals with Beckwith-Wiedemann syndrome (BWS), an overgrowth disorder with features that are the opposite of IMAGe syndrome. Here, we investigate the effects of IMAGe-associated mutations on protein stability, cell cycle progression and cell proliferation. Mutations in the PCNA-binding site of CDKN1C significantly increase CDKN1C protein stability and prevent cell cycle progression into the S phase. Overexpression of either wild-type or BWS-mutant CDKN1C inhibited cell proliferation. However, the IMAGe-mutant CDKN1C protein decreased cell growth significantly more than both the wild-type or BWS protein. These findings bring new insights into the molecular events underlying IMAGe syndrome.

No MeSH data available.


Related in: MedlinePlus