Limits...
Stress-induced alternative splice forms of MDM2 and MDMX modulate the p53-pathway in distinct ways.

Jacob AG, Singh RK, Comiskey DF, Rouhier MF, Mohammad F, Bebee TW, Chandler DS - PLoS ONE (2014)

Bottom Line: We show here that MDM2-ALT1 is capable of binding full-length MDMX as well as full-length MDM2.Moreover, MDM2-ALT1 expression causes cell cycle arrest in the G1 phase in a p53 and p21 dependent manner, which is consistent with the increased levels of p21.In summary, our study shows that the stress-inducible alternative splice forms MDM2-ALT1 and MDMX-ALT2 are important modifiers of the p53 pathway and present a potential mechanism to tailor the p53-mediated cellular stress response.

View Article: PubMed Central - PubMed

Affiliation: From the Center for Childhood Cancer at the Research Institute at Nationwide Children's Hospital, Columbus, Ohio, United States of America; The Department of Pediatrics, and Molecular, Cellular and Developmental Biology (MCDB) program, The Ohio State University, Columbus, Ohio, United States of America; Center for RNA Biology, Wexner Medical Center, The Ohio State University, Columbus, Ohio, United States of America.

ABSTRACT
MDM2 and MDMX are the chief negative regulators of the tumor-suppressor protein p53 and are essential for maintaining homeostasis within the cell. In response to genotoxic stress and also in several cancer types, MDM2 and MDMX are alternatively spliced. The splice variants MDM2-ALT1 and MDMX-ALT2 lack the p53-binding domain and are incapable of negatively regulating p53. However, they retain the RING domain that facilitates dimerization of the full-length MDM proteins. Concordantly, MDM2-ALT1 has been shown to lead to the stabilization of p53 through its interaction with and inactivation of full-length MDM2. The impact of MDM2-ALT1 expression on the p53 pathway and the nature of its interaction with MDMX remain unclear. Also, the role of the architecturally similar MDMX-ALT2 and its influence of the MDM2-MDMX-p53 axis are yet to be elucidated. We show here that MDM2-ALT1 is capable of binding full-length MDMX as well as full-length MDM2. Additionally, we demonstrate that MDMX-ALT2 is able to dimerize with both full-length MDMX and MDM2 and that the expression of MDM2-ALT1 and MDMX-ALT2 leads to the upregulation of p53 protein, and also of its downstream target p21. Moreover, MDM2-ALT1 expression causes cell cycle arrest in the G1 phase in a p53 and p21 dependent manner, which is consistent with the increased levels of p21. Finally we present evidence that MDM2-ALT1 and MDMX-ALT2 expression can activate subtly distinct subsets of p53-transcriptional targets implying that these splice variants can modulate the p53 tumor suppressor pathway in unique ways. In summary, our study shows that the stress-inducible alternative splice forms MDM2-ALT1 and MDMX-ALT2 are important modifiers of the p53 pathway and present a potential mechanism to tailor the p53-mediated cellular stress response.

Show MeSH

Related in: MedlinePlus

MDM2-ALT1 over-expression leads to G1 phase cell cycle arrest in a p53-dependent and p21-dependent manner.A. HCT116 wild-type (wt), B. HCT116 p53−/− and C. HCT116 p21−/− cells were transfected with myc-tagged GFP or MDM2-ALT1 (2Alt1) or MDMX-ALT2 (XAlt2), harvested 24 hours post-transfection and stained with propidium iodide solution and sorted for DNA content. The bar graphs represent the percentage of cells in the various phases of the cell cycle. Error bars represent the standard error mean from at least 3 independent experiments. HCT116 cells that are wildtype (A) show significantly higher percentage of cells in G1-phase upon MDM2-ALT1 expression (31.44% ±2.45 SEM) compared to GFP-expressing cells (24.40% ±1.40 SEM; n = 5, p = 0.0369). In case of MDMX-ALT2 over-expression in HCT116 wildtype cells, there is no significant change in percentage of cells in G1 phase compared to negative control GFP-expressing cells (p =  0.4389). HCT116 cells that are  for p53 (B) or p21 (C) do not show any differences in the percentage of cells in any of the cell cycle phases upon over-expression of GFP or MDM2-ALT1 or MDMX-ALT2. D. Representative immuno blot showing expression of p53, p21 and loading control GAPDH in HCT116 wt, p53−/− and p21−/− cells.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4126728&req=5

pone-0104444-g003: MDM2-ALT1 over-expression leads to G1 phase cell cycle arrest in a p53-dependent and p21-dependent manner.A. HCT116 wild-type (wt), B. HCT116 p53−/− and C. HCT116 p21−/− cells were transfected with myc-tagged GFP or MDM2-ALT1 (2Alt1) or MDMX-ALT2 (XAlt2), harvested 24 hours post-transfection and stained with propidium iodide solution and sorted for DNA content. The bar graphs represent the percentage of cells in the various phases of the cell cycle. Error bars represent the standard error mean from at least 3 independent experiments. HCT116 cells that are wildtype (A) show significantly higher percentage of cells in G1-phase upon MDM2-ALT1 expression (31.44% ±2.45 SEM) compared to GFP-expressing cells (24.40% ±1.40 SEM; n = 5, p = 0.0369). In case of MDMX-ALT2 over-expression in HCT116 wildtype cells, there is no significant change in percentage of cells in G1 phase compared to negative control GFP-expressing cells (p =  0.4389). HCT116 cells that are for p53 (B) or p21 (C) do not show any differences in the percentage of cells in any of the cell cycle phases upon over-expression of GFP or MDM2-ALT1 or MDMX-ALT2. D. Representative immuno blot showing expression of p53, p21 and loading control GAPDH in HCT116 wt, p53−/− and p21−/− cells.

Mentions: As p21 is a strong inhibitor of the transition from G1 to S phase during the cell cycle, we examined the effects of p21 upregulation on cell cycle progression in MCF7 cells expressing MDM2-ALT1 or MDMX-ALT2 in comparison with cells expressing LacZ. Propidium iodide staining of the transfected cells was used to identify the number of cells populating the various phases of the cell cycle. We observed that the percentage of cells in G1 phase was significantly higher in samples expressing MDM2-ALT1 compared to LacZ expressing samples in a manner concordant with the upregulation of p21 levels upon MDM2-ALT1 expression (Fig S3A). However, this difference was not significant in the cells expressing MDMX-ALT2 (Fig S3A). These results indicate that upon MDM2-ALT1 expression at least, the upregulation of p21 resulting from the stabilization of p53 can have functional consequences as evidenced by the G1-phase cell cycle arrest in cells expressing MDM2-ALT1. To determine whether the effect on cell cycle progression is p53-dependent, we utilized H1299 cells that are p53- and show that the over-expression of neither MDM2-ALT1 nor MDMX-ALT2 results in G1 phase cell cycle arrest (Fig S3B). These experiments establish the p53-dependence of MDM2-ALT1 mediated G1 phase cell cycle arrest (compare the effects on MCF7 cells with wild-type p53 and H1299 cells, which are p53-negative). However, it is possible that these effects arise from genetic differences between the breast carcinoma (MCF7) and non small-cell lung carcinoma (H1299) cell lines. To rule out such a possibility, we over-expressed GFP or MDM2-ALT1 or MDMX-ALT2 in HCT116 cells that are either wild-type (Fig 3A) or for p53 (Fig 3B). Indeed, even in these isotype matched cell-lines, we observed that the MDM2-ALT1 mediated G1 phase cell cycle arrest is dependent on the presence of p53 as only HCT116 cells wild-type for p53 (Fig 3A) and not those for p53 (Fig 3B), showed significantly higher percentage of cells stalled in G1 phase compared to GFP expressing cells. Here also the expression of MDMX-ALT2 did not show significant changes in cell cycle progression compared to GFP (Fig 3).


Stress-induced alternative splice forms of MDM2 and MDMX modulate the p53-pathway in distinct ways.

Jacob AG, Singh RK, Comiskey DF, Rouhier MF, Mohammad F, Bebee TW, Chandler DS - PLoS ONE (2014)

MDM2-ALT1 over-expression leads to G1 phase cell cycle arrest in a p53-dependent and p21-dependent manner.A. HCT116 wild-type (wt), B. HCT116 p53−/− and C. HCT116 p21−/− cells were transfected with myc-tagged GFP or MDM2-ALT1 (2Alt1) or MDMX-ALT2 (XAlt2), harvested 24 hours post-transfection and stained with propidium iodide solution and sorted for DNA content. The bar graphs represent the percentage of cells in the various phases of the cell cycle. Error bars represent the standard error mean from at least 3 independent experiments. HCT116 cells that are wildtype (A) show significantly higher percentage of cells in G1-phase upon MDM2-ALT1 expression (31.44% ±2.45 SEM) compared to GFP-expressing cells (24.40% ±1.40 SEM; n = 5, p = 0.0369). In case of MDMX-ALT2 over-expression in HCT116 wildtype cells, there is no significant change in percentage of cells in G1 phase compared to negative control GFP-expressing cells (p =  0.4389). HCT116 cells that are  for p53 (B) or p21 (C) do not show any differences in the percentage of cells in any of the cell cycle phases upon over-expression of GFP or MDM2-ALT1 or MDMX-ALT2. D. Representative immuno blot showing expression of p53, p21 and loading control GAPDH in HCT116 wt, p53−/− and p21−/− cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4126728&req=5

pone-0104444-g003: MDM2-ALT1 over-expression leads to G1 phase cell cycle arrest in a p53-dependent and p21-dependent manner.A. HCT116 wild-type (wt), B. HCT116 p53−/− and C. HCT116 p21−/− cells were transfected with myc-tagged GFP or MDM2-ALT1 (2Alt1) or MDMX-ALT2 (XAlt2), harvested 24 hours post-transfection and stained with propidium iodide solution and sorted for DNA content. The bar graphs represent the percentage of cells in the various phases of the cell cycle. Error bars represent the standard error mean from at least 3 independent experiments. HCT116 cells that are wildtype (A) show significantly higher percentage of cells in G1-phase upon MDM2-ALT1 expression (31.44% ±2.45 SEM) compared to GFP-expressing cells (24.40% ±1.40 SEM; n = 5, p = 0.0369). In case of MDMX-ALT2 over-expression in HCT116 wildtype cells, there is no significant change in percentage of cells in G1 phase compared to negative control GFP-expressing cells (p =  0.4389). HCT116 cells that are for p53 (B) or p21 (C) do not show any differences in the percentage of cells in any of the cell cycle phases upon over-expression of GFP or MDM2-ALT1 or MDMX-ALT2. D. Representative immuno blot showing expression of p53, p21 and loading control GAPDH in HCT116 wt, p53−/− and p21−/− cells.
Mentions: As p21 is a strong inhibitor of the transition from G1 to S phase during the cell cycle, we examined the effects of p21 upregulation on cell cycle progression in MCF7 cells expressing MDM2-ALT1 or MDMX-ALT2 in comparison with cells expressing LacZ. Propidium iodide staining of the transfected cells was used to identify the number of cells populating the various phases of the cell cycle. We observed that the percentage of cells in G1 phase was significantly higher in samples expressing MDM2-ALT1 compared to LacZ expressing samples in a manner concordant with the upregulation of p21 levels upon MDM2-ALT1 expression (Fig S3A). However, this difference was not significant in the cells expressing MDMX-ALT2 (Fig S3A). These results indicate that upon MDM2-ALT1 expression at least, the upregulation of p21 resulting from the stabilization of p53 can have functional consequences as evidenced by the G1-phase cell cycle arrest in cells expressing MDM2-ALT1. To determine whether the effect on cell cycle progression is p53-dependent, we utilized H1299 cells that are p53- and show that the over-expression of neither MDM2-ALT1 nor MDMX-ALT2 results in G1 phase cell cycle arrest (Fig S3B). These experiments establish the p53-dependence of MDM2-ALT1 mediated G1 phase cell cycle arrest (compare the effects on MCF7 cells with wild-type p53 and H1299 cells, which are p53-negative). However, it is possible that these effects arise from genetic differences between the breast carcinoma (MCF7) and non small-cell lung carcinoma (H1299) cell lines. To rule out such a possibility, we over-expressed GFP or MDM2-ALT1 or MDMX-ALT2 in HCT116 cells that are either wild-type (Fig 3A) or for p53 (Fig 3B). Indeed, even in these isotype matched cell-lines, we observed that the MDM2-ALT1 mediated G1 phase cell cycle arrest is dependent on the presence of p53 as only HCT116 cells wild-type for p53 (Fig 3A) and not those for p53 (Fig 3B), showed significantly higher percentage of cells stalled in G1 phase compared to GFP expressing cells. Here also the expression of MDMX-ALT2 did not show significant changes in cell cycle progression compared to GFP (Fig 3).

Bottom Line: We show here that MDM2-ALT1 is capable of binding full-length MDMX as well as full-length MDM2.Moreover, MDM2-ALT1 expression causes cell cycle arrest in the G1 phase in a p53 and p21 dependent manner, which is consistent with the increased levels of p21.In summary, our study shows that the stress-inducible alternative splice forms MDM2-ALT1 and MDMX-ALT2 are important modifiers of the p53 pathway and present a potential mechanism to tailor the p53-mediated cellular stress response.

View Article: PubMed Central - PubMed

Affiliation: From the Center for Childhood Cancer at the Research Institute at Nationwide Children's Hospital, Columbus, Ohio, United States of America; The Department of Pediatrics, and Molecular, Cellular and Developmental Biology (MCDB) program, The Ohio State University, Columbus, Ohio, United States of America; Center for RNA Biology, Wexner Medical Center, The Ohio State University, Columbus, Ohio, United States of America.

ABSTRACT
MDM2 and MDMX are the chief negative regulators of the tumor-suppressor protein p53 and are essential for maintaining homeostasis within the cell. In response to genotoxic stress and also in several cancer types, MDM2 and MDMX are alternatively spliced. The splice variants MDM2-ALT1 and MDMX-ALT2 lack the p53-binding domain and are incapable of negatively regulating p53. However, they retain the RING domain that facilitates dimerization of the full-length MDM proteins. Concordantly, MDM2-ALT1 has been shown to lead to the stabilization of p53 through its interaction with and inactivation of full-length MDM2. The impact of MDM2-ALT1 expression on the p53 pathway and the nature of its interaction with MDMX remain unclear. Also, the role of the architecturally similar MDMX-ALT2 and its influence of the MDM2-MDMX-p53 axis are yet to be elucidated. We show here that MDM2-ALT1 is capable of binding full-length MDMX as well as full-length MDM2. Additionally, we demonstrate that MDMX-ALT2 is able to dimerize with both full-length MDMX and MDM2 and that the expression of MDM2-ALT1 and MDMX-ALT2 leads to the upregulation of p53 protein, and also of its downstream target p21. Moreover, MDM2-ALT1 expression causes cell cycle arrest in the G1 phase in a p53 and p21 dependent manner, which is consistent with the increased levels of p21. Finally we present evidence that MDM2-ALT1 and MDMX-ALT2 expression can activate subtly distinct subsets of p53-transcriptional targets implying that these splice variants can modulate the p53 tumor suppressor pathway in unique ways. In summary, our study shows that the stress-inducible alternative splice forms MDM2-ALT1 and MDMX-ALT2 are important modifiers of the p53 pathway and present a potential mechanism to tailor the p53-mediated cellular stress response.

Show MeSH
Related in: MedlinePlus