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p53-Independent cell cycle and erythroid differentiation defects in murine embryonic stem cells haploinsufficient for Diamond Blackfan anemia-proteins: RPS19 versus RPL5.

Singh SA, Goldberg TA, Henson AL, Husain-Krautter S, Nihrane A, Blanc L, Ellis SR, Lipton JM, Liu JM - PLoS ONE (2014)

Bottom Line: When embryoid bodies were further differentiated to primitive erythroid colonies, both mutants exhibited a marked reduction in colony formation, which was again nonspecifically rescued by p53 inhibition.Concordantly, Rpl5 mutant ES cells had a more pronounced growth defect in liquid culture compared to the Rps19 mutant cells.We conclude that the defects in our RPS19 and RPL5 haploinsufficient mouse ES cells are not adequately explained by p53 stabilization, as p53 knockdown appears to increase the growth and differentiation potential of both parental and mutant cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Hofstra North Shore-LIJ School of Medicine, Hempstead, New York, United States of America ; The Feinstein Institute for Medical Research, Manhasset, New York, United States of America ; Division of Hematology/Oncology, Steven and Alexandra Cohen Children's Medical Center of New York, New Hyde Park, New York, United States of America ; Department of Pediatrics, Hofstra North Shore-LIJ School of Medicine, Hempstead, New York, United States of America.

ABSTRACT
Diamond Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome caused by ribosomal protein haploinsufficiency. DBA exhibits marked phenotypic variability, commonly presenting with erythroid hypoplasia, less consistently with non-erythroid features. The p53 pathway, activated by abortive ribosome assembly, is hypothesized to contribute to the erythroid failure of DBA. We studied murine embryonic stem (ES) cell lines harboring a gene trap mutation in a ribosomal protein gene, either Rps19 or Rpl5. Both mutants exhibited ribosomal protein haploinsufficiency and polysome defects. Rps19 mutant ES cells showed significant increase in p53 protein expression, however, there was no similar increase in the Rpl5 mutant cells. Embryoid body formation was diminished in both mutants but nonspecifically rescued by knockdown of p53. When embryoid bodies were further differentiated to primitive erythroid colonies, both mutants exhibited a marked reduction in colony formation, which was again nonspecifically rescued by p53 inhibition. Cell cycle analyses were normal in Rps19 mutant ES cells, but there was a significant delay in the G2/M phase in the Rpl5 mutant cells, which was unaffected by p53 knockdown. Concordantly, Rpl5 mutant ES cells had a more pronounced growth defect in liquid culture compared to the Rps19 mutant cells. We conclude that the defects in our RPS19 and RPL5 haploinsufficient mouse ES cells are not adequately explained by p53 stabilization, as p53 knockdown appears to increase the growth and differentiation potential of both parental and mutant cells. Our studies demonstrate that gene trap mouse ES cells are useful tools to study the pathogenesis of DBA.

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Rpl5 mutant ES cells exhibit a p53-independent cell cycle arrest.Cell cycle analyses were performed by fixing ES cells with 70% ethanol, followed by staining with PI solution containing RNase A. Quantification of cell cycle phases (A), along with flow cytometry profiles (B) of Rps19 mutant ES cells show no difference, compared to the parent. In contrast, the cell cycle profile of the Rpl5 mutant ES cells exhibited a three-fold increase in the G2 phase with a concomitant decrease in the G1 and S phases, consistent with a delayed G2 phase transition (A, C) (three independent pooled experiments). Stable transfection of the Rpl5 mutant with a vector containing Rpl5 cDNA showed complete correction of the cell cycle defect; however, siRNA knockdown of p53 was unable to rescue the defect (D).
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pone-0089098-g005: Rpl5 mutant ES cells exhibit a p53-independent cell cycle arrest.Cell cycle analyses were performed by fixing ES cells with 70% ethanol, followed by staining with PI solution containing RNase A. Quantification of cell cycle phases (A), along with flow cytometry profiles (B) of Rps19 mutant ES cells show no difference, compared to the parent. In contrast, the cell cycle profile of the Rpl5 mutant ES cells exhibited a three-fold increase in the G2 phase with a concomitant decrease in the G1 and S phases, consistent with a delayed G2 phase transition (A, C) (three independent pooled experiments). Stable transfection of the Rpl5 mutant with a vector containing Rpl5 cDNA showed complete correction of the cell cycle defect; however, siRNA knockdown of p53 was unable to rescue the defect (D).

Mentions: Cell cycle analyses at the ES cell stage showed a difference between the Rps19 mutant and the Rpl5 mutant. The cell cycle status of the Rps19 mutant was essentially unchanged, compared to its parent cells (Figure 5A &B). On the other hand, the Rpl5 mutant exhibited a significant increase in the percentage of cells in the G2/M phase with a concomitant decrease of cells in the G1 and S phases, consistent with a G2 cell cycle delay (Figure 5C &D). The delay observed in the cell cycle was rescued by transfection of the mutant with wild type Rpl5 cDNA. To get further insights into the putative involvement of p53 in the observed cell cycle defect, p53 was knocked down in the Rpl5 mutant. However, despite the high efficiency of the knockdown (97%), as evaluated by qRT-PCR, no difference was observed in the cell cycle. This data strongly suggests that the G2 phase defect observed is due to a mechanism independent of p53.


p53-Independent cell cycle and erythroid differentiation defects in murine embryonic stem cells haploinsufficient for Diamond Blackfan anemia-proteins: RPS19 versus RPL5.

Singh SA, Goldberg TA, Henson AL, Husain-Krautter S, Nihrane A, Blanc L, Ellis SR, Lipton JM, Liu JM - PLoS ONE (2014)

Rpl5 mutant ES cells exhibit a p53-independent cell cycle arrest.Cell cycle analyses were performed by fixing ES cells with 70% ethanol, followed by staining with PI solution containing RNase A. Quantification of cell cycle phases (A), along with flow cytometry profiles (B) of Rps19 mutant ES cells show no difference, compared to the parent. In contrast, the cell cycle profile of the Rpl5 mutant ES cells exhibited a three-fold increase in the G2 phase with a concomitant decrease in the G1 and S phases, consistent with a delayed G2 phase transition (A, C) (three independent pooled experiments). Stable transfection of the Rpl5 mutant with a vector containing Rpl5 cDNA showed complete correction of the cell cycle defect; however, siRNA knockdown of p53 was unable to rescue the defect (D).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3928369&req=5

pone-0089098-g005: Rpl5 mutant ES cells exhibit a p53-independent cell cycle arrest.Cell cycle analyses were performed by fixing ES cells with 70% ethanol, followed by staining with PI solution containing RNase A. Quantification of cell cycle phases (A), along with flow cytometry profiles (B) of Rps19 mutant ES cells show no difference, compared to the parent. In contrast, the cell cycle profile of the Rpl5 mutant ES cells exhibited a three-fold increase in the G2 phase with a concomitant decrease in the G1 and S phases, consistent with a delayed G2 phase transition (A, C) (three independent pooled experiments). Stable transfection of the Rpl5 mutant with a vector containing Rpl5 cDNA showed complete correction of the cell cycle defect; however, siRNA knockdown of p53 was unable to rescue the defect (D).
Mentions: Cell cycle analyses at the ES cell stage showed a difference between the Rps19 mutant and the Rpl5 mutant. The cell cycle status of the Rps19 mutant was essentially unchanged, compared to its parent cells (Figure 5A &B). On the other hand, the Rpl5 mutant exhibited a significant increase in the percentage of cells in the G2/M phase with a concomitant decrease of cells in the G1 and S phases, consistent with a G2 cell cycle delay (Figure 5C &D). The delay observed in the cell cycle was rescued by transfection of the mutant with wild type Rpl5 cDNA. To get further insights into the putative involvement of p53 in the observed cell cycle defect, p53 was knocked down in the Rpl5 mutant. However, despite the high efficiency of the knockdown (97%), as evaluated by qRT-PCR, no difference was observed in the cell cycle. This data strongly suggests that the G2 phase defect observed is due to a mechanism independent of p53.

Bottom Line: When embryoid bodies were further differentiated to primitive erythroid colonies, both mutants exhibited a marked reduction in colony formation, which was again nonspecifically rescued by p53 inhibition.Concordantly, Rpl5 mutant ES cells had a more pronounced growth defect in liquid culture compared to the Rps19 mutant cells.We conclude that the defects in our RPS19 and RPL5 haploinsufficient mouse ES cells are not adequately explained by p53 stabilization, as p53 knockdown appears to increase the growth and differentiation potential of both parental and mutant cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Hofstra North Shore-LIJ School of Medicine, Hempstead, New York, United States of America ; The Feinstein Institute for Medical Research, Manhasset, New York, United States of America ; Division of Hematology/Oncology, Steven and Alexandra Cohen Children's Medical Center of New York, New Hyde Park, New York, United States of America ; Department of Pediatrics, Hofstra North Shore-LIJ School of Medicine, Hempstead, New York, United States of America.

ABSTRACT
Diamond Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome caused by ribosomal protein haploinsufficiency. DBA exhibits marked phenotypic variability, commonly presenting with erythroid hypoplasia, less consistently with non-erythroid features. The p53 pathway, activated by abortive ribosome assembly, is hypothesized to contribute to the erythroid failure of DBA. We studied murine embryonic stem (ES) cell lines harboring a gene trap mutation in a ribosomal protein gene, either Rps19 or Rpl5. Both mutants exhibited ribosomal protein haploinsufficiency and polysome defects. Rps19 mutant ES cells showed significant increase in p53 protein expression, however, there was no similar increase in the Rpl5 mutant cells. Embryoid body formation was diminished in both mutants but nonspecifically rescued by knockdown of p53. When embryoid bodies were further differentiated to primitive erythroid colonies, both mutants exhibited a marked reduction in colony formation, which was again nonspecifically rescued by p53 inhibition. Cell cycle analyses were normal in Rps19 mutant ES cells, but there was a significant delay in the G2/M phase in the Rpl5 mutant cells, which was unaffected by p53 knockdown. Concordantly, Rpl5 mutant ES cells had a more pronounced growth defect in liquid culture compared to the Rps19 mutant cells. We conclude that the defects in our RPS19 and RPL5 haploinsufficient mouse ES cells are not adequately explained by p53 stabilization, as p53 knockdown appears to increase the growth and differentiation potential of both parental and mutant cells. Our studies demonstrate that gene trap mouse ES cells are useful tools to study the pathogenesis of DBA.

Show MeSH
Related in: MedlinePlus