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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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Embryoid body (EB) formation is impaired in both Rps19 and Rpl5 mutants.ES cells were differentiated into EBs and scored on day 4 to assess total number of EBs formed. Both mutants showed a reduction in EB formation when compared to the parental cells (3 independent experiments).
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pone-0089098-g002: Embryoid body (EB) formation is impaired in both Rps19 and Rpl5 mutants.ES cells were differentiated into EBs and scored on day 4 to assess total number of EBs formed. Both mutants showed a reduction in EB formation when compared to the parental cells (3 independent experiments).

Mentions: Functional defects intrinsic to these mutant cells were assessed by in vitro differentiation assays. Primary differentiation experiments were first used to measure the efficiency of embryoid body (EB) formation, defined as the number of EBs formed per ES cell plated, an in vitro representation of the differentiation potential of the mutant cells. The efficiency of embryoid body generation from ES cells was significantly reduced in both Rps19 and Rpl5 mutant cells (Figure 2).


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)

Embryoid body (EB) formation is impaired in both Rps19 and Rpl5 mutants.ES cells were differentiated into EBs and scored on day 4 to assess total number of EBs formed. Both mutants showed a reduction in EB formation when compared to the parental cells (3 independent experiments).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0089098-g002: Embryoid body (EB) formation is impaired in both Rps19 and Rpl5 mutants.ES cells were differentiated into EBs and scored on day 4 to assess total number of EBs formed. Both mutants showed a reduction in EB formation when compared to the parental cells (3 independent experiments).
Mentions: Functional defects intrinsic to these mutant cells were assessed by in vitro differentiation assays. Primary differentiation experiments were first used to measure the efficiency of embryoid body (EB) formation, defined as the number of EBs formed per ES cell plated, an in vitro representation of the differentiation potential of the mutant cells. The efficiency of embryoid body generation from ES cells was significantly reduced in both Rps19 and Rpl5 mutant cells (Figure 2).

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