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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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Primitive erythropoiesis is defective in Rps19 and Rpl5 mutants.Day 4–5 EBs were harvested, made into single cell suspension, and added to primitive erythroid differentiation media. Colonies were scored on day 7. Both Rps19 mutant (A) and Rpl5 mutant (B) cell lines exhibited a severe defect in primitive erythroid colony formation. (Rpl5-5 independent pooled experiments, Rps19-3 independent pooled experiments).
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pone-0089098-g003: Primitive erythropoiesis is defective in Rps19 and Rpl5 mutants.Day 4–5 EBs were harvested, made into single cell suspension, and added to primitive erythroid differentiation media. Colonies were scored on day 7. Both Rps19 mutant (A) and Rpl5 mutant (B) cell lines exhibited a severe defect in primitive erythroid colony formation. (Rpl5-5 independent pooled experiments, Rps19-3 independent pooled experiments).

Mentions: Primitive erythropoiesis assays, performed on day 4–5 EBs, and definitive erythropoiesis assays, performed on day 7–11 EBs, produced morphologically distinct erythroid colonies (Figure S2A). Hemoglobin qRT-PCR was used to confirm the identity of these colonies. As expected, primitive erythroid colonies showed a markedly higher ratio of mouse embryonic hemoglobin (Hbb-βh1) mRNA expression to mouse adult hemoglobin (Hbb-β1) mRNA expression, compared with definitive BFU-Es (Figure S2B). There were less definitive erythroid colonies formed in the Rps19 and Rpl5 mutants compared to the parental lines (Figure S3), consistent with the failure of definitive erythropoiesis in the majority of DBA patients. Primitive erythropoiesis, assessed by the total number of colonies formed, was markedly decreased in both the Rps19 and Rpl5 mutants (Figure 3A and B). The significance of this failure of primitive erythropoiesis is unclear, as the majority of DBA patients present postnatally.


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)

Primitive erythropoiesis is defective in Rps19 and Rpl5 mutants.Day 4–5 EBs were harvested, made into single cell suspension, and added to primitive erythroid differentiation media. Colonies were scored on day 7. Both Rps19 mutant (A) and Rpl5 mutant (B) cell lines exhibited a severe defect in primitive erythroid colony formation. (Rpl5-5 independent pooled experiments, Rps19-3 independent pooled experiments).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0089098-g003: Primitive erythropoiesis is defective in Rps19 and Rpl5 mutants.Day 4–5 EBs were harvested, made into single cell suspension, and added to primitive erythroid differentiation media. Colonies were scored on day 7. Both Rps19 mutant (A) and Rpl5 mutant (B) cell lines exhibited a severe defect in primitive erythroid colony formation. (Rpl5-5 independent pooled experiments, Rps19-3 independent pooled experiments).
Mentions: Primitive erythropoiesis assays, performed on day 4–5 EBs, and definitive erythropoiesis assays, performed on day 7–11 EBs, produced morphologically distinct erythroid colonies (Figure S2A). Hemoglobin qRT-PCR was used to confirm the identity of these colonies. As expected, primitive erythroid colonies showed a markedly higher ratio of mouse embryonic hemoglobin (Hbb-βh1) mRNA expression to mouse adult hemoglobin (Hbb-β1) mRNA expression, compared with definitive BFU-Es (Figure S2B). There were less definitive erythroid colonies formed in the Rps19 and Rpl5 mutants compared to the parental lines (Figure S3), consistent with the failure of definitive erythropoiesis in the majority of DBA patients. Primitive erythropoiesis, assessed by the total number of colonies formed, was markedly decreased in both the Rps19 and Rpl5 mutants (Figure 3A and B). The significance of this failure of primitive erythropoiesis is unclear, as the majority of DBA patients present postnatally.

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