title: p53-Independent cell cycle and erythroid differentiation defects in murine embryonic stem cells haploinsufficient for Diamond Blackfan anemia-proteins: RPS19 versus RPL5.
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.
authors.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.
authors.forename: Sharon A
authors.initials: SA
authors.lastname: Singh
authors.affiliation: 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.
authors.forename: Tracie A
authors.initials: TA
authors.lastname: Goldberg
authors.affiliation: Department of Biochemistry, University of Louisville, Louisville, Kentucky, United States of America.
authors.forename: Adrianna L
authors.initials: AL
authors.lastname: Henson
authors.affiliation: The Feinstein Institute for Medical Research, Manhasset, New York, United States of America.
authors.forename: Sehba
authors.initials: S
authors.lastname: Husain-Krautter
authors.affiliation: The Feinstein Institute for Medical Research, Manhasset, New York, United States of America.
authors.forename: Abdallah
authors.initials: A
authors.lastname: Nihrane
authors.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.
authors.forename: Lionel
authors.initials: L
authors.lastname: Blanc
authors.affiliation: Department of Biochemistry, University of Louisville, Louisville, Kentucky, United States of America.
authors.forename: Steven R
authors.initials: SR
authors.lastname: Ellis
authors.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.
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Lipton
authors.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.
authors.forename: Johnson M
authors.initials: JM
authors.lastname: Liu
mesh_terms: D029503:Anemia, Diamond-Blackfan / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002453:Cell Cycle / Q000502:physiology; D002454:Cell Differentiation / Q000502:physiology*; D017931:DNA Primers / Q000235:genetics; D004195:Disease Models, Animal; D053595:Embryonic Stem Cells / Q000502:physiology*; D041905:Erythroid Cells / Q000166:cytology*; D057895:Haploinsufficiency; D051379:Mice; D060888:Real-Time Polymerase Chain Reaction; D012269:Ribosomal Proteins / Q000235:genetics* / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: D017931:DNA Primers; D012269:Ribosomal Proteins; C531064:Rps19 protein, mouse; D016159:Tumor Suppressor Protein p53; C029742:ribosomal protein L5