title: Reduction in levels of the cyclin-dependent kinase inhibitor p27(kip-1) coupled with transforming growth factor beta neutralization induces cell-cycle entry and increases retroviral transduction of primitive human hematopoietic cells.
abstract: Successful gene therapy depends on stable transduction of hematopoietic stem cells. Target cells must cycle to allow integration of Moloney-based retroviral vectors, yet hematopoietic stem cells are quiescent. Cells can be held in quiescence by intracellular cyclin-dependent kinase inhibitors. The cyclin-dependent kinase inhibitor p15(INK4B) blocks association of cyclin-dependent kinase (CDK)4/cyclin D and p27(kip-1) blocks activity of CDK2/cyclin A and CDK2/cyclin E, complexes that are mandatory for cell-cycle progression. Antibody neutralization of beta transforming growth factor (TGFbeta) in serum-free medium decreased levels of p15(INK4B) and increased colony formation and retroviral-mediated transduction of primary human CD34(+) cells. Although TGFbeta neutralization increased colony formation from more primitive, noncycling hematopoietic progenitors, no increase in M-phase-dependent, retroviral-mediated transduction was observed. Transduction of the primitive cells was augmented by culture in the presence of antisense oligonucleotides to p27(kip-1) coupled with TGFbeta-neutralizing antibodies. The transduced cells engrafted immune-deficient mice with no alteration in human hematopoietic lineage development. We conclude that neutralization of TGFbeta, plus reduction in levels of the cyclin-dependent kinase inhibitor p27, allows transduction of primitive and quiescent hematopoietic progenitor populations.
authors.affiliation: Division of Research Immunology/Bone Marrow Transplantation, Childrens Hospital Los Angeles, CA 90027, USA.
authors.forename: M A
authors.initials: MA
authors.lastname: Dao
authors.forename: N
authors.initials: N
authors.lastname: Taylor
authors.forename: J A
authors.initials: JA
authors.lastname: Nolta
mesh_terms: D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D015703:Antigens, CD / Q000032:analysis; D018952:Antigens, CD34 / Q000032:analysis; D001854:Bone Marrow Cells / Q000166:cytology; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology*; D018797:Cell Cycle Proteins; D002999:Clone Cells; D003114:Colony-Forming Units Assay; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D018189:Immunomagnetic Separation; D051379:Mice; D008819:Mice, Nude; D016513:Mice, SCID; D008869:Microtubule-Associated Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008979:Moloney murine leukemia virus; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D013601:T-Lymphocytes / Q000166:cytology; D013873:Thionucleotides; D014161:Transduction, Genetic; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000276:immunology / Q000494:pharmacology*; D025521:Tumor Suppressor Proteins
chemical_list: D000906:Antibodies; D015703:Antigens, CD; D018952:Antigens, CD34; C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; D008869:Microtubule-Associated Proteins; D016376:Oligonucleotides, Antisense; D013873:Thionucleotides; D016212:Transforming Growth Factor beta; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases