title: Retroviral-mediated gene transduction of c-kit into single hematopoietic progenitor cells from cord blood enhances erythroid colony formation and decreases sensitivity to inhibition by tumor necrosis factor-alpha and transforming growth factor-beta1.
abstract: The c-kit receptor and its ligand, steel factor (SLF), are critical for optimal hematopoiesis. We evaluated effects of transducing cord blood (CB) progenitor cells with a retrovirus encoding human c-kit cDNA. CD34(+) cells were sorted as a population or as 1 cell/well for cells expressing high levels of CD34 and different levels of c-kit (++,+,Lo/-), transduced and then cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), IL-6, erythropoietin (Epo) +/- SLF in the absence of serum. At a single-cell level, transduction with c-kit, but not with control (neo only), virus significantly increased colony formation, especially by erythroid and multipotential progenitors. The enhancing effect of c-kit transduction was inversely correlated with expression of c-kit protein before transduction. The greatest enhancing effects were noted in CD34KitLo+/- cells transduced with c-kit. The stimulating effect was apparent even in the absence of exogenously added SLF, but in the presence of GM-CSF, IL-3, IL-6, and Epo. Enzyme-linked immunosorbent assay (ELISA) of SLF protein, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of SLF mRNA expression in CD34+ cells, and use of neutralizing antibodies to SLF and/or c-kit suggested the presence of endogenous, although probably very low level, expression of SLF by these progenitor cells. Transduction of c-kit significantly decreased sensitivity of progenitor cells to the inhibitory effects of transforming growth factor-beta1 and tumor necrosis factor-alpha. c-kit-transduced cells had increased expression of c-kit protein and decreased spontaneous or cytokine-induced apoptosis. Our results suggest that transduced c-kit into selected progenitor cells can enhance proliferation and decrease apoptosis and that endogenous SLF may mediate this effect.
authors.affiliation: Department of Microbiology, Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN, USA.
authors.forename: L
authors.initials: L
authors.lastname: Lu
authors.forename: M C
authors.initials: MC
authors.lastname: Heinrich
authors.forename: L S
authors.initials: LS
authors.lastname: Wang
authors.forename: M S
authors.initials: MS
authors.lastname: Dai
authors.forename: A J
authors.initials: AJ
authors.lastname: Zigler
authors.forename: L
authors.initials: L
authors.lastname: Chai
authors.forename: H E
authors.initials: HE
authors.lastname: Broxmeyer
mesh_terms: D015703:Antigens, CD / Q000097:blood; D018952:Antigens, CD34 / Q000097:blood; D017209:Apoptosis; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D016207:Cytokines / Q000494:pharmacology*; D015672:Erythroid Precursor Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D004921:Erythropoietin / Q000494:pharmacology; D005312:Fetal Blood; D005822:Genetic Vectors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D015850:Interleukin-6 / Q000494:pharmacology; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D012190:Retroviridae; D019089:Stem Cell Factor / Q000494:pharmacology; D014162:Transfection / Q000379:methods*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D015703:Antigens, CD; D018952:Antigens, CD34; D016207:Cytokines; D007377:Interleukin-3; D015850:Interleukin-6; D011994:Recombinant Proteins; D019089:Stem Cell Factor; D016212:Transforming Growth Factor beta; D014409:Tumor Necrosis Factor-alpha; D004921:Erythropoietin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D019009:Proto-Oncogene Proteins c-kit