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Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2319-2332
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- and Transforming Growth Factor- 1
Li Lu,
Michael C. Heinrich,
Li-Sheng Wang,
Mu-Shui Dai,
Amy J. Zigler,
Lin Chai, and
Hal E. Broxmeyer
From the Departments of Microbiology and Immunology, Medicine
(Hematology/Oncology), the Walther Oncology Center, Indiana University
School of Medicine, Indianapolis, IN; the Walther Cancer Institute,
Indianapolis, IN; and the Division of Hematology and Medical Oncology,
Department of Medicine, and Portland Veterans Affairs Medical Center,
Portland, OR.
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 CD34+++
kitLo/ 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- 1 and tumor necrosis factor- .
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.
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