Retroviral mediated gene transfer of Flt3 ligand enhances proliferation and MAP kinase activity of AML5 cells

S. E. Braun, S. M. Aronica, Y. Ge, H. Takahira, M. Etienne-Julan, L. Lu, M. D. Minden, S. D. Lyman, H. E. Broxmeyer

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Abstract

Flt3/flk-2 ligand (Flt3-L) co-stimulates and synergizes with cytokines such as granulocyte-macrophage colony stimulating factor, granulocyte colony stimulating factor (G-CSF), interleukin-3 (IL-3), and erythropoietin in the proliferation of bone marrow and cord blood hematopoietic stem and progenitor cells. To study the biological effects of Flt3-L on the Flt3-L responsive AML5 cell line, the retroviral vector L(Flt3-L)SN was constructed based on the vector LXSN, but containing the human Flt3-L cDNA transcriptionally regulated by the Mo-MLV LTR. High-titer amphotropic producer cells that generated 106 cfu/mL after shuttle packaging through ecotropic packaging cells were isolated. AML5 cells were cultured overnight with L(Flt3-L)SN retroviral supernatant, 8 μg/mL polybrene, and 100 U/mL G-CSF, and expanded 1 week in medium with G-CSF. Transduced cells were selected in medium containing 0.4 mg/mL G418 and then in medium with 1.0 mg/mL G418. Retroviral mediated gene transfer in G418-resistant cells was confirmed after amplification by PCR of neo-specific sequences in genomic DNA. Northern blot analysis demonstrated L(Flt3-L)SN mRNA expression. Soluble Flt3-L was undetectable (<100 μg/mL) by ELISA assay of conditioned medium from transduced cells, but Flt3-F was detected on the surface of AML5 cells by FACS analysis. Cells were plated in colony assay with and without 100 ng/mL Flt3-L, 100 U/mL G-CSF, and the combination. Gene transfer or growth factor treatment increased somewhat the clonogenicity of the nontransduced AML5 cells. More strikingly, L(Flt3-L)SN and each growth factor combination greatly increased the size of the resultant colonies such that the size of colonies from AML5/Flt3-L cells without added growth factor approximated that of the AML5 cells stimulated by exogenous soluble Flt3-L. Moreover, MAP kinase activity in L(Flt3-L)SN-transduced cells cultured without soluble Flt3-L was increased to the level induced in control cells by soluble Flt3-L. These results indicate that retroviral mediated gene transfer and autologous expression of the Flt3-L enhances growth and intracellular signaling of AML5 cells, information that should be of value for studying the effects of Flt3-L on immature subsets of primary hematopoietic stem and progenitor cells.

Original languageEnglish (US)
Pages (from-to)51-56
Number of pages6
JournalExperimental Hematology
Volume25
Issue number1
StatePublished - Feb 17 1997

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Keywords

  • AML5 cells
  • cell proliferation
  • Flt3 ligand
  • gene transfer
  • MAP kinase

ASJC Scopus subject areas

  • Cancer Research
  • Cell Biology
  • Genetics
  • Hematology
  • Oncology
  • Transplantation

Cite this

Braun, S. E., Aronica, S. M., Ge, Y., Takahira, H., Etienne-Julan, M., Lu, L., Minden, M. D., Lyman, S. D., & Broxmeyer, H. E. (1997). Retroviral mediated gene transfer of Flt3 ligand enhances proliferation and MAP kinase activity of AML5 cells. Experimental Hematology, 25(1), 51-56.