title: Retroviral mediated gene transfer of Flt3 ligand enhances proliferation and MAP kinase activity of AML5 cells.
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 10(6) cfu/mL after shuttle packaging through ecotropic packaging cells were isolated. AML5 cells were cultured overnight with L(Flt3-L)SN retroviral supernatant, 8 micrograms/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 pg/mL) by ELISA assay of conditioned medium from transduced cells, but Flt3-L 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.
authors.affiliation: Walther Oncology Center, Department of Medicine, Indiana University School of Medicine, Indianapolis 46202-5121, USA.
authors.forename: S E
authors.initials: SE
authors.lastname: Braun
authors.forename: S M
authors.initials: SM
authors.lastname: Aronica
authors.forename: Y
authors.initials: Y
authors.lastname: Ge
authors.forename: H
authors.initials: H
authors.lastname: Takahira
authors.forename: M
authors.initials: M
authors.lastname: Etienne-Julan
authors.forename: L
authors.initials: L
authors.lastname: Lu
authors.forename: M D
authors.initials: MD
authors.lastname: Minden
authors.forename: S D
authors.initials: SD
authors.lastname: Lyman
authors.forename: H E
authors.initials: HE
authors.lastname: Broxmeyer
mesh_terms: D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics*; D002455:Cell Division / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D008565:Membrane Proteins / Q000235:genetics*; D012190:Retroviridae; D014407:Tumor Cells, Cultured
chemical_list: D008565:Membrane Proteins; C084881:flt3 ligand protein; D017871:Calcium-Calmodulin-Dependent Protein Kinases