Specific pharmacological dimerization of KDR in lentivirally transduced human hematopoietic cells activates anti-apoptotic and proliferative mechanisms

FASEB J. 2005 Oct;19(12):1752-4. doi: 10.1096/fj.05-4006fje. Epub 2005 Aug 2.

Abstract

Selective and regulatable expansion of transduced cells could augment gene therapy for many disorders. The activation of modified growth factor receptors via synthetic chemical inducers of dimerization allows for the coordinated growth of transduced cells. This system can also provide information on specific receptor-mediated signaling without interference from other family members. Although several receptor subunits have been investigated in this context, little is known about the precise molecular events associated with dimerizer-initiated signaling. We have constructed and expressed an AP20187-regulated KDR chimeric receptor in human TF1 cells and analyzed activation of this gene switch using functional, biochemical, and microarray analyses. When deprived of natural ligands, GM-CSF, interleukin-3, or erythropoietin, AP20187 prevented apoptosis of transduced TF1 cells, induced dose-dependent proliferation, and supported long-term growth. In addition, AP20187 stimulation activated the signaling molecules associated with mitogen-activated protein kinase and phosphatidyl-inositol 3-kinase/Akt pathways. Microarray analysis determined that a number of transcripts involved in a variety of cellular processes were differentially expressed. Notably, mRNAs affiliated with heat stress, including Hsp70 and Hsp105, were up-regulated. Functional assays showed that Hsp70 and Hsp105 protected transduced TF1 cells from apoptosis and premature senescence, in part through regulation of Akt. These observations delineate specific roles for kinase insert domain-containing receptor, or KDR, signaling and suggest strategies to endow genetically modified cells with a survival advantage enabling the generation of adequate cell numbers for therapeutic outcomes.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Apoptosis*
  • Cell Line
  • Cell Line, Tumor
  • Cell Proliferation
  • Cell Transformation, Viral
  • Dimerization
  • Dose-Response Relationship, Drug
  • Down-Regulation
  • Erythropoietin / metabolism
  • Flow Cytometry
  • Gene Transfer Techniques
  • Genetic Therapy
  • Genetic Vectors
  • Granulocyte-Macrophage Colony-Stimulating Factor / metabolism
  • Green Fluorescent Proteins / metabolism
  • HSP110 Heat-Shock Proteins / metabolism
  • HSP70 Heat-Shock Proteins / metabolism
  • HeLa Cells
  • Hematopoietic Stem Cells / cytology*
  • Humans
  • Immunoblotting
  • Interleukin-3 / metabolism
  • Lentivirus / genetics*
  • Oligonucleotide Array Sequence Analysis
  • Phosphorylation
  • Protein Structure, Tertiary
  • RNA, Messenger / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Signal Transduction
  • Tacrolimus / analogs & derivatives
  • Tacrolimus / pharmacology
  • Transfection
  • Up-Regulation
  • Vascular Endothelial Growth Factor Receptor-2 / chemistry*
  • Vascular Endothelial Growth Factor Receptor-2 / metabolism*

Substances

  • AP20187
  • HSP110 Heat-Shock Proteins
  • HSP70 Heat-Shock Proteins
  • HSPH1 protein, human
  • Interleukin-3
  • RNA, Messenger
  • Erythropoietin
  • Green Fluorescent Proteins
  • Granulocyte-Macrophage Colony-Stimulating Factor
  • Vascular Endothelial Growth Factor Receptor-2
  • Tacrolimus