ap20187 and Cell-Transformation--Viral

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.

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

Transient phosphorylation of the alpha-subunit of translation initiation factor 2 (eIF2alpha) represses translation and activates select gene expression under diverse stressful conditions. Defects in the eIF2alpha phosphorylation-dependent integrated stress response impair resistance to accumulation of malfolded proteins in the endoplasmic reticulum (ER stress), to oxidative stress and to nutrient deprivations. To study the hypothesized protective role of eIF2alpha phosphorylation in isolation of parallel stress signaling pathways, we fused the kinase domain of pancreatic endoplasmic reticulum kinase (PERK), an ER stress-inducible eIF2alpha kinase that is normally activated by dimerization, to a protein module that binds a small dimerizer molecule. The activity of this artificial eIF2alpha kinase, Fv2E-PERK, is subordinate to the dimerizer and is uncoupled from upstream stress signaling. Fv2E-PERK activation enhanced the expression of numerous stress-induced genes and protected cells from the lethal effects of oxidants, peroxynitrite donors and ER stress. Our findings indicate that eIF2alpha phosphorylation can initiate signaling in a cytoprotective gene expression pathway independently of other parallel stress-induced signals and that activation of this pathway can single-handedly promote a stress-resistant preconditioned state.

Topics: Activating Transcription Factor 4; Animals; Cell Line, Transformed; Cell Survival; Cell Transformation, Viral; Cytoprotection; Dimerization; eIF-2 Kinase; Endoplasmic Reticulum; Enzyme Activation; Enzyme Inhibitors; Eukaryotic Initiation Factor-2; Gene Expression Regulation; Glutamates; Mice; Oxidative Stress; Pancreas; Phosphorylation; Protein Biosynthesis; Protein Structure, Tertiary; Reactive Oxygen Species; Recombinant Fusion Proteins; Retroviridae; Signal Transduction; Tacrolimus; Thapsigargin; Trans-Activators; Tunicamycin