tacrolimus 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

Chronic active Epstein-Barr virus infection (CAEBV) is a heterogeneous EBV-related disorder, ranging from mild/moderate forms to rapidly lethal disorders. The lethal form of CAEBV is characterized by multiple organ failure, hemophagocytic syndrome, and development of lymphomas. Allogeneic stem cell transplantation is considered as the only potentially curative treatment for the lethal form of CAEBV, but it is not always desirable because of the high incidence of regimen-related toxicities. A 17-year-old female with CAEBV, who was refractory to conventional therapies and considered to be unable to receive a myeloablative regimen because of multiple organ dysfunction, underwent allogeneic nonmyeloablative stem cell transplantation (allo-NST) before developing a hematological malignancy. She has been well without any signs of CAEBV for 27 months after allo-NST, and we confirmed that specific cytotoxic T lymphocyte activity against EBV was reconstituted. This outcome suggests that allo-NST can control CAEBV by reconstituting the host immunity against EBV.

Topics: Adolescent; B-Lymphocytes; Cell Line, Transformed; Cell Transformation, Viral; Chronic Disease; Cyclosporine; DNA, Viral; Drug Resistance; Epstein-Barr Virus Infections; Etoposide; Female; Hepatitis, Viral, Human; Herpesvirus 4, Human; Humans; Immunosuppressive Agents; Multiple Organ Failure; Peripheral Blood Stem Cell Transplantation; T-Lymphocytes, Cytotoxic; Tacrolimus; Transplantation Conditioning; Transplantation, Homologous

Two intracellular signal pathways mediated by cAMP and protein kinase C (PKC) were involved in the regulation of FN gene expression (Lee et al., Exp. Mol. Med. 30: 240, 1998). In this study, a possible involvement of protein phosphatase-dependent pathways in the regulation of FN gene expression was investigated by using protein phosphatase type 2B (PP2B) inhibitors, cyclosporin A and ascomycin. Both cyclosporin A and ascomycin increased the levels of FN mRNA in WI-38 human lung fibroblasts and the SV40-transformed WI-38 cells but not in MC3T3-E1 osteoblasts. The expression of FN appears to increase from six hours up to 48 hours after treatment suggesting that it is not an immediate effect. In addition, this effect required a new protein synthesis. Neither cyclosporin A nor ascomycin affects the phorbol myristate acetate (PMA)-induced stimulation of FN gene expression and the same result occurred in vice versa suggesting the mechanism of PMA and cyclosporin A/ascomycin in the regulation of FN gene expression may share a common downstream pathway. Taken together, this study suggests that PP2B is involved in the regulation of FN gene expression in normal and transformed fibroblasts but not in osteoblasts.

Topics: Animals; Calcineurin Inhibitors; Cell Line, Transformed; Cell Transformation, Viral; Cyclosporine; Enzyme Inhibitors; Fibroblasts; Fibronectins; Gene Expression Regulation; Humans; Lung; Mice; Osteoblasts; Tacrolimus

A new lymphoma cell line, designated SUBL, was established from a Japanese patient with Epstein-Barr virus (EBV)-associated lymphoma, which developed during FK 506 therapy after liver transplantation. This cell line has undergone 80 passages over a period of 22 months. The cultured cells were positive for CD19, CD20, CD21, CD22, CD23, and HLA-DR, and negative for CD10 and surface immunoglobulins. Immunoglobulin gene analysis revealed rearrangements of JH and JK. T-cell antigens or T-cell receptor gene rearrangements were not observed on the cell line. The SUBL cells were positive for Epstein-Barr virus nuclear antigen (EBNA). The EBV genome was detected in the original tissue and the cell line by the in situ hybridization method. These data indicate that this cell line represents the B-cell lineage at a pre-B-cell stage. SUBL cells showed successful heterotransplantation to mice with severe combined immunodeficiency (SCID). Chromosomal analysis revealed the karyotype 46,XY,t(2;3)(p11;q27). Molecular studies showed that c-myc, N-myc, and bcl-2 were not rearranged. This cell line will provide a useful in vitro system to study the relationship between chromosomal abnormalities and the activation of cellular oncogenes.

Topics: Animals; Antigens, CD; Antigens, Viral, Tumor; Burkitt Lymphoma; Cell Transformation, Viral; Child; Chromosomes, Human, Pair 2; Chromosomes, Human, Pair 3; Gene Rearrangement; Herpesvirus 4, Human; Humans; Immunophenotyping; In Situ Hybridization; Liver Transplantation; Lymphoma, B-Cell; Male; Mice; Mice, SCID; Neoplasm Transplantation; Pleural Effusion; Sigmoid Neoplasms; Tacrolimus; Translocation, Genetic; Tumor Cells, Cultured