sirolimus and Ataxia-Telangiectasia

Immune system-related pathology is common in ataxia-telangiectasia (A-T) patients and mice that lack the protein kinase, A-T mutated (ATM). However, it has not been studied how ATM influences immune responses to a viral infection. Using the lymphocytic choriomeningitis virus (LCMV) infection model, we show that ATM(-/-) mice, despite having fewer naïve CD8⁺ T cells, effectively clear the virus. However, aberrant CD8⁺ T-cell responses are observed, including defective expansion and contraction, effector-to-memory differentiation, and a switch in viral-epitope immunodominance. T-cell receptor-activated, but not naïve, ATM(-/-) splenic CD8⁺ T cells have increased ribosomal protein S6 and Akt phosphorylation and do not proliferate well in response to IL-15, a cytokine important for memory T-cell development. Accordingly, pharmacological Akt or mammalian target of rapamycin complex 1 (mTORC1) inhibition during T-cell receptor activation alone rescues the IL-15 proliferation defect. Finally, rapamycin treatment during LCMV infection in vivo increases the number of memory T cells in ATM(-/-) mice. Altogether, these results show that CD8⁺T cells lacking ATM have hyperactive Akt and mTORC1 signaling in response to T-cell receptor activation, which results in aberrant cytokine responses and memory T-cell development. We speculate that similar signaling defects contribute to the immune system pathology of A-T, and that inhibition of Akt and/or mTORC1 may be of therapeutic value.

Topics: Animals; Ataxia Telangiectasia; Ataxia Telangiectasia Mutated Proteins; CD8-Positive T-Lymphocytes; Cell Cycle Proteins; Cell Differentiation; Cell Proliferation; Disease Models, Animal; DNA-Binding Proteins; Enzyme Activation; Immunologic Memory; Interleukin-15; Lymphocyte Activation; Lymphocytic Choriomeningitis; Lymphocytic choriomeningitis virus; Mechanistic Target of Rapamycin Complex 1; Mice; Multiprotein Complexes; Phosphorylation; Protein Serine-Threonine Kinases; Proteins; Proto-Oncogene Proteins c-akt; Receptors, Antigen, T-Cell; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Suppressor Proteins

Rapamycin induces chromosome malsegregation in mammalian cell lines and yeast. Previous studies indicate that the function impaired in ataxia-telangiectasia (A-T) patients is necessary for both the growth inhibition and the chromosome malsegregation induced by rapamycin, and that treating the non-tumorigenic Chinese hamster cell line CHEF/18 with rapamycin results in supernumerary centrosomes and multipolar spindles. In this paper we report that lymphoblastoid cell lines established from A-T patients as well as hamster A-T-like cells are more resistant to rapamycin than the respective normal cell lines. Two cell lines derived from Nijmegen Breakage Syndrome (NBS) patients, who have clinical symptoms similar to those of A-T but a different molecular defect, were not resistant to rapamycin. Both A-T lymphoblastoid cells and A-T-like fibroblasts had giant centrosomes formed by more than two areas of gamma-tubulin-reacting material. Such giant centrosomes were also observed in CHEF/18 cells after prolonged treatment with rapamycin. Formation of giant centrosomes, possibly due to the coalescence of supernumerary centrosomes, was associated with increased aneuploidy in treated cells. Expression analysis of cell-cycle regulatory genes in rapamycin-treated human lymphoblastoid cells indicated that rapamycin decreased the expression of the tumor suppressor gene GADD45. The levels of RB, p21 and p53 mRNA were also decreased, although to a lesser extent. As rapamycin is often used as an immunosuppressant in pediatric transplant patients, these data indicate that caution should be taken, especially when the drug is given for prolonged periods of time.

Topics: Animals; Ataxia Telangiectasia; Cell Cycle; Cell Line; Centrosome; Chromosome Aberrations; Chromosomes; Cricetinae; Cyclin-Dependent Kinase Inhibitor p21; Dose-Response Relationship, Drug; Drug Resistance; Fibroblasts; GADD45 Proteins; Gene Expression Regulation; Humans; Intracellular Signaling Peptides and Proteins; Lymphocytes; Metaphase; Micronucleus Tests; Microscopy, Confocal; Models, Statistical; Nijmegen Breakage Syndrome; Phenotype; Retinoblastoma Protein; Sirolimus; Spindle Apparatus; Time Factors; Tumor Suppressor Protein p53

Topics: Ataxia Telangiectasia; Caspase 3; Caspases; Cell Cycle; Cyclosporine; DNA Repair; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fibroblasts; HIV Protease Inhibitors; Humans; Lymphocytes; Peptide Hydrolases; Proteasome Endopeptidase Complex; Protein Kinases; Ribosomal Protein S6 Kinases; Ritonavir; Signal Transduction; Sirolimus

The antifungal and immunosuppressive drug rapamycin arrests the cell cycle in G1-phase in both yeast and mammalian cells. In mammalian cells, rapamycin selectively inhibits phosphorylation and activation of p70 S6 kinase (p70(S6K)), a protein involved in the translation of a subset of mRNAs, without affecting other known kinases. We now report that rapamycin causes chromosome malsegregation in mammalian and yeast cells. Chromosome malsegregation was determined by metaphase chromosome analysis of human lymphocytes and lymphoblasts, detection of CREST-positive micronuclei in human lymphoblasts and Chinese hamster embryonic fibroblast (CHEF) cells, and selection of doubly prototrophic cells in a specially constructed yeast strain. The number of ana-telophases with displaced chromosomes and interphase and mitotic cells with an irregular number of centrosomes was also determined in CHEF cells. In quiescent mammalian cells (human lymphocytes and CHEF cells) induced with growth factor to re-enter the cell cycle, rapamycin was effective when cells were exposed at the time of p70(S6K) activation. In yeast, rapamycin was more effective when treatment was started in G1- than in G2-synchronized cells. Cells from ataxia telangiectasia (A-T) patients are characterized by chromosome instability and have recently been found to be resistant to the growth-inhibiting effect of rapamycin. We found that an A-T lymphoblastoid cell line was also resistant to the induction of chromosome malsegregation by rapamycin, but the level of spontaneous aneuploidy was higher than in normal cells. In yeast, the induction of chromosome malsegregation was dependent on the presence of a wild-type TUB2 gene, encoding the beta-subunit of tubulin. The finding that rapamycin acts in different cell types and organisms suggests that the drug affects a conserved step important for proper segregation of chromosomes. One or more proteins required for chromosome segregation could be under the control of the rapamycin-sensitive pathway.

Topics: Aneuploidy; Animals; Ataxia Telangiectasia; Chromosome Segregation; Cricetinae; Dose-Response Relationship, Drug; Fibroblasts; Humans; Lymphocytes; Metaphase; Micronuclei, Chromosome-Defective; Phosphorylation; Protein Kinases; Ribosomal Protein S6 Kinases; Saccharomyces cerevisiae; Sirolimus; Tubulin

The gene mutated in the human genetic disorder ataxia-telangiectasia (A-T) has been described recently (Savitsky et al., 1995a) and the complete coding sequence of this gene, ATM, has been reported (Savitsky et al., 1995b). The derived amino acid sequence demonstrates significant homologies to several proteins containing a phosphatidylinositol 3-kinase (PI3-kinase) domain, including the yeast TOR proteins and the human protein FRAP. Since the TOR and FRAP proteins are targets for the immunosuppressive drug rapamycin, we have investigated the effects of this compound on A-T cells. We report here that 3 A-T cell lines are more resistant than control cells to rapamycin's growth inhibiting effects but were more sensitive to the PI3-kinase inhibitor wortmannin. As expected rapamycin (1 nM) inhibited the rate of exit of control cells from G1 phase but failed to perturb the progression of A-T cells. This difference in cell cycle progress after rapamycin treatment is reflected in ribosomal S6 protein kinase (p70S6k) by both a downward mobility shift on SDS-PAGE and inhibition of activity. Furthermore, the G1 phase cyclin-dependent kinase, cyclin E-cdk2, was rapidly inhibited in control cells post-treatment, whereas in A-T cells it took considerably longer to observe inhibition. There was no evidence that a GST-FKBP12 fusion protein specifically precipitated the ATM protein in the presence of rapamycin in either cell type. These results demonstrate that the ATM protein is not a direct target for rapamycin but its functional loss renders cells more resistant to this compound.

Topics: Amino Acid Sequence; Androstadienes; Antibiotics, Antineoplastic; Antifungal Agents; Ataxia Telangiectasia; Ataxia Telangiectasia Mutated Proteins; Binding Sites; Carrier Proteins; Cell Cycle; Cell Cycle Proteins; Cells, Cultured; DNA-Binding Proteins; Dose-Response Relationship, Drug; Drug Resistance; Enzyme Inhibitors; Heat-Shock Proteins; Humans; Immunosuppressive Agents; Molecular Sequence Data; Mutation; Nocodazole; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor); Polyenes; Protein Serine-Threonine Kinases; Proteins; Recombinant Fusion Proteins; Ribosomal Protein S6 Kinases; Sirolimus; Tacrolimus Binding Proteins; Tumor Cells, Cultured; Tumor Suppressor Proteins; Wortmannin