sirolimus and Lymphoma--T-Cell

There have been only five reported cases of primary posttransplant T-cell lymphoma. We report the first case associated with the use of sirolimus (Rapamycin, Wyeth-Ayerst, Philadelphia, PA). The patient, receiving prednisone, cyclosporine, and sirolimus treatment, developed ascites, diarrhea, and weight loss 7 months after his second renal transplant. Tissue obtained at laparotomy established the diagnosis of primary T-cell lymphoma. Latent membrane protein-1 for Epstein-Barr virus was negative, but in-site hybridization test for Epstein-Barr-encoded RNA was positive. Despite aggressive chemotherapy, the patient died 8 months posttransplant. This is the sixth reported case of primary intestinal posttransplant T-cell lymphoma, but it is the first case associated with the use of sirolimus. The incidence of posttransplant lymphoproliferative disease in patients receiving sirolimus should be studied.

Topics: Fatal Outcome; Humans; Immunosuppressive Agents; Intestinal Neoplasms; Kidney Transplantation; Lymphoma, T-Cell; Male; Middle Aged; Postoperative Complications; Sirolimus

The immunosuppressant drug, rapamycin (RAP), is a potent inhibitor of IL-2-dependent T-cell proliferation. The antiproliferative effect of RAP is mediated through the formation of an active complex with its cytosolic receptor protein, FKBP12. The molecular target of the FKBP12.RAP complex is a putative lipid kinase termed the mammalian Target Of Rapamycin (mTOR). This review will discuss recent findings suggesting that mTOR is a novel regulator of G1- to S-phase progression in eukaryotic cells.

Topics: Animals; Carrier Proteins; Cell Cycle Proteins; Cloning, Molecular; Cyclins; Cyclosporine; DNA-Binding Proteins; DNA, Complementary; Eukaryotic Cells; Fungal Proteins; G1 Phase; Heat-Shock Proteins; Immunosuppressive Agents; Interleukin-2; Lymphocyte Activation; Lymphoma, T-Cell; Mammals; Models, Immunological; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor); Polyenes; Protein Synthesis Inhibitors; Ribosomal Protein S6 Kinases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sirolimus; T-Lymphocytes; Tacrolimus; Tacrolimus Binding Proteins; Tumor Cells, Cultured

Everolimus is an oral agent that targets the mammalian target of rapamycin (mTOR) pathway. This study investigated mTOR pathway activation in T-cell lymphoma (TCL) cell lines and assessed antitumor activity in patients with relapsed/refractory TCL in a phase 2 trial. The mTOR pathway was activated in all 6 TCL cell lines tested and everolimus strongly inhibited malignant T-cell proliferation with minimal cytotoxic effects. Everolimus completely inhibited phosphorylation of ribosomal S6, a raptor/mTOR complex 1 (mTORC1) target, without a compensatory activation of the rictor/mTORC2 target Akt (S475). In the clinical trial, 16 patients with relapsed TCL were enrolled and received everolimus 10 mg by mouth daily. Seven patients (44%) had cutaneous (all mycosis fungoides); 4 (25%) had peripheral T cell not otherwise specified; 2 (13%) had anaplastic large cell; and 1 each had extranodal natural killer/T cell, angioimmunoblastic, and precursor T-lymphoblastic leukemia/lymphoma types. The overall response rate was 44% (7/16; 95% confidence interval [CI]: 20% to 70%). The median progression-free survival was 4.1 months (95% CI, 1.5-6.5) and the median overall survival was 10.2 months (95% CI, 2.6-44.3). The median duration of response for the 7 responders was 8.5 months (95% CI, 1.0 to not reached). These studies indicate that everolimus has antitumor activity and provide proof-of-concept that targeting the mTORC1 pathway in TCL is clinically relevant. This trial was registered at www.clinicaltrials.gov as #NCT00436618.

Topics: Adult; Aged; Aged, 80 and over; Animals; Apoptosis; Blotting, Western; Cell Proliferation; Cytokines; Everolimus; Female; Flow Cytometry; Humans; Immunosuppressive Agents; In Vitro Techniques; Lymphoma, T-Cell; Male; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Middle Aged; Multiprotein Complexes; Neoplasm Recurrence, Local; Phosphorylation; Prognosis; Sirolimus; Survival Rate; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

mTOR drives tumor growth but also supports T-cell function, rendering the applications of mTOR inhibitors complex especially in T-cell malignancies. Here, we studied the effects of the mTOR inhibitor rapamycin in mouse EL4 T-cell lymphoma. Typical pharmacologic rapamycin (1-8 mg/kg) significantly reduced tumor burden via direct suppression of tumor cell proliferation and improved survival in EL4 challenge independent of antitumor immunity. Denileukin diftitox (DD)-mediated depletion of regulatory T cells significantly slowed EL4 growth in vivo in a T-cell-dependent fashion. However, typical rapamycin inhibited T-cell activation and tumor infiltration in vivo and failed to boost DD treatment effects. Low-dose (LD) rapamycin (75 μg/kg) increased potentially beneficial CD44hiCD62L

Topics: Animals; Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cell Proliferation; Diphtheria Toxin; Disease Models, Animal; Flow Cytometry; Humans; Immunotherapy; Interleukin-2; Jurkat Cells; Lymphocyte Activation; Lymphoma, T-Cell; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Recombinant Fusion Proteins; Sirolimus; T-Lymphocytes

The mTOR pathway is a master regulator of cellular growth and metabolism. The biosynthetic and energetic demand of rapidly proliferating cells such as cancer cells is met by metabolic adaptations such as an increased glycolytic rate known as the Warburg effect. Herein, we characterize the anti-tumor effect of rapamycin in a mouse model of T-cell lymphoma and examine the metabolic effects in vitro. The murine T-cell lymphoma line, MBL2, and human cutaneous T-cell lymphoma (CTCL) lines, HH and Hut78, were used in syngeneic or standard NSG mouse models to demonstrate a marked suppression of tumor growth by rapamycin accompanied by inhibition of mTORC1/2. Analysis of the metabolic phenotype showed a substantial reduction in the glycolytic rate and glucose utilization in rapamycin-treated lymphoma cells. This was associated with reduced expression of glucose transporters and glycolytic enzymes in cultured cells and xenograft tumors. As a result of the decrease in glycolytic state, rapamycin-treated cells displayed reduced sensitivity to low-glucose conditions but continued to rely on mitochondrial oxidative phosphorylation (OXPHOS) with sensitivity to inhibition of OXPHOS. Taken together, we demonstrate that rapamycin suppresses growth of T-cell lymphoma tumors and leads to a reduction in aerobic glycolysis counteracting the Warburg effect of cancer cells.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Gene Expression Regulation, Neoplastic; Glycolysis; Heterografts; Humans; Lymphoma, T-Cell; Mice; Mice, Inbred C57BL; Molecular Targeted Therapy; Oxidative Phosphorylation; Phenotype; Sensitivity and Specificity; Sirolimus; TOR Serine-Threonine Kinases

In this issue of Blood, Witzig et al report on the promising in vitro and in vivo activity of everolimus in T-cell lymphoma (TCL) and pave the way for future combination studies.

Topics: Animals; Female; Humans; Immunosuppressive Agents; Lymphoma, T-Cell; Male; Multiprotein Complexes; Neoplasm Recurrence, Local; Sirolimus; TOR Serine-Threonine Kinases

Epstein-Barr virus (EBV) infects B cells, as well as T cells and natural killer (NK) cells, and is associated with T or NK cell lymphoid malignancies. In various tumor cells, mTOR performs an essential function together with Akt with regard to cell growth. We investigated the effects of mTOR inhibitors on EBV-associated T- and NK-cell lymphomas.. We investigated the Akt/mTOR activation pathway in EBV-positive and -negative T- and NK-cell lines (SNT13, SNT16, Jurkat, SNK6, KAI3, and KHYG1). We evaluated the antitumor effects of mTOR inhibitors (rapamycin and its analogue, CCI-779) against these cell lines in culture and in a murine xenograft model that was established by subcutaneous injection of SNK6 cells into NOG mice.. All EBV-positive and -negative T- and NK-cell lines tested displayed activation of the Akt/mTOR pathway, and treatment with mTOR inhibitors suppressed mTOR activation. The inhibitors induced G1 cell-cycle arrest and inhibited cell proliferation in T- and NK-cell lines. Overall, T cell lines were more sensitive to rapamycin, but there were no significant differences between EBV-positive and -negative cell lines. Treatment with rapamycin did not affect lytic or latent EBV gene expression. Intraperitoneal treatment with CCI-779 significantly inhibited the growth of established tumors in NOG mice and reduced the EBV load in peripheral blood.. These results suggest that inhibition of mTOR signaling is a promising new strategy for improving treatment of EBV-associated T- and NK-cell lymphoma.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Epstein-Barr Virus Infections; G1 Phase Cell Cycle Checkpoints; Herpesvirus 4, Human; Humans; Jurkat Cells; Killer Cells, Natural; Lymphoma, T-Cell; Mice; Sirolimus; T-Lymphocytes; TOR Serine-Threonine Kinases

Topics: Angiomyolipoma; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Cyclophosphamide; Doxorubicin; Everolimus; Humans; Immunosuppressive Agents; Kidney Neoplasms; Lymphoma, T-Cell; Male; Middle Aged; Positron-Emission Tomography; Prednisone; Radiography; Sirolimus; Vincristine

Everolimus (RAD001) is a novel mammalian target of rapamycin (mTOR) inhibitor, and anti-proliferative activity in various malignancies has been reported. This study evaluated the anti-tumor effects and schedule-dependent synergism of everolimus in combination with other chemotherapeutic agents in T-cell lymphoma cell lines.. Human T-cell lymphoma cell lines Hut-78 and Jurkat were treated with increasing doses of everolimus, alone or in combination with doxorubicin, etoposide, vincristine, or bortezomib, using different dosing schedules. Anti-tumor effects were measured by assays for cell proliferation, apoptosis, and cell cycle distribution. Drug interactions were determined by median effect analysis.. Exposure to everolimus alone induced G1 phase cell cycle arrest without significant apoptosis. With certain dosing schedules, everolimus showed synergism with doxorubicin, etoposide, and bortezomib, but antagonism with vincristine. Cytotoxic synergism was observed following cotreatment with doxorubicin and everolimus, bortezomib and everolimus, doxorubicin followed by everolimus, and bortezomib followed by everolimus. By contrast, cell exposure to everolimus followed by doxorubicin or followed by bortezomib resulted in antagonistic effects. Sequential exposure to doxorubicin or bortezomib followed by everolimus effectively prevented potential negative interactions, and resulted in drug synergism. Drug combination synergisms or antagonisms were associated with variable effects on the cell cycle distribution.. Everolimus effectively inhibited the growth of T-cell lymphoma cells in vitro. Specific schedule-dependent combinations of everolimus with other anti-tumor agents which avoid potential drug antagonism and produce effective synergism may lead to clinically effective treatments for T-cell lymphoma.

Topics: Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Boronic Acids; Bortezomib; Cell Cycle; Cell Proliferation; Dose-Response Relationship, Drug; Doxorubicin; Drug Synergism; Etoposide; Everolimus; Humans; Jurkat Cells; Lymphoma, T-Cell; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Pyrazines; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Vincristine

Adenosine monophosphate-activated protein kinase (AMPK) acts as a major sensor of cellular energy status in cancers and is critically involved in cell sensitivity to anticancer agents. Here, we showed that AMPK was inactivated in lymphoma and related to the upregulation of the mammalian target of rapamycin (mTOR) pathway. AMPK activator metformin potentially inhibited the growth of B- and T-lymphoma cells. Strong antitumor effect was also observed on primary lymphoma cells while sparing normal hematopoiesis ex vivo. Metformin-induced AMPK activation was associated with the inhibition of the mTOR signaling without involving AKT. Moreover, lymphoma cell response to the chemotherapeutic agent doxorubicin and mTOR inhibitor temsirolimus was significantly enhanced when co-treated with metformin. Pharmacologic and molecular knock-down of AMPK attenuated metformin-mediated lymphoma cell growth inhibition and drug sensitization. In vivo, metformin induced AMPK activation, mTOR inhibition and remarkably blocked tumor growth in murine lymphoma xenografts. Of note, metformin was equally effective when given orally. Combined treatment of oral metformin with doxorubicin or temsirolimus triggered lymphoma cell autophagy and functioned more efficiently than either agent alone. Taken together, these data provided first evidence for the growth-inhibitory and drug-sensitizing effect of metformin on lymphoma. Selectively targeting mTOR pathway through AMPK activation may thus represent a promising new strategy to improve treatment of lymphoma patients.

Topics: AMP-Activated Protein Kinase Kinases; Animals; Antineoplastic Agents; Autophagy; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Doxorubicin; Drug Synergism; Enzyme Activation; Humans; Lymphoma, B-Cell; Lymphoma, T-Cell; Metformin; Mice; Protein Kinase Inhibitors; Protein Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Human cancers, including acute myeloid leukemia (AML), commonly display constitutive phosphoinositide 3-kinase (PI3K) AKT signaling. However, the exact role of AKT activation in leukemia and its effects on hematopoietic stem cells (HSCs) are poorly understood. Several members of the PI3K pathway, phosphatase and tensin homolog (Pten), the forkhead box, subgroup O (FOXO) transcription factors, and TSC1, have demonstrated functions in normal and leukemic stem cells but are rarely mutated in leukemia. We developed an activated allele of AKT1 that models increased signaling in normal and leukemic stem cells. In our murine bone marrow transplantation model using a myristoylated AKT1 (myr-AKT), recipients develop myeloproliferative disease, T-cell lymphoma, or AML. Analysis of the HSCs in myr-AKT mice reveals transient expansion and increased cycling, associated with impaired engraftment. myr-AKT-expressing bone marrow cells are unable to form cobblestones in long-term cocultures. Rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR) rescues cobblestone formation in myr-AKT-expressing bone marrow cells and increases the survival of myr-AKT mice. This study demonstrates that enhanced AKT activation is an important mechanism of transformation in AML and that HSCs are highly sensitive to excess AKT/mTOR signaling.

Topics: Animals; Antibiotics, Antineoplastic; Bone Marrow Cells; Bone Marrow Transplantation; Cell Division; Cell Line; Hematopoietic Stem Cells; Humans; Intracellular Signaling Peptides and Proteins; Kidney; Leukemia, Myeloid, Acute; Lymphoma, T-Cell; Mice; Mice, Inbred C57BL; Myeloproliferative Disorders; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Signal Transduction; Sirolimus; Spleen; TOR Serine-Threonine Kinases

Topics: Everolimus; Humans; Immunosuppressive Agents; Killer Cells, Natural; Lymphoma, Extranodal NK-T-Cell; Lymphoma, Non-Hodgkin; Lymphoma, T-Cell; Prognosis; Sirolimus; Stem Cell Transplantation

Recurrent chromosomal aberrations in hematopoietic tumors target genes involved in pathogenesis. Their identification and functional characterization are therefore important for the establishment of rational therapies. Here, we investigated genomic amplification at 7q22 in the T-cell lymphoma cell line SU-DHL-1 belonging to the subtype of anaplastic large-cell lymphoma (ALCL). Cytogenetic analysis mapped this amplicon to 86-95 Mb. Copy-number determination quantified the amplification level at 5- to 6-fold. Expression analysis of genes located within this region identified cyclin-dependent kinase 6 (CDK6) as a potential amplification target. In comparison with control cell lines, SU-DHL-1 expressed considerably higher levels of CDK6. Functionally, SU-DHL-1 cells exhibited reduced sensitivity to rapamycin treatment, as indicated by cell growth and cell cycle analysis. Rapamycin reportedly inhibits degradation of the CDK inhibitor p27 with concomitant downregulation of cyclin D3, implying a proliferative advantage for CDK6 overexpression. Amplification of the CDK6 locus was analyzed in primary T-cell lymphoma samples and, while detected infrequently in those classified as ALCL (1%), was detected in 23% of peripheral T-cell lymphomas not otherwise specified. Taken together, analysis of the 7q22 amplicon identified CDK6 as an important cell cycle regulator in T-cell lymphomas, representing a novel potential target for rational therapy.

Topics: Cell Line, Tumor; Chromosome Aberrations; Chromosomes, Human, Pair 7; Cyclin-Dependent Kinase 6; Cytogenetic Analysis; Drug Resistance; Gene Dosage; Gene Expression Regulation, Neoplastic; Humans; Lymphoma, Large-Cell, Anaplastic; Lymphoma, T-Cell; Lymphoma, T-Cell, Peripheral; Sirolimus; Tumor Cells, Cultured

Topics: Adult; Epstein-Barr Virus Infections; Humans; Immunosuppressive Agents; Kidney Transplantation; Lymphoma, Large B-Cell, Diffuse; Lymphoma, T-Cell; Lymphoproliferative Disorders; Male; Middle Aged; Protein Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The mechanisms of cell transformation mediated by the nucleophosmin (NPM)/anaplastic lymphoma kinase (ALK) tyrosine kinase are only partially understood. Here, we report that cell lines and native tissues derived from the NPM/ALK-expressing T-cell lymphoma display persistent activation of mammalian target of rapamycin (mTOR) as determined by phosphorylation of mTOR targets S6rp and 4E-binding protein 1 (4E-BP1). The mTOR activation is serum growth factor-independent but nutrient-dependent. It is also dependent on the expression and enzymatic activity of NPM/ALK as demonstrated by cell transfection with wild-type and functionally deficient NPM/ALK, small interfering RNA (siRNA)-mediated NPM/ALK depletion and kinase activity suppression using the inhibitor WHI-P154. The NPM/ALK-induced mTOR activation is transduced through the mitogen-induced extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway and, to a much lesser degree, through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. Accordingly, whereas the low-dose PI3K inhibitor wortmannin and Akt inhibitor III profoundly inhibited Akt phosphorylation, they had a very modest effect on S6rp and 4E-BP1 phosphorylation. In turn, MEK inhibitors U0126 and PD98059 and siRNA-mediated depletion of either ERK1 or ERK2 inhibited S6rp phosphorylation much more effectively. Finally, the mTOR inhibitor rapamycin markedly decreased proliferation and increased the apoptotic rate of ALK+TCL cells. These findings identify mTOR as a novel key target of NPM/ALK and suggest that mTOR inhibitors may prove effective in therapy of ALK-induced malignancies.

Topics: Anaplastic Lymphoma Kinase; Animals; Blotting, Western; Cell Line; Cell Line, Tumor; Cell Proliferation; Cell Survival; Extracellular Signal-Regulated MAP Kinases; Humans; Immunohistochemistry; Lymphoma, T-Cell; Mitogen-Activated Protein Kinases; Models, Biological; Nuclear Proteins; Nucleophosmin; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Protein Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-akt; Quinazolines; Receptor Protein-Tyrosine Kinases; RNA, Small Interfering; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transfection

The inhibitors of cyclin-dependent kinase (CDK) 4 (INK4) bind CDK4/6 to prevent their association with D-cyclins and G(1) cell cycle initiation and progression. We report here that among the seven CDK inhibitors, p18(INK4c) played an important role in modulating TCR-mediated T cell proliferation. Loss of p18(INK4c) in T cells led to hyperproliferation in response to CD3 stimulation. p18(INK4c)-null mice developed lymphoproliferative disorder and T cell lymphomas. Expression of IL-2, IL-2R-alpha, and the major G(1) cell cycle regulatory proteins was not altered in p18-null T cells. Both FK506 and rapamycin efficiently inhibited proliferation of p18-null T cells. In activated T cells, p18(INK4c) remained constant, and preferentially associated with and inhibited CDK6 but not CDK4. We propose that p18(INK4c) sets an inhibitory threshold in T cells and one function of CD28 costimulation is to counteract the p18(INK4c) inhibitory activity on CDK6-cyclin D complexes. The p18(INK4c) protein may provide a novel target to modulate T cell immunity.

Topics: Animals; CD28 Antigens; CD3 Complex; CDC2-CDC28 Kinases; Cell Cycle; Cell Cycle Proteins; Cell Division; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p18; Cyclin-Dependent Kinases; Cytokines; Enzyme Inhibitors; G1 Phase; Immunosuppressive Agents; Lymphocyte Activation; Lymphoma, T-Cell; Lymphoproliferative Disorders; Mice; Mice, Knockout; Mice, SCID; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Receptors, Antigen, T-Cell; Sirolimus; T-Lymphocyte Subsets; Tacrolimus; Tumor Suppressor Proteins

The immunosuppressant, rapamycin, inhibits cell growth by interfering with the function of a novel kinase, termed mammalian target of rapamycin (mTOR). The putative catalytic domain of mTOR is similar to those of mammalian and yeast phosphatidylinositol (PI) 3-kinases. This study demonstrates that mTOR is a component of a cytokine-triggered protein kinase cascade leading to the phosphorylation of the eukaryotic initiation factor-4E (eIF-4E) binding protein, PHAS-1, in activated T lymphocytes. This event promotes G1 phase progression by stimulating eIF-4E-dependent translation initiation. A mutant YAC-1 T lymphoma cell line, which was selected for resistance to the growth-inhibitory action of rapamycin, was correspondingly resistant to the suppressive effect of this drug on PHAS-1 phosphorylation. In contrast, the PI 3-kinase inhibitor, wortmannin, reduced the phosphorylation of PHAS-1 in both rapamycin-sensitive and -resistant T cells. At similar drug concentrations (0.1-1 microM), wortmannin irreversibly inhibited the serine-specific autokinase activity of mTOR. The autokinase activity of mTOR was also sensitive to the structurally distinct PI 3-kinase inhibitor, LY294002, at concentrations (1-30 microM) nearly identical to those required for inhibition of the lipid kinase activity of the mammalian p85-p110 heterodimer. These studies indicate that the signaling functions of mTOR, and potentially those of other high molecular weight PI 3-kinase homologs, are directly affected by cellular treatment with wortmannin or LY294002.

Topics: Adaptor Proteins, Signal Transducing; Adenosine Triphosphate; Androstadienes; Animals; Brain Chemistry; Carrier Proteins; Cell Cycle Proteins; Cell Line; Chromones; Dithiothreitol; Enzyme Inhibitors; Eukaryotic Initiation Factors; Immunosuppressive Agents; Interleukin-2; Intracellular Signaling Peptides and Proteins; Lymphocyte Activation; Lymphoma, T-Cell; Mice; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoproteins; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Polyenes; Protein Kinases; Rats; Recombinant Fusion Proteins; Signal Transduction; Sirolimus; T-Lymphocytes; T-Lymphocytes, Cytotoxic; TOR Serine-Threonine Kinases; Tumor Cells, Cultured; Wortmannin

Rapamycin (RAP) disrupts signaling events implicated in cytokine-dependent proliferation of lymphocytes and other cells. This action is known to involve the formation of molecular complexes between the drug and intracellular binding proteins, termed FKBPs. However, the biochemical target(s) for the effector RAP-FKBP complexes remain uncharacterized. As an approach to explore the mechanism of action of RAP, we have isolated three independent sets of somatic mutants of the YAC-1 murine T cell line with markedly reduced sensitivity to the drug's inhibitory effects on proliferation and on IL-1-induced IFN-gamma production. These mutants were still fully sensitive to FK-506, an immunosuppressant structurally related to RAP whose mode of action also involves an interaction with FKBPs. Furthermore, the 12-kDa FKBP, FKBP12, was detectable in immunoblots from cytosolic extracts and eluates from RAP-affinity matrix in the mutants as in wild-type cells, suggesting that the resistance to RAP in the mutants is not due to a lack of FKBP12 expression. Cell fusion experiments were conducted to further define the nature of the alterations imparting RAP resistance in these mutants. Clones deficient in either thymidine kinase or hypoxanthine-guanine phosphoribosyltransferase, suitable as fusion partners for aminopterin-based selection of hybrids were generated from the wild-type or mutant lines. In most instances, the hybrids derived from the fusion between RAP-sensitive clones and RAP-resistant clones exhibited a RAP-resistant phenotype. Similar results were obtained with hybrids between RAP-resistant YAC-1 clones and the RAP-sensitive EL-4 cell line. Therefore, the mutations that confer resistance to RAP in the present system are dominant. Altogether, our observations are consistent with a model where pharmacologically relevant targets for the RAP-FKBP complex, rather than FKBP, might be altered in the mutants such that the inactivation of these targets by the effector complex is prevented.

Topics: Animals; Carrier Proteins; Cell Division; Cell Fusion; DNA-Binding Proteins; Drug Resistance; Flow Cytometry; Heat-Shock Proteins; Immunosuppressive Agents; Lymphoma, T-Cell; Mice; Mutation; Polyenes; Protein Serine-Threonine Kinases; Ribosomal Protein S6 Kinases; RNA, Messenger; Sirolimus; Tacrolimus; Tacrolimus Binding Proteins; Tumor Cells, Cultured

The immunosuppressive macrolide, rapamycin, impedes the G1 to S cell cycle progression in cytokine-stimulated normal lymphocytes and in certain autonomously proliferating cell lines. Here, we found that the rapamycin-induced growth arrest augments homotypic aggregation in the YAC-1 T cell lymphoma. The growth arrest and increased aggregation were both blocked by the rapamycin antagonist, L-685,818, which interacts with the intracellular binding proteins mediating rapamycin's biochemical action. Moreover, rapamycin-induced aggregation was not seen in YAC-1 cells mutants selected for resistance to the drug's antiproliferative effect. Although the inhibition of G1/S progression induced by serum deprivation also resulted in increased cellular aggregation, cell cycle blockade in late G1 by mimosine, early S phase by hydroxyurea, or G2/M by nocodazole all failed to do so. Furthermore, the aggregation induced by rapamycin was blocked by antibodies to the alpha (CD11a) or beta (CD18) subunits of the integrin, LFA-1, or to its ligands, ICAM-1 and ICAM-2, and did not occur in LFA-1-deficient YAC mutants. However, the surface expression of LFA-1, ICAM-1, or ICAM-2 was not augmented in cells aggregated by rapamycin. Finally, the serine/threonine protein phosphatase inhibitor, okadaic acid, was found to abrogate rapamycin-induced aggregation. Therefore, rapamycin's impairment of YAC-1 cell growth in G1 is accompanied by enhanced LFA-1-mediated homotypic cell adhesion that may reflect an increase of the integrin's avidity for its ligands and may involve protein phosphorylation/dephosphorylation events. This suggests the existence of a link between cell cycle progression and "inside-out" LFA-1 signaling, possibly regulated by rapamycin's biochemical targets.

Topics: Animals; Antibodies; CD11 Antigens; CD18 Antigens; Cell Adhesion; Cell Cycle; Cell Division; Cell Line; Culture Media, Serum-Free; Flow Cytometry; G1 Phase; Hydroxyurea; Immunosuppressive Agents; Lymphocyte Function-Associated Antigen-1; Lymphoma, T-Cell; Mimosine; Nocodazole; Polyenes; Sirolimus; Tacrolimus; Tumor Cells, Cultured

The immunosuppressive drug, rapamycin, interferes with an undefined signaling pathway required for the progression of G1-phase T-cells into S phase. Genetic analyses in yeast indicate that binding of rapamycin to its intracellular receptor, FKBP12, generates a toxic complex that inhibits cell growth in G1 phase. These analyses implicated two related proteins, TOR1 and TOR2, as targets of the FKBP12-rapamycin complex in yeast. In this study, we have used a glutathione S-transferase (GST)-FKBP12-rapamycin affinity matrix to isolate putative mammalian targets of rapamycin (mTOR) from tissue extracts. In the presence of rapamycin, immobilized GST-FKBP12 specifically precipitates similar high molecular mass proteins from both rat brain and murine T-lymphoma cell extracts. Binding experiments performed with rapamycin-sensitive and -resistant mutant clones derived from the YAC-1 T-lymphoma cell line demonstrate that the GST-FKBP12-rapamycin complex recovers significantly lower amounts of the candidate mTOR from rapamycin-resistant cell lines. The latter results suggest that mTOR is a relevant target of rapamycin in these cells. Finally, we report the isolation of a full-length mTOR cDNA that encodes a direct ligand for the FKBP12-rapamycin complex. The deduced amino acid sequence of mTOR displays 42 and 45% identity to those of yeast TOR1 and TOR2, respectively. These results strongly suggest that the FKBP12-rapamycin complex interacts with homologous ligands in yeast and mammalian cells and that the loss of mTOR function is directly related to the inhibitory effect of rapamycin on G1- to S-phase progression in T-lymphocytes and other sensitive cell types.

Topics: Amino Acid Sequence; Animals; Base Sequence; Carrier Proteins; Cell Cycle Proteins; DNA Primers; DNA-Binding Proteins; DNA, Complementary; Fungal Proteins; Heat-Shock Proteins; Humans; Lymphoma, T-Cell; Molecular Sequence Data; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor); Polyenes; Protein Kinases; Rats; Rats, Sprague-Dawley; Saccharomyces cerevisiae Proteins; Sequence Homology, Amino Acid; Sirolimus; Tacrolimus Binding Proteins; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

Transforming growth factor beta 1 (TGF-beta 1) is a multifunctional cytokine whose potent immunomodulatory activity is well documented. To explore the mechanisms of this activity we examined the effect of TGF-beta 1 on the production of IFN-gamma measured at the mRNA and protein levels in the YAC-1 T cell lymphoma. In previous studies, this model proved useful to characterize the mode of action of the immunosuppressant rapamycin (RAP). Here, we found that when induced by IL-1 or IL-1 + PMA, the production of IFN-gamma is suppressed by both TGF-beta 1 (ED50 = 1.9 pM) and RAP (ED50 = 0.2 nM). In contrast, when induced by the calcium ionophore ionomycin, in the absence or in the presence of PMA, this production is enhanced up to 10-fold by TGF-beta 1 (ED50 = 1.8 pM) and 1.5-3-fold by RAP. Therefore, in YAC-1 cells, TGF-beta 1 exerts opposite effects on IFN-gamma production depending on the mode of activation, and these effects parallel those of RAP. To further analyze the mode of action of TGF-beta 1 in this system, we used okadaic acid (OA), an inhibitor of serine/threonine protein phosphatases. Treatment with OA rendered the expression of IFN-gamma mRNA induced by IL-1 insensitive to TGF-beta 1 or RAP, indicating that activation of a phosphatase may play a role in the suppressive effect of both agents. However, OA did not prevent the augmentation of ionomycin-mediated induction of IFN-gamma mRNA by either TGF-beta 1 or RAP. Hence, the up-regulation of IFN-gamma production by TGF-beta 1 and RAP may involve a different biochemical mechanism than that mediating their suppressive action. These observations also favor the hypothesis that the two agents act on the same regulatory pathways. This was further supported by the finding that TGF-beta 1 and RAP modulate IFN-gamma production in an additive rather than synergistic fashion. However, their effects could be dissociated in mutants of YAC-1 cells selected for resistance to the inhibition of IL-1-mediated IFN-gamma induction by RAP. Moreover, the IFN-gamma modulatory action of RAP in YAC-1 cells was accompanied by an antiproliferative effect, whereas TGF-beta 1 failed to alter the growth of these cells. Therefore, the immunomodulatory action of TGF-beta 1 may result from the disruption of biochemical processes related to, although distinct from, those affected by RAP.

Topics: Animals; Cell Division; Drug Interactions; Drug Synergism; Ethers, Cyclic; Immunosuppressive Agents; Interferon-gamma; Interleukin-1; Ionomycin; Lymphoma, T-Cell; Mice; Okadaic Acid; Phosphoprotein Phosphatases; Polyenes; RNA, Messenger; Sirolimus; Swine; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta

Rapamycin (RAP) inhibits several biologic responses in the YAC-1 T cell lymphoma, including the serum-driven proliferation and cyclin A mRNA expression, the induction of Ly-6E Ag expression by IFN, and the induction of IFN-gamma production by IL-1. RAP also suppresses the enzymatic activity of the 70 kDa S6 protein kinase (pp70s6k). To define the mechanistic relationship between these multiple effects of RAP, we have generated stable somatic mutants with altered sensitivities to this drug. A first series of mutants, represented by the R19, 4R16, and 10R13 clones, showed markedly reduced sensitivity to the inhibitory effect of RAP on all biologic responses tested and on pp70s6k activity. Two other mutant types, R103 and R125, were both highly sensitive to RAP-mediated suppression of proliferation, of IL-1-induced IFN-gamma production, and of pp70s6k activity but differed in their Ly-6E response. This response was not affected by RAP in the R125 clone and was enhanced in the R103 clone. Therefore, the inhibitory effects of RAP on proliferation and IL-1-mediated IFN-gamma induction both appear associated with the inhibition of pp70s6k activity, whereas the modulation of Ly-6E induction is independent from the latter. Moreover, the cellular binding of [3H]dihydro-FK-506 was found to be blocked by RAP in all mutant types to the same extent as in wild-type YAC-1 cells, suggesting that the altered sensitivity to the effects of RAP in these mutants is not due to an inability of the drug to enter the cells or to interact with FKBP. Further biochemical characterization of the mutant cells described here is expected to help clarify the mechanisms of RAP action.

Topics: Animals; Antigens, Ly; Clone Cells; Cyclins; Gene Expression; Interferon-gamma; Interleukin-1; Lymphocyte Activation; Lymphoma, T-Cell; Mice; Polyenes; Protein Serine-Threonine Kinases; Recombinant Proteins; Ribosomal Protein S6 Kinases; RNA, Messenger; Sirolimus; Tetradecanoylphorbol Acetate