First-in-human study demonstrating pharmacological activation of heme oxygenase-1 in humans.

Heme oxygenase (HO)-1 degrades heme and protects against oxidative stress, but it has not been pharmacologically induced in humans. In this randomized study of 10 healthy volunteers, hemin (3 mg/kg intravenously in 25% albumin) was shown to increase plasma HO-1 protein concentration four- to fivefold and HO-1 activity ~15-fold relative to baseline at 24 and 48 h (placebo -56.41 +/- 6.31 (baseline), 69.79 +/- 13.00 (24 h), 77.44 +/- 10.62 (48 h) vs. hemin -71.70 +/- 9.20 (baseline), 1,126.20 +/- 293.30 (24 h), 1,192.20 +/- 333.30 (48 h)) in four of five subjects as compared with albumin alone (P

Mammalian target of rapamycin-dependent phosphorylation of PHAS-I in four (S/T)P sites detected by phospho-specific antibodies.

The role and control of the four rapamycin-sensitive phosphorylation sites that govern the association of PHAS-I with the mRNA cap-binding protein, eukaryotic initiation factor 4E (eIF4E), were investigated by using newly developed phospho-specific antibodies. Thr(P)-36/45 antibodies reacted with all three forms of PHAS-I that were resolved when cell extracts were subjected to SDS-polyacrylamide gel electrophoresis. Thr(P)-69 antibodies bound the forms of intermediate and lowest mobility, and Ser(P)-64 antibodies reacted only with the lowest mobility form. A portion of PHAS-I that copurified with eIF4E reacted with Thr(P)-36/45 and Thr(P)-69 antibodies but not with Ser(P)-64 antibodies. Insulin and/or amino acids increased, and rapamycin decreased, the reactivity of all three antibodies with PHAS-I in both HEK293 cells and 3T3-L1 adipocytes. Immunoprecipitated epitope-tagged mammalian target of rapamycin (mTOR) phosphorylated Thr-36/45. mTOR also phosphorylated Thr-69 and Ser-64 but only when purified immune complexes were incubated with the activating antibody, mTAb1. Interestingly, the phosphorylation of Thr-69 and Ser-64 was much more sensitive to inhibition by rapamycin-FKBP12 than the phosphorylation of Thr-36/45, and the phosphorylation of Ser-64 by mTOR was facilitated by phosphorylation of Thr-36, Thr-45, and Thr-69. In these respects the phosphorylation of PHAS-I by mTOR in vitro resembles the ordered phosphorylation of PHAS-I in cells.

Mutational analysis of sites in the translational regulator, PHAS-I, that are selectively phosphorylated by mTOR.

Results obtained with PHAS-I proteins having Ser to Ala mutations in the five known phosphorylation sites indicate that mTOR preferentially phosphorylates Thr36 and Thr45. The effects of phosphorylating these sites on eIF4E binding were assessed in a far-Western analysis with a labeled eIF4E probe. Phosphorylation of Thr36 only slightly attenuated binding of PHAS-I to eIF4E, while phosphorylation of Thr45 markedly inhibited binding. Phosphorylation of neither site affected the electrophoretic mobility of the protein, indicating that results of studies that rely solely on a gel-shift assay to assess changes in PHAS-I phosphorylation must be interpreted with caution.

Evidence of insulin-stimulated phosphorylation and activation of the mammalian target of rapamycin mediated by a protein kinase B signaling pathway.

The effects of insulin on the mammalian target of rapamycin, mTOR, were investigated in 3T3-L1 adipocytes. mTOR protein kinase activity was measured in immune complex assays with recombinant PHAS-I as substrate. Insulin-stimulated kinase activity was clearly observed when immunoprecipitations were conducted with the mTOR antibody, mTAb2. Insulin also increased by severalfold the 32P content of mTOR that was determined after purifying the protein from 32P-labeled adipocytes with rapamycin.FKBP12 agarose beads. Insulin affected neither the amount of mTOR immunoprecipitated nor the amount of mTOR detected by immunoblotting with mTAb2. However, the hormone markedly decreased the reactivity of mTOR with mTAb1, an antibody that activates the mTOR protein kinase. The effects of insulin on increasing mTOR protein kinase activity and on decreasing mTAb1 reactivity were abolished by incubating mTOR with protein phosphatase 1. Interestingly, the epitope for mTAb1 is located near the COOH terminus of mTOR in a 20-amino acid region that includes consensus sites for phosphorylation by protein kinase B (PKB). Experiments were performed in MER-Akt cells to investigate the role of PKB in controlling mTOR. These cells express a PKB-mutant estrogen receptor fusion protein that is activated when the cells are exposed to 4-hydroxytamoxifen. Activating PKB with 4-hydroxytamoxifen mimicked insulin by decreasing mTOR reactivity with mTAb1 and by increasing the PHAS-I kinase activity of mTOR. Our findings support the conclusion that insulin activates mTOR by promoting phosphorylation of the protein via a signaling pathway that contains PKB.

Phosphorylation of the translational repressor PHAS-I by the mammalian target of rapamycin.

The immunosuppressant rapamycin interferes with G1-phase progression in lymphoid and other cell types by inhibiting the function of the mammalian target of rapamycin (mTOR). mTOR was determined to be a terminal kinase in a signaling pathway that couples mitogenic stimulation to the phosphorylation of the eukaryotic initiation factor (eIF)-4E-binding protein, PHAS-I. The rapamycin-sensitive protein kinase activity of mTOR was required for phosphorylation of PHAS-I in insulin-stimulated human embryonic kidney cells. mTOR phosphorylated PHAS-I on serine and threonine residues in vitro, and these modifications inhibited the binding of PHAS-I to eIF-4E. These studies define a role for mTOR in translational control and offer further insights into the mechanism whereby rapamycin inhibits G1-phase progression in mammalian cells.

The mammalian target of rapamycin phosphorylates sites having a (Ser/Thr)-Pro motif and is activated by antibodies to a region near its COOH terminus.

The eukaryotic initiation factor 4E (eIF4E)-binding protein, PHAS-I, was phosphorylated rapidly and stoichiometrically when incubated with [gamma-32P]ATP and the mammalian target of rapamycin (mTOR) that had been immunoprecipitated with an antibody, mTAb1, directed against a region near the COOH terminus of mTOR. PHAS-I was phosphorylated more slowly by mTOR obtained either by immunoprecipitation with other antibodies or by affinity purification using a rapamycin/FKBP12 resin. Adding mTAb1 to either of these preparations of mTOR increased PHAS-I phosphorylation severalfold, indicating that mTAb1 activates the mTOR protein kinase. mTAb1-activated mTOR phosphorylated Thr36, Thr45, Ser64, Thr69, and Ser82 in PHAS-I. All five of these sites fit a (Ser/Thr)-Pro motif and are dephosphorylated in response to rapamycin in rat adipocytes. Thus, our findings indicate that Pro is a determinant of the mTOR protein kinase specificity and that mTOR contributes to the phosphorylation of PHAS-I in cells.

Direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3-kinase inhibitors, wortmannin and LY294002.

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.

Protein-tyrosine kinase-dependent activation of STAT transcription factors in interleukin-2- or interleukin-4-stimulated T lymphocytes.

The proliferation of activated T lymphocytes is critically dependent on the binding of the T-cell growth factors, interleukin (IL)-2 and IL-4, to distinct but evolutionarily related cell surface receptors. Previous results suggest that the IL-2 receptor (IL-2R) and IL-4R are coupled to both overlapping and distinct intracellular signaling pathways in T lymphocytes. In this study, we demonstrate that activation of Janus tyrosine kinases (JAKs) and STAT transcription factors is rapidly induced by exposure of factor-dependent murine T-cell lines to IL-2 or IL-4. Both IL-2 and IL-4 stimulated the rapid activation of JAK1 and JAK3, whereas JAK2 activity was unaffected by either cytokine. These responses were accompanied by the appearance in cell nuclei of 3 DNA binding activities that recognized a high-affinity binding site for STAT factors. In transient transfection assays, this STAT factor target sequence conferred IL-2 and IL-4 inducibility on a synthetic luciferase reporter gene. Antibody supershifting experiments indicated that IL-2 induces the formation of STAT dimers containing STAT3 and STAT1 alpha. Although IL-4 also activated STAT1 alpha, the major IL4-induced STAT factor is not STAT3 and remains undefined. Pretreatment of the T-cells with the protein-tyrosine kinase inhibitor herbimycin A blocked both the nuclear translocation of STAT factors and STAT-dependent reporter gene transcription. Immunoblot analyses confirmed that cytoplasmic STAT3 was heavily phosphorylated on tyrosine in IL-2-stimulated cells, and that phosphorylated STAT3 appeared in the nuclei of these cells. These results indicate that identical JAKs and partially overlapping sets of STATs are activated by IL-2 and IL-4 in T lymphocytes.

Isolation of a protein target of the FKBP12-rapamycin complex in mammalian cells.

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.

Mechanism of action of rapamycin: new insights into the regulation of G1-phase progression in eukaryotic cells.

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.

Rapamycin inhibition of interleukin-2-dependent p33cdk2 and p34cdc2 kinase activation in T lymphocytes.

The immunosuppressant rapamycin (RAP) is a potent inhibitor of the entry of interleukin (IL)-2-stimulated T cells into S-phase. Earlier results indicated that RAP treatment arrested the growth of the murine IL-2-dependent T cell line CTLL-2 in late G1-phase. To explore further the interactions of RAP with the cell cycle control machinery in T cells, we examined the effects of RAP treatment on the activation of the cyclin-dependent kinases p34cdc2 and p33cdk2 in G1-phase CTLL-2 cells. Stimulation of factor-deprived cells with IL-2 led to the assembly of high molecular weight complexes containing active p34cdc2 and p33cdk2. The appearance of these complexes was explained, at least in part, by the association of both cyclin-dependent kinases with IL-2-induced cyclin A. RAP treatment profoundly inhibited both cyclin A expression and the appearance of active cyclin A-cyclin-dependent kinase complexes in IL-2-stimulated, late G1-phase CTLL-2 cells. Although p34cdc2 activation was largely dependent on association with cyclin A, a significant proportion of the active p33cdk2 pool was complexed with cyclin E. In contrast to cyclin A, the IL-2-induced accumulation of cyclin E in G1-phase cells was only partially suppressed by RAP, and cyclin E-p33cdk2 complexes were readily detected in drug-treated cells. These cyclin E-cyclin-dependent kinase complexes were nonetheless devoid of histone H1 kinase activity. The inhibitory effects of RAP on the activation of cyclin E- and cyclin A-associated cyclin-dependent kinases suggest that one or both events participate in the regulation of T cell entry into S-phase.

Rapamycin-induced inhibition of p34cdc2 kinase activation is associated with G1/S-phase growth arrest in T lymphocytes.

The macrolide rapamycin (RAP) is a potent inhibitor of interleukin-2 (IL-2)-induced T-cell proliferation. Current models suggest that RAP, when complexed to its intracellular receptor, FK506-binding protein, interferes with an IL-2 receptor-coupled signaling pathway required for cell-cycle progression from G1- to S-phase. Here we show that RAP treatment inhibits the growth of an IL-2-dependent cytotoxic T-cell line, CTLL-2, in late G1-phase, just prior to entry of the cells into S-phase. In contrast, RAP-treated CTLL-2 cells retained the ability to respond to IL-2 with enhanced cytolytic activity, indicating that RAP was not a general suppressant of cellular responsiveness to IL-2. Subsequent studies revealed that IL-2 stimulation triggered a delayed activation of the p34cdc2 kinase, the timing of which correlated with the G1- to S-phase transition. The IL-2-dependent increase in p34cdc2 kinase activity was blocked by RAP. The RAP sensitivity of the p34cdc2 activation mechanism implicates this signaling pathway in the control of S-phase commitment in IL-2-stimulated T-cells.

Reversal of desipramine toxicity in rats using drug-specific antibody Fab' fragment: effects on hypotension and interaction with sodium bicarbonate.

The effect of drug-specific antibody Fab' fragment on desipramine (DMI) toxicity was studied in anesthetized rats to determine 1) whether DMI-induced hypotension can be reversed, and 2) whether the effect of this Fab' fragment can be enhanced by the concurrent administration of hypertonic NaHCO3. DMI (60 mg/kg) was administered i.p. to produce marked hypotension. Antitricyclic antidepressant (TCA) Fab' (molar Fab'/DMI ratio = 0.11) or control Fab' was administered 15 min later as a 10 min i.v. infusion. The mean arterial pressure was higher at the end of anti-TCA Fab' infusion than after control Fab' (58 +/- 8 vs. 17 +/- 7 mm Hg, P less than .001). In a second protocol, DMI (30 mg/kg) was administered to prolong QRS duration. Anti-TCA Fab' alone (molar Fab'/DMI ratio = 0.09) and NaHCO3 alone both reduced QRS prolongation compared to control treatment, and combined therapy was more effective than either one alone. In both protocols, anti-TCA Fab' markedly increased the total DMI concentration and the bound fraction of DMI in serum, but did not alter the unbound DMI concentration. In the low DMI dose protocol, anti-TCA Fab' also reduced the cardiac DMI concentration. Concurrent treatment with anti-TCA Fab' and NaHCO3 substantially increased urinary DMI and anti-TCA Fab' excretion compared to treatment with anti-TCA Fab' alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Pretreatment with drug-specific antibody reduces desipramine cardiotoxicity in rats.

The effect of a drug-specific antibody on desipramine (DMI) cardiotoxicity was studied in rats. Animals were pretreated i.v. with 4.2 g/kg of a monoclonal antibody (anti-TCA) followed by DMI HCl 30 mg/kg i.p. (molar ratio of anti-TCA binding sites to DMI = 0.56). Peak QRS complex prolongation was substantially lower after pretreatment with anti-TCA than after control antibody (70 +/- 14 v. 21 +/- 4%, p less than 0.001). Time to peak toxicity was the same in both groups. Binding of DMI by anti-TCA was demonstrated by a higher serum total DMI concentration and increased DMI binding in serum after anti-TCA compared to controls. The DMI concentration in anti-TCA treated animals was lower in some organs (brain, lung, liver, spleen), but not in others (heart, muscle, kidney, fat). The calculated fraction of the DMI dose bound by anti-TCA was 19.9%. The steepness of the DMI dose-response curve was examined by administering DMI alone (without antibody) at various doses to rats. Compared to 30 mg/kg DMI, a dose reduction of 30-50% was needed to reduce QRS duration to the same extent as anti-TCA pretreatment. We conclude that DMI cardiotoxicity was markedly reduced by the binding of a relative small fraction of the DMI body burden to anti-TCA. This disproportionate effect of DMI binding was not due to the steepness of the DMI dose-response curve, nor to slowing of the rate of DMI distribution to tissues.

Drug-specific F(ab')2 fragment reduces desipramine cardiotoxicity in rats.

Current therapy for cardiotoxicity due to tricyclic antidepressant (TCA) overdose is often ineffective in seriously poisoned patients. We studied the effect of a drug-specific antibody fragment on TCA cardiotoxicity in rats. Animals received anti-TCA F(ab')2 2.0 g/kg i.v. over 10 min starting 15 min after administration of a toxic dose of desipramine (DMI). This anti-TCA F(ab')2 dose was 36.9% of the molar DMI dose in terms of binding sites. Anti-TCA F(ab')2 infusion had no adverse effects and rapidly reduced DMI induced QRS prolongation compared with control F(ab')2 (23 +/- 14 vs 71 +/- 11% QRS prolongation at the end of infusion, P less than 0.001). This beneficial effect lasted for the 45 min duration of the study. Markedly enhanced DMI binding in serum after anti-TCA F(ab')2 was demonstrated by a 48-fold increase in the total DMI concentration over controls and a reduction in the fraction of unbound DMI (44.5 +/- 19.4 vs 0.7 +/- 0.2%). Anti-TCA F(ab')2 reduced the DMI concentration in brain but not in other organs. We conclude that anti-TCA F(ab')2 substantially reduces DMI cardiotoxicity in rats, and does so rapidly enough to be of potential clinical benefit for patients with DMI overdose. Because only a small fraction of the DMI dose was bound by antibody, these data suggest that antibody fragment doses considerably less than equimolar to the DMI dose may be effective in treating DMI cardiotoxicity.

Redistribution of tricyclic antidepressants in rats using a drug-specific monoclonal antibody: dose-response relationship.

A monoclonal antibody was used to study the dose-response relationship for antibody-mediated redistribution of tricyclic antidepressants (TCA) in rats. The antibody (anti-TCA) was an IgG1 with Ka = 3.0 x 10(8) M-1 for desipramine (DMI) and 2.2 x 10(8) M-1 for imipramine (IMI). Anesthetized rats received 1 mg DMI (10% of the toxic dose), followed in 15 min by anti-TCA iv at doses representing anti-TCA/DMI molar ratios of 0.003, 0.013, and 0.07. Anti-TCA produced prompt, dose-related increases in the serum DMI concentration of 33, 164, and 776%. Similar results were obtained in rats treated with IMI. The highest dose of anti-TCA reduced the concentration of IMI in the heart. In a second protocol, the anti-TCA dose was kept constant and the DMI or IMI dose varied. The increase in serum drug concentration was 1750, 1260, and 150% (DMI) and 1460, 1200, and 170% (IMI) at drug doses of 0.1, 10, and 1000 micrograms. Thus, the percentage increase in serum drug concentration was diminished only 12-fold (DMI) or 9-fold (IMI) by a 10,000-fold increase in drug dose. At the highest anti-TCA/DMI ratio (lowest DMI dose), tissue DMI concentrations were significantly reduced. We conclude that 1) anti-TCA can effect substantial redistribution of a subtoxic dose of DMI or IMI, even when the antibody dose is less than equimolar to the TCA dose, and 2) the extent of TCA redistribution depends upon the doses of both antibody and drug; anti-TCA is most effective when the body burden of TCA is high. These data support the potential therapeutic use of anti-TCA for DMI or IMI toxicity, and should be useful in anticipating the dose and affinity of anti-TCA required.