tacrolimus and Neuroblastoma

Tacrolimus (FK506)/mycophenolate mofetil (MMF) has been demonstrated to be an effective salvage therapy for steroid-resistant chronic graft-versus-host disease (GVHD), but its effectiveness as prophylaxis for acute GVHD (aGVHD) is unknown. We investigated the safety and efficacy of FK506/MMF in preventing aGVHD and sparing the use of methotrexate and methylprednisolone in childhood and adolescent allogeneic stem cell transplant (AlloSCT) recipients. Thirty-four childhood and adolescent patients (median age, 7 years; range, 0.5-21 years; 24 males and 10 females) undergoing 37 AlloSCTs for malignant (n = 22) and nonmalignant (n = 12) disorders received FK506 (0.03 mg/kg/d by continuous intravenous infusion) and MMF (15 mg/kg per dose orally or intravenously twice daily). Stem cell sources included 22 umbilical cord blood donors (21 unrelated and 1 related), 6 related bone marrow donors, and 9 related peripheral blood donors. Malignant diagnoses included 7 acute lymphoblastic leukemias, 3 acute myeloid leukemias, 1 acute promyelocytic leukemia, 2 non-Hodgkin lymphomas, 4 Hodgkin diseases, 3 chronic myeloid leukemias, and 2 neuroblastomas; nonmalignant diagnoses included 2 beta-thalassemias, 1 sickle cell disease, 4 aplastic anemias, 1 Wiskott-Aldrich syndrome, 1 Hurler syndrome, 2 hemophagocytic lymphohistiocytoses, and 1 myelodysplastic syndrome. The probability of developing grade > or =II aGVHD was 45.4% +/- 9.7% (7 related bone marrow/related peripheral blood; 5 umbilical cord blood), and for chronic GVHD it was 38.1% +/- 19.7%. FK506/MMF was well tolerated. Three patients had grade III to IV neurotoxicity (disorientation and leukoencephalopathy); 4 patients developed grade III to IV nephrotoxicity (all received concomitant nephrotoxins). Patients who achieved target mycophenolic acid levels (1.0-3.5 microg/mL) before day +30 had a significantly reduced incidence of developing grade >/=II aGVHD (16.7% +/- 15.2% versus 100%; P <.02). These results suggest that FK506/MMF is well tolerated and may be a safe and effective methotrexate- and methylprednisolone-sparing alternative GVHD prophylaxis regimen after AlloSCT. Further pharmacokinetic and pharmacodynamic studies are ongoing in pediatric and adolescent AlloSCT recipients to define optimal MMF dosing.

Topics: Adolescent; Adult; Child; Child, Preschool; Drug Resistance; Drug Therapy, Combination; Female; Graft vs Host Disease; Hematologic Diseases; Humans; Immunosuppressive Agents; Infant; Male; Mucopolysaccharidosis I; Mycophenolic Acid; Neuroblastoma; Pilot Projects; Prognosis; Stem Cell Transplantation; Tacrolimus; Transplantation Conditioning; Transplantation, Homologous; Treatment Outcome

Neurotoxicity is one of the major side effects caused by calcineurin inhibitors such as tacrolimus in clinical practice. The underlying mechanisms remain unclear, and no potential protective agents have been identified yet. Here, we aimed to investigate tacrolimus-induced neurotoxicity and assess the protective effects of ibudilast, a nonselective phosphodiesterase inhibitor with neuroprotective effects, against tacrolimus-induced neurotoxicity. An in vitro assay of human neuroblastoma SH-SY5Y cells showed that ibudilast reduced tacrolimus-induced cell death. Subsequently, using in vivo studies, we assessed the pathological mechanism of neurotoxicity and evaluated the protective effect of ibudilast. Wistar rats were subcutaneously administered tacrolimus (2.5 or 5.0 mg/kg/day) for 14 d, and ibudilast (7.5 mg/kg/day) was intraperitoneally administered once a day beginning 2 d prior to tacrolimus (5 mg/kg/day) administration. We observed that ibudilast significantly reduced the tacrolimus-induced neurotoxic events. From the assessment of excised brains, we found that tacrolimus was penetrated to brain and the brain concentration was correlated with the neurotoxicity-score, although ibudilast had no effect on this pharmacokinetics. Tacrolimus-induced neuronal damage was histopathologically evaluated using Nissl and TUNEL staining, where only the cerebral cortex and CA1 region in hippocampus exhibited neuronal death, but not the CA3 region, dendrite gyrus, and cerebellum. Co-administration of ibudilast significantly attenuated these histopathological changes. In conclusion, these results suggest that tacrolimus translocation into the brain and neuronal damage in the cerebral cortex and CA1 are the underlying mechanisms of tacrolimus-induced neurotoxicity and that ibudilast could be a protective agent against this adverse event.

Topics: Animals; Humans; Neuroblastoma; Neuroprotective Agents; Neurotoxicity Syndromes; Pyridines; Rats; Rats, Wistar; Tacrolimus

The vast majority of therapeutic approaches tested so far for prion diseases, transmissible neurodegenerative disorders of human and animals, tackled PrP

Topics: Animals; Casein Kinase II; Cell Line, Tumor; Cell Membrane; Cell Survival; Drug Evaluation, Preclinical; Fluorescent Dyes; Gene Expression; Green Fluorescent Proteins; Harmaline; HEK293 Cells; Hematoxylin; Humans; Mice; Neuroblastoma; Prions; PrPC Proteins; Quinacrine; Tacrolimus

Trafficking of G protein-coupled receptors plays a crucial role in controlling the precise signalling of the receptor as well as its proper regulation. Metabotropic glutamate receptor 1 (mGluR1), a G protein-coupled receptor, is a member of the group I mGluR family. mGluR1 plays a critical role in neuronal circuit formation and also in multiple types of synaptic plasticity. This receptor has also been reported to be involved in various neuropsychiatric diseases. Other than the central nervous system, mGluR1 plays crucial roles in various non-neuronal cells like hepatocytes, skin cells, etc. Although it has been reported that mGluR1 gets endocytosed on ligand application, the events after the internalization of the receptor has not been studied. We show here that mGluR1 internalizes on ligand application. Subsequent to endocytosis, majority of the receptors localize at the recycling compartment and no significant presence of the receptor was noticed in the lysosome. Furthermore, mGluR1 returned to the cell membrane subsequent to ligand-mediated internalization. We also show here that the recycling of mGluR1 is dependent on the activity of protein phosphatase 2A. Thus, our data suggest that the ligand-mediated internalized receptors recycle back to the cell surface in protein phosphatase 2A-dependent manner.

Topics: Ammonium Chloride; Animals; Calcineurin Inhibitors; Cell Line, Tumor; Cell Membrane; Cyclosporine; Endocytosis; HEK293 Cells; Humans; Methoxyhydroxyphenylglycol; Mice; Neuroblastoma; Protein Phosphatase 2; Receptors, Metabotropic Glutamate; RNA, Small Interfering; Tacrolimus; Time Factors; Transfection

FKBP51 and FKBP52 (FK506-binding protein 51 and 52) are tetratricopeptide repeat-domain immunophilins belonging to the tetratricopeptide-protein•hsp90•hsp70•p23 heterocomplex bound to steroid receptors. Immunophilins are related to receptor folding, subcellular localization, and hormone-dependent transcription. Also, they bind the immunosuppressant macrolide FK506, which shows neuroregenerative and neuroprotective actions by a still unknown mechanism. In this study, we demonstrate that in both, undifferentiated neuroblastoma cells and embryonic hippocampal neurons, the FKBP52•hsp90•p23 heterocomplex concentrates in a perinuclear structure. Upon cell stimulation with FK506, this structure disassembles and this perinuclear area becomes transcriptionally active. The acquisition of a neuronal phenotype is accompanied by increased expression of βIII-tubulin, Map-2, Tau-1, but also hsp90, hsp70, p23, and FKBP52. During the early differentiation steps, the perinuclear heterocomplex redistributes along the cytoplasm and nascent neurites, p23 binds to intermediate filaments and microtubules acquired higher filamentary organization. While FKBP52 moves towards neurites and concentrates in arborization bodies and terminal axons, FKBP51, whose expression remains constant, replaces FKBP52 in the perinuclear structure. Importantly, neurite outgrowth is favored by FKBP52 over-expression or FKBP51 knock-down, and is impaired by FKBP52 knock-down or FKBP51 over-expression, indicating that the balance between these FK506-binding proteins plays a key role during the early mechanism of neuronal differentiation.

Topics: Animals; Cell Differentiation; Cell Line, Tumor; Female; Fluorescent Antibody Technique, Indirect; Hippocampus; HSP90 Heat-Shock Proteins; Immunophilins; Immunosuppressive Agents; Neurites; Neuroblastoma; Neurons; Pregnancy; Rats; RNA, Messenger; Signal Transduction; Subcellular Fractions; Tacrolimus; Tacrolimus Binding Proteins; Transfection; Uridine Triphosphate

This study examined the role of calcineurin, a major calcium-dependent protein phosphatase, in dephosphorylating Ser-9 and activating glycogen synthase kinase-3beta (GSK-3beta). Treatment with calcineurin inhibitors increased phosphorylation of GSK-3beta at Ser-9 in SH-SY5Y human neuroblastoma cells. The over-expression of a constitutively active calcineurin mutant, calcineurin A beta (1-401), led to a significant decrease in phosphorylation at Ser-9, an increase in the activity of GSK-3beta, and an increase in the phosphorylation of tau. K(m) of calcineurin for a GSK-3beta phosphopeptide was 469.3 microM, and specific activity of calcineurin was 15.2 nmol/min/mg. In addition, calcineurin and GSK-3beta were co-immunoprecipitated in neuron-derived cells and brain tissues, and calcineurin formed a complex only with dephosphorylated GSK-3beta. We conclude that in vitro, calcineurin can dephosphorylate GSK-3beta at Ser-9 and form a stable complex with GSK-3beta, suggesting the possibility that calcineurin regulates the dephosphorylation and activation of GSK-3betain vivo.

Topics: Animals; Calcineurin; Calcineurin Inhibitors; Cell Line, Tumor; Cyclosporine; Enzyme Activation; Enzyme Inhibitors; Glioma; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Male; Mice; Mutation; Neuroblastoma; Neurons; Phosphorylation; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Serine; Tacrolimus; Transfection

Prion diseases or transmissible spongiform encephalopathies (TSEs) are infectious and fatal neurodegenerative disorders in humans and animals. Pathological features of TSEs include the conversion of cellular prion protein (PrP(C)) into an altered disease-associated conformation generally designated PrP(Sc), abnormal deposition of PrP(Sc) aggregates, and spongiform degeneration of the brain. The molecular steps leading to PrP(C) aggregation are unknown. Here, we have utilized an inducible oligomerization strategy to test if, in the absence of any infectious prion particles, the encounter between PrP(C) molecules may trigger its aggregation in neuronal cells. A chimeric PrP(C) composed of one (Fv1) or two (Fv2) modified FK506-binding protein (Fv) fused with PrP(C) were created, and transfected in N2a cells. Similar to PrP(C), Fv1-PrP and Fv2-PrP were glycosylated, displayed normal localization, and anti-apoptotic function. When cells were treated with the dimeric Fv ligand AP20187, to induce dimerization (Fv1) or oligomerization (Fv2) of PrP(C), both dimerization and oligomerization of PrP(C) resulted in the de novo production, release and deposition of extracellular PrP aggregates. Aggregates were insoluble in non-ionic detergents and partially resistant to proteinase K. These findings demonstrate that homologous interactions between PrP(C) molecules may constitute a minimal and sufficient molecular event leading to PrP(C) aggregation and extracellular deposition.

Topics: Animals; Binding Sites; Cell Line, Tumor; Dimerization; Dose-Response Relationship, Drug; Endopeptidase K; Gene Expression Regulation; Glioma; Humans; Ligands; Mice; Microscopy, Electron, Transmission; Mutation; Neuroblastoma; Prions; Protein Binding; Tacrolimus; Transfection

Neurite outgrowth plays a key role in neuronal development and regeneration, and is the hallmark assay for the effects of neurotrophic factors such as nerve growth factor (NGF). However, measuring neurite outgrowth is a slow and resource-intensive process. We therefore wanted to identify surrogate biomarkers for neurite outgrowth activity by gene expression analysis in SH-O10 cells, a subclone of the human SH-SY5Y neuroblastoma cell line but with much higher NGF-induced neurite outgrowth activity. Microarray analysis identified seven genes where mRNA levels were changed. NGF-induced decreases in levels of two genes, CyclinB2 and BIRC5, were confirmed by quantitative real-time RT-PCR. Levels of NGF-induced decreases in CyclinB2 and BIRC5 mRNA in several SH-SY5Y subclones with different neurite outgrowth responses correlated with their neurite outgrowth activities. Decreases in CyclinB2 and BIRC5 mRNA induced by FK506 or retinoic acid, both of which exert potentiation of NGF-induced neurite outgrowth effects but with different mechanisms, also correlated with their neurite outgrowth activities. In conclusion, decreasing levels of CyclinB2 and BIRC5 mRNA strongly correlate with neurite outgrowth activities in terms of NGF-related effect in SH-SY5Y subclonal cells, and have potential to become quantitative surrogate biomarkers for measuring NGF-related neurite outgrowth.

Topics: Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Cyclin B; Dose-Response Relationship, Drug; Drug Interactions; Gene Expression; Humans; Immunosuppressive Agents; Inhibitor of Apoptosis Proteins; Linear Models; Microtubule-Associated Proteins; Neoplasm Proteins; Nerve Growth Factor; Neurites; Neuroblastoma; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Survivin; Tacrolimus; Tretinoin

The mechanism of action of the neurotoxin 6-hydroxydopamine (6-OHDA) is thought to involve the generation of free radicals and subsequent apoptotic processes. We have demonstrated in vitro that the neuroimmunophilin, FK506 (10-100 nM), dose dependently and significantly restored the ROS production to the control level, increased the Bcl-2 protein level, partly inhibited the cytochrome C release from mitochondria and reduced the caspase-3 activation in SH-SY5Y cells. On the other hand, there was no significant restoration of the ATP level by FK506 and the toxin activated proteins, p53 and Bax, were not normalized by FK506. In support of these latter results, daily administration of FK506 for 7 days to rats (0.5, 1 and 3 mg/kg i.p.) did not significantly prevent the apomorphine-induced contralateral circling, measured 2 weeks after unilateral nigral lesioning. Moreover, FK506 pretreatment did not significantly lower the toxin elevated lipid peroxidation levels, indicating that oxidative stress was present even after the FK506 treatment in the lesioned striatum. Taken together, our results with FK506 are inconsistent. We confirm the antioxidant nature of FK506, that is, it blocks ROS production in SH-SY5Y cells. However, there were no significant protective effects in any apoptotic analyses in SH-SY5Y cells and in animal studies, a 7-day FK506 pre-treatment was not able to reverse the toxic effect of 6-OHDA in a rat model of Parkinson's disease.

Topics: Animals; Apomorphine; Behavior, Animal; Caspase 3; Caspases; Cell Death; Cell Line, Tumor; Disease Models, Animal; Dopamine; Dopamine Agonists; Dose-Response Relationship, Drug; Immunophilins; Male; Motor Activity; Nerve Degeneration; Neuroblastoma; Neurons; Neuroprotective Agents; Oxidopamine; Parkinson Disease; Random Allocation; Rats; Rats, Wistar; Tacrolimus

The mechanism of the neuroprotective effect of FK506 in relation to nitric oxide (NO) production has not been clarified in vivo. We have investigated the effect of FK506 on ischemia-induced NO production in association with the pathogenesis of delayed neuronal death (DND) in rats.. In vivo microdialysis was performed in the hippocampus of male Sprague-Dawley rats (250-350 g). Dialysate samples were collected every 3 min. In the ischemia group (n=16), global ischemia was induced for 21 min and reperfusion was achieved. In the FK506 treatment group (n=25), FK506 (1 mg/kg, i.v.) was administered 21 min prior to the onset of global ischemia. Sham operations were done (n=15). The levels of NO(2)(-) in the dialysate samples were determined by the Griess reaction. The animals were decapitated 7 days after ischemia. Coronal brain sections were stained with hematoxylin and eosin.. In the ischemia group, the NO(2)(-) level significantly increased during ischemia. In the FK506 treatment group, there was no significant change in the NO(2)(-) level during ischemia. In histological examinations, FK506 treatment showed a neuroprotective effect against DND.. The effect of FK506 inhibiting NO production contributes to the neuro-protective effect of FK506 on DND in the hippocampus.

Topics: Analysis of Variance; Animals; Brain Ischemia; Cell Count; Cell Death; Cell Line; Cell Survival; Dose-Response Relationship, Drug; Hippocampus; Humans; L-Lactate Dehydrogenase; Male; Microdialysis; Neuroblastoma; Neurons; Neuroprotective Agents; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Rats; Rats, Sprague-Dawley; Reperfusion; Staining and Labeling; Tacrolimus; Tetrazolium Salts; Thiazoles

The immunophilin FKBP12 associates with intracellular Ca2+ channels and this interaction can be disrupted by the immunosuppressant FK506. We have investigated the effect of FK506 on Ca2+ release and Ca2+ uptake in permeabilized cell types. Changes in medium free [Ca2+] were detected by the fluorescent Ca2+ indicator fluo-3 in digitonin-permeabilized SH-SY5Y human neuroblastoma cells, DT40 and R23-11 (i.e. triple inositol 1,4,5-trisphosphate (IP3) receptor knockout cells) chicken B lymphocytes and differentiated and undifferentiated BC3H1 skeletal muscle cells. 45Ca2+ fluxes were studied in saponin-permeabilized A7r5 rat smooth muscle cells. Addition of FK506 to permeabilized SH-SY5Y cells led to a sustained elevation of the medium [Ca2+] corresponding to approximately 30 % of the Ca2+ ionophore A23187-induced [Ca2+] rise. This rise in [Ca2+] was not dependent on mitochondrial activity. This FK506-induced [Ca2+] rise was related to the inhibition of the sarcoplasmic/endoplasmic reticulum Ca2+-Mg2+-ATPase (SERCA) Ca2+ pump. Oxalate-facilitated 45Ca2+ uptake in SH-SY5Y microsomes was inhibited by FK506 with an IC50 of 19 microM. The inhibition of the SERCA Ca2+ pump was not specific since several macrocyclic lactone compounds (ivermectin > FK506, ascomycin and rapamycin) were able to inhibit Ca2+ uptake activity. FK506 (10 microM) did not affect IP3-induced Ca2+ release in permeabilized SH-SY5Y and A7r5 cells, but enhanced caffeine-induced Ca2+ release via the ryanodine receptor (RyR) in differentiated BC3H1 cells. In conclusion, FK506 inhibited active Ca2+ uptake by the SERCA Ca2+ pump; in addition, FK506 enhanced intracellular Ca2+ release through the RyR, but it had no direct effect on IP3-induced Ca2+ release.

Topics: Animals; Antiprotozoal Agents; Aorta; B-Lymphocytes; Biological Transport; Caffeine; Calcimycin; Calcium; Calcium Channels; Calcium Signaling; Calcium-Transporting ATPases; Chickens; Enzyme Inhibitors; Humans; Immunosuppressive Agents; Inositol 1,4,5-Trisphosphate Receptors; Ionophores; Ivermectin; Mice; Microsomes; Muscle, Smooth, Vascular; Neuroblastoma; Oxalates; Phosphodiesterase Inhibitors; Rats; Receptors, Cytoplasmic and Nuclear; Sirolimus; Spermine; Tacrolimus; Thapsigargin; Tumor Cells, Cultured

The Ca2+/calmodulin-dependent protein phosphatase 2B (PP2B) also known as calcineurin (CN) has been implicated in the Ca2+-dependent inactivation of Ca2+ channels in several cell types. To study the role of calcineurin in the regulation of Ca2+-channel activity, phosphatase expression was altered in NG108-15 cells by transfection of sense and antisense plasmid constructs carrying the catalytic subunit of human PP2Bbeta3. Relative to mock-transfected (wild-type) controls, cells overexpressing calcineurin showed dramatically reduced high-voltage-activated Ca2+ currents which were recoverable by the inclusion of 1 microM FK506 in the patch pipette. Conversely, in cells with reduced calcineurin expression, high-voltage-activated Ca2+ currents were larger relative to controls. Additionally in these cells, low-voltage-activated currents were significantly reduced. Analysis of high-voltage-activated Ca2+ currents revealed that the kinetics of inactivation were significantly accelerated in cells overexpressing calcineurin. Following the delivery of a train of depolarizing pulses in experiments designed to produce large-scale Ca2+ influx across the cell membrane, Ca2+-dependent inactivation of high-voltage-activated Ca2+ currents was increased in sense cells, and this increase could be reduced by intracellular application of 1 mM BAPTA or 1 microM FK506. These data support a role of calcineurin in the negative feedback regulation of Ca2+ entry through voltage-operated Ca2+ channels.

Topics: Animals; Antisense Elements (Genetics); Calcineurin; Calcium; Calcium Channels; Chelating Agents; Egtazic Acid; Gene Expression; Glioma; Humans; Immunoblotting; Immunosuppressive Agents; Membrane Potentials; Neuroblastoma; Neurons; Patch-Clamp Techniques; Tacrolimus; Transfection; Tumor Cells, Cultured

Ceramide is a key mediator of apoptosis during the cellular stress response which is also involved in stroke-induced death. Transient occlusion of the middle cerebral artery (MCA) in rats led to a strong generation of ceramide as measured in thalamus and entorhinal cortex of the ischemic brain tissue. Enhanced levels of ceramide may be involved in apoptosis signaling following stroke since exogenously added synthetic C2-ceramide increased expression of c-jun and the death-inducing ligands (DILs) CD95-L, TRAIL and TNF-alpha in neuroblastoma cells. DILs in turn mediated death via binding to their respective receptors as concluded from diminished apoptosis upon blocking of the common pathway by dominant negative FADD. C2-ceramide induced both necrosis and apoptosis in a concentration-dependent manner corresponding to the situation present in the ischemic brain. The immunosuppressant FK506 inhibited the release of ceramide, expression of CD95-L and apoptosis in an in vitro and in vivo model for ischemia/reperfusion. These data suggest that ceramide is a crucial initiator of death, e.g., by induction of DILs following stroke.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Brain Chemistry; Cerebrovascular Disorders; Enzyme Inhibitors; Fas Ligand Protein; Gene Expression; Humans; Immunosuppressive Agents; Ischemic Attack, Transient; Male; Membrane Glycoproteins; Necrosis; Neuroblastoma; Neurons; Proto-Oncogene Proteins c-jun; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction; Sphingosine; Tacrolimus; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Up-Regulation

The neurotrophic property of the immunosuppressant drug FK506 (tacrolimus) is believed to depend on the 12-kDa FK506-binding protein (FKBP-12). Here, we show that FK506 maintains its neurotrophic activity in primary hippocampal cell cultures from FKBP-12 knockout mice. In human neuroblastoma SH-SY5Y cells, the neurotrophic action of FK506 (10 pM to 10 nM) is completely prevented by the addition of a monoclonal antibody (50-100 nM) to the immunophilin FKBP-52 (also known as FKBP-59 or heat shock protein 56), a component of mature steroid receptor complexes. By itself, the FKBP-52 antibody is also neurotrophic. The neurotrophic activity of dexamethasone (50 nM) is potentiated by FK506, whereas that of beta-estradiol (50 nM) is not altered, suggesting a common mechanisms of action. Geldanamycin (which disrupts mature steroid receptor complexes) is also neurotrophic (0.1-10 nM), whereas it reduces the neurotrophic activity of FK506 and steroid hormones (dexamethasone and beta-estradiol). Conversely, 20 mM molybdate (which prevents the disruption of mature steroid receptor complexes) decreases the neurotrophic activity of FK506, FKBP-52 antibody, dexamethasone, and beta-estradiol. In rats, FK506 (10 mg/kg s.c.) augments the regenerative response of regenerating motor and sensory neurons to nerve injury as shown by its ability to increase the axotomy-induced induction of c-jun expression. A model is proposed to account for the neurotrophic action of both neuroimmunophilin ligands (FK506) and steroid hormones. Components of steroid receptor complexes represent novel targets for the rational design of new neurotrophic drugs.

Topics: Animals; Antibodies; Benzoquinones; Cells, Cultured; Cysteine Proteinase Inhibitors; Dexamethasone; Embryo, Mammalian; Estradiol; Hippocampus; Humans; Immunophilins; Lactams, Macrocyclic; Mice; Mice, Knockout; Molybdenum; Motor Neurons; Nerve Growth Factors; Nerve Regeneration; Neurites; Neuroblastoma; Neurons; Neurons, Afferent; Neuroprotective Agents; Proto-Oncogene Proteins c-jun; Quinones; Rats; Rats, Sprague-Dawley; Sciatic Nerve; Tacrolimus; Tacrolimus Binding Proteins; Tumor Cells, Cultured

Programmed cell death (PCD) is a prominent feature of the development of the immune and nervous systems. In both systems, widespread PCD occurs in primitive progenitor cells during development. In this study, we demonstrated that Ewing's sarcoma (ES) cells, undifferentiated neural precursors, underwent apoptosis upon engagement of CD99 with anti-CD99 monoclonal antibody. Apoptosis via CD99 occurred only in the undifferentiated state of ES cells, but not in differentiated ES cells. CD99-induced apoptosis in ES cells appeared to require de novo synthesis of RNA and protein as well as caspase activation. Cyclosporin A, known to be a potent inhibitor of both calcineurin activation and mitochondrial permeability transition pore opening, inhibited CD99-mediated apoptosis, whereas FK-506, a specific calcineurin inhibitor, did not, indicating the induction of CD99-mediated apoptosis through a calcineurin-independent pathway. Furthermore, the dying cells displayed the reduction of mitochondrial transmembrane potential (delta psi m). These results suggest that CD99 engagement induce CsA-inhibitable mitochondrial permeability transition pore opening, followed by a reduction of delta psi m and caspase activation, thereby leading to apoptosis. Based on these results, we suggest the possible involvement of CD99 in the apoptotic processes that occur during nervous system development and also its application in immunotherapeutic trials for ES cases.

Topics: 12E7 Antigen; Antibodies, Monoclonal; Antigens, CD; Apoptosis; Calcineurin; Calcium; Caspase Inhibitors; Caspases; Cell Adhesion Molecules; Cell Differentiation; Cyclosporine; Dactinomycin; Fluorescent Antibody Technique, Indirect; Humans; Membrane Potentials; Microscopy, Confocal; Microscopy, Electron; Neuroblastoma; Neuroectodermal Tumors, Primitive; Sarcoma, Ewing; Tacrolimus; Tumor Cells, Cultured

The immunosuppressant drugs FK506 and cyclosporin A inhibit T-cell proliferation via a common mechanism: calcineurin inhibition following binding to their respective binding proteins, the peptidyl prolyl isomerases FKBP-12 and cyclophilin A. In contrast, FK506, but not cyclosporin A, accelerates nerve regeneration. In the present study, we show that the potent FKBP-12 inhibitor V-10,367, which lacks the structural components of FK506 required for calcineurin inhibition, increases neurite outgrowth in SH-SY5Y neuroblastoma cells and speeds nerve regeneration in the rat sciatic nerve crush model. In SH-SY5Y cells, V-10,367 increased the lengths of neurite processes in a concentration-dependent (between 1 and 10 nM) fashion over time (up to 168 h). Daily subcutaneous injections of V-10,367 accelerated the onset of clinical signs of functional recovery in the hind feet compared to vehicle-treated control animals. Interdigit distances (between the first and fifth digits) measured on foot prints obtained during walking showed an increase in toe spread in V-10,367-treated rats compared to vehicle-treated controls. Electron microscopy demonstrated larger regenerating axons distal to the crush site in the sciatic nerve from V-10,367-treated rats. Quantitation of axonal areas in the soleus nerve revealed a shift to larger axonal calibers in V-10,367-treated rats (400 or 200 mg/kg/day); mean axonal areas were increased by 52 and 59%, respectively, compared to vehicle-treated controls. FKBP-12 ligands lacking calcineurin inhibitory activity represent a new class of potential drugs for the treatment of human peripheral nerve disorders.

Topics: Animals; Calcineurin Inhibitors; Carrier Proteins; DNA-Binding Proteins; Drug Evaluation, Preclinical; Heat-Shock Proteins; Humans; Injections, Subcutaneous; Locomotion; Male; Molecular Structure; Nerve Crush; Nerve Regeneration; Neurites; Neuroblastoma; Neuroprotective Agents; Pyridines; Rats; Rats, Sprague-Dawley; Sciatic Nerve; Stimulation, Chemical; Structure-Activity Relationship; Tacrolimus; Tacrolimus Binding Proteins; Tumor Cells, Cultured

Topics: Animals; Antibodies, Monoclonal; Blotting, Western; Brain Chemistry; Brain Neoplasms; Carrier Proteins; Cerebellum; Cerebral Cortex; DNA-Binding Proteins; Heat-Shock Proteins; Hippocampus; Humans; Neuroblastoma; Olfactory Bulb; Organ Specificity; Rats; Recombinant Proteins; Tacrolimus; Tacrolimus Binding Proteins; Tumor Cells, Cultured