ABSTRACT
Accumulation of transactive response DNA binding protein (TDP-43) fragments in motor neurons is a post mortem hallmark of different neurodegenerative diseases. TDP-43 fragments are the products of the apoptotic caspases-3 and -7. Either excessive or insufficient cellular Ca(2+) availability is associated with activation of apoptotic caspases. However, as far as we know, it is not described whether activation of caspases, due to restricted intracellular Ca(2+), affects TDP-43 cleavage. Here we show that in various cell lineages with restricted Ca(2+) availability, TDP-43 is initially cleaved by caspases-3 and -7 and then, also by caspases-6 and -8 once activated by caspase-3. Furthermore, we disclose the existence of a TDP-43 caspase-mediated fragment of 15kDa, in addition to the well-known fragments of 35 and 25kDa. Interestingly, with respect to the other two fragments this novel fragment is the major product of caspase activity on murine TDP-43 whereas in human cell lines the opposite occurs. This outcome should be considered when murine models are used to investigate TDP-43 proteinopathies.
Subject(s)
Apoptosis/genetics , Calcium/metabolism , Caspases/metabolism , DNA-Binding Proteins/metabolism , Animals , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Gene Expression Regulation , HeLa Cells , Humans , MiceABSTRACT
Lapatinib is a highly selective reversible inhibitor of the tyrosine kinase domains of HER2 and EGFR, approved for the treatment of advanced stage HER2-overexpressing breast cancers. Although targeted therapy with lapatinib provides initial clinical advantage, cancer cells' adaptive responses can overcome the inhibitory effects of lapatinib. HER3 upregulation and autocrine induction of HER3 ligand neuregulin-1 (NRG), have been implicated in the restoration of AKT and ERK1/2 activity and rescue of cell proliferation. In this study we evaluated the effects of lapatinib alone and in combination with AMPK activator GSK-621 in HER2-overexpressing breast cancer cell lines SKBR3 and BT474. Our results show that in cells exposed to lapatinib and GSK-621 in combination, lapatinib-mediated HER3 upregulation was reduced and reactivation of AKT and ERK1/2 kinases was prevented. The two drugs in combination decreased cell viability in a synergistic manner and greatly reduced the ability of NRG to rescue cell proliferation. Finally, we provide evidence that in cells exposed to lapatinib and GSK-621 in combination the establishment of a transcriptionally permissive chromatin structure at the HER3 promoter is hampered. The results of this study highlight a potential role for AMPK activation in counteracting lapatinib-induced adaptive responses of HER2-overexpressing breast cancer cells.
Subject(s)
Antineoplastic Agents , Breast Neoplasms , AMP-Activated Protein Kinases/metabolism , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Proliferation , Chromatin , Female , Humans , Lapatinib/pharmacology , Ligands , Neuregulin-1/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Quinazolines/pharmacology , Receptor, ErbB-2/genetics , Receptor, ErbB-2/metabolism , Signal Transduction , Up-RegulationABSTRACT
In recent years, human dihydroorotate dehydrogenase inhibitors have been associated with acute myelogenous leukemia as well as studied as potent host targeting antivirals. Starting from MEDS433 (IC50 1.2 nM), we kept improving the structure-activity relationship of this class of compounds characterized by 2-hydroxypyrazolo[1,5-a]pyridine scaffold. Using an in silico/crystallography supported design, we identified compound 4 (IC50 7.2 nM), characterized by the presence of a decorated aryloxyaryl moiety that replaced the biphenyl scaffold, with potent inhibition and pro-differentiating abilities on AML THP1 cells (EC50 74 nM), superior to those of brequinar (EC50 249 nM) and boosted when in combination with dipyridamole. Finally, compound 4 has an extremely low cytotoxicity on non-AML cells as well as MEDS433; it has shown a significant antileukemic activity in vivo in a xenograft mouse model of AML.
Subject(s)
Leukemia, Myeloid, Acute , Oxidoreductases Acting on CH-CH Group Donors , Animals , Humans , Mice , Antiviral Agents/pharmacology , Dihydroorotate Dehydrogenase , Dipyridamole/therapeutic use , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Leukemia, Myeloid, Acute/drug therapy , Pyridines/pharmacology , Pyridines/therapeutic use , Structure-Activity RelationshipABSTRACT
TDP-43, encoded by TARDBP, is a ubiquitously expressed, primarily nuclear protein. In recent years, TDP-43 has been identified as the major pathological protein in ALS due to its mislocalisation in the cytoplasm of motor neurons of patients with and without TARDBP mutations and expression in forms that do not match its predicted molecular weight. In this study, the TDP-43 profile was investigated using western immunoblot analysis in whole lysates, nuclei and cytoplasm of circulating lymphomonocytes from 16 ALS patients, 4 with (ALS/TDP+) and 12 without (ALS/TDP-) TARDBP mutations in the protein C-terminal domain, and thirteen age-matched, healthy donors (controls). Three disease-unaffected first-degree relatives of an ALS/TDP+ patient were also included: one carried the parent mutation (Rel/TDP+) whereas the other two did not (Rel/TDP-). In all ALS patients, relatives and controls, TDP-43 retained the predicted molecular weight in whole cell lysates and nuclei, but in the cytoplasm its molecular weight was slightly smaller than expected. In quantitative terms, TDP-43 was expressed at approximately the same levels in whole cell lysates of ALS patients, relatives and controls. In contrast, TDP-43 accumulated in the cytoplasm with concomitant nuclear depletion in all ALS/TDP+ patients, in about 50% of ALS/TDP- patients and in the Rel/TDP+ subject compared to the controls. In the remaining ALS/TDP- patients and in the two Rel/TDP- subjects, TDP-43 matched the control levels in both subcellular compartments. Were these findings further confirmed, circulating lymphomonocytes could be informative of TDP-43 mislocalisation in nervous tissue and used as a biomarker for future disease risk.
Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Cytoplasm/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Lymphocyte Subsets/metabolism , Lymphocyte Subsets/pathology , Monocytes/metabolism , Monocytes/pathology , Adult , Aged , Amyotrophic Lateral Sclerosis/genetics , Cytoplasm/pathology , DNA-Binding Proteins/chemistry , Female , Humans , Inclusion Bodies/metabolism , Inclusion Bodies/pathology , Male , Middle Aged , Mutation/geneticsABSTRACT
The connection with acute myelogenous leukemia (AML) of dihydroorotate dehydrogenase (hDHODH), a key enzyme in pyrimidine biosynthesis, has attracted significant interest from pharma as a possible AML therapeutic target. We recently discovered compound 1, a potent hDHODH inhibitor (IC50 = 1.2 nM), able to induce myeloid differentiation in AML cell lines (THP1) in the low nM range (EC50 = 32.8 nM) superior to brequinar's phase I/II clinical trial (EC50 = 265 nM). Herein, we investigate the 1 drug-like properties observing good metabolic stability and no toxic profile when administered at doses of 10 and 25 mg/kg every 3 days for 5 weeks (Balb/c mice). Moreover, in order to identify a backup compound, we investigate the SAR of this class of compounds. Inside the series, 17 is characterized by higher potency in inducing myeloid differentiation (EC50 = 17.3 nM), strong proapoptotic properties (EC50 = 20.2 nM), and low cytotoxicity toward non-AML cells (EC30(Jurkat) > 100 µM).
Subject(s)
Biphenyl Compounds/chemistry , Enzyme Inhibitors/chemistry , Oxidoreductases Acting on CH-CH Group Donors/antagonists & inhibitors , Pyrazoles/chemistry , Pyridines/chemistry , Animals , Apoptosis/drug effects , Binding Sites , Cell Differentiation/drug effects , Cell Line, Tumor , Dihydroorotate Dehydrogenase , Drug Design , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Female , Half-Life , Humans , Leukemia, Myeloid, Acute/drug therapy , Male , Mice , Mice, Inbred BALB C , Microsomes, Liver/metabolism , Molecular Docking Simulation , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Pyrazoles/metabolism , Pyrazoles/pharmacology , Pyrazoles/therapeutic use , Pyridines/metabolism , Pyridines/pharmacology , Pyridines/therapeutic use , Rats , Rats, Sprague-Dawley , Structure-Activity RelationshipABSTRACT
Stimulation of naïve CD4(+) T cells through engagement of the T-cell receptor (TCR) and the CD28 co-receptor initiates cell proliferation which critically depends on interleukin (IL)-2 secretion and subsequent autocrine signalling via the IL-2 receptor. However, several studies indicate that in CD28-costimulated T cells additional IL-2-independent signals are also required for cell proliferation. In this study, using a neutralizing anti-human IL-2 antibody and two selective, structurally unrelated, cell-permeable I-κB kinase (IKK) inhibitors, BMS-345541 and PS-1145, we show that in human naïve CD4(+) T cells stimulated through a short engagement of the TCR and the CD28 co-receptor, IKK controls the expression of the cell cycle regulatory proteins cyclin D3, cyclin E and cyclin-dependent kinase 2 (CDK2) and the stability of the F-box protein S-phase kinase-associated protein 2 (SKP2) and its co-factor CDC28 protein kinase regulatory subunit 1B (CKS1B), through IL-2-independent mechanisms.
Subject(s)
CD28 Antigens/immunology , CD4-Positive T-Lymphocytes/metabolism , Cell Cycle Proteins/metabolism , Gene Expression Regulation/immunology , I-kappa B Kinase/antagonists & inhibitors , Interleukin-2/metabolism , Lymphocyte Activation/immunology , Active Transport, Cell Nucleus , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , CD3 Complex/immunology , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/immunology , CDC2-CDC28 Kinases , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Cycle/drug effects , Cell Cycle Proteins/genetics , Cell Proliferation/drug effects , Cyclin D3/genetics , Cyclin D3/metabolism , Cyclin E/genetics , Cyclin E/metabolism , Cyclin-Dependent Kinase 2/genetics , Cyclin-Dependent Kinase 2/metabolism , Cyclin-Dependent Kinase Inhibitor p27 , Cyclin-Dependent Kinases/genetics , Cyclin-Dependent Kinases/metabolism , Gene Expression/drug effects , Gene Expression/genetics , Gene Expression Regulation/drug effects , Heterocyclic Compounds, 3-Ring/pharmacology , Humans , I-kappa B Proteins/metabolism , Imidazoles/pharmacology , Interleukin-2/antagonists & inhibitors , Interleukin-2/genetics , Interleukin-2/immunology , Interleukin-2 Receptor alpha Subunit/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Lymphocyte Activation/drug effects , NF-KappaB Inhibitor alpha , NF-kappa B/metabolism , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , Protein Kinase Inhibitors/pharmacology , Pyridines/pharmacology , Quinoxalines/pharmacology , S-Phase Kinase-Associated Proteins/genetics , S-Phase Kinase-Associated Proteins/metabolism , Signal Transduction/drug effects , Signal Transduction/immunology , Up-Regulation/drug effectsABSTRACT
Niemann-Pick disease (NPD) type A is a neurodegenerative disorder caused by sphingomyelin (SM) accumulation in lysosomes relying on reduced or absent acid sphingomyelinase (ASM) activity. NPD-A patients develop progressive neurodegeneration including cerebral and cerebellar atrophy, relevant Purkinje cell and myelin deficiency with death within 3 years. ASM'knock-out' (ASMKO) mice, an animal model of NPD-A, develop a phenotype largely mimicking that of NPD-A. The mechanisms underlying myelin formation are poorly documented in ASMKO mice. In this study we determined the content of four myelin-specific proteins, myelin basic protein (MBP), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), myelin associated glycoprotein (MAG) and proteolipid protein (PLP), and that of myelin-enriched sphingolipids in the brains of ASMKO and wild-type mice in early stages of post-natal (pn) life. Protein and mRNA analysis revealed that in ASMKO mice beginning from 4 post-natal weeks (wk-pn), the expression levels of MAG, CNP, and MBP were below those observed in wild-type mice and the same applied to PLP at 10 wk-pn. Moreover, at 4 wk-pn the expression of SOX10, one of the transcription factors involved in oligodendrocyte development and maintenance was lower in ASMKO mice. Lipid analysis showed that SM and the gangliosides GM3 and GM2 accumulated in the brains of ASMKO mice, as opposed to galactocerebroside and galactosulfocerebroside that, in parallel with the mRNAs of UDP-galactose ceramide galactosyltransferase and galactose-3-O-sulfotransferase 1, the two transferases involved in their synthesis, decreased. Myelin lipid analysis showed a progressive sphingomyelin accumulation in ASMKO mice; noteworthy, of the two sphingomyelin species known to be resolved by TLC, only that with the lower Rf accumulated. The immunohistochemical analysis showed that the reduced expression of myelin specific proteins in ASMKO mice at 10 wk-pn was not restricted to the Purkinje layer of the cerebellar cortex but involved the cerebral cortex as well. In conclusion, reduced oligodendrocyte metabolic activity is likely to be the chief cause of myelin deficiency in ASMKO mice, thus shedding light on the molecular dysfunctions underlying neurodegeneration in NPD-A.
Subject(s)
Brain/metabolism , Myelin Proteins/metabolism , Niemann-Pick Disease, Type A/metabolism , SOXE Transcription Factors/deficiency , Sphingolipids/metabolism , Sphingomyelin Phosphodiesterase/deficiency , Animals , Brain/enzymology , Disease Models, Animal , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Mutant Strains , Myelin Proteins/genetics , Niemann-Pick Disease, Type A/genetics , SOXE Transcription Factors/biosynthesis , SOXE Transcription Factors/genetics , Sphingolipids/genetics , Sphingomyelin Phosphodiesterase/geneticsABSTRACT
Human dihydroorotate dehydrogenase ( hDHODH) catalyzes the rate-limiting step in de novo pyrimidine biosynthesis, the conversion of dihydroorotate to orotate. hDHODH has recently been found to be associated with acute myelogenous leukemia, a disease for which the standard of intensive care has not changed over decades. This work presents a novel class of hDHODH inhibitors, which are based on an unusual carboxylic group bioisostere 2-hydroxypyrazolo[1,5- a]pyridine, that has been designed starting from brequinar, one of the most potent hDHODH inhibitors. A combination of structure-based and ligand-based strategies produced compound 4, which shows brequinar-like hDHODH potency in vitro and is superior in terms of cytotoxicity and immunosuppression. Compound 4 also restores myeloid differentiation in leukemia cell lines at concentrations that are one log digit lower than those achieved in experiments with brequinar. This Article reports the design, synthesis, SAR, X-ray crystallography, biological assays, and physicochemical characterization of the new class of hDHODH inhibitors.
Subject(s)
Cell Differentiation/drug effects , Drug Design , Myeloid Cells/cytology , Myeloid Cells/drug effects , Oxidoreductases Acting on CH-CH Group Donors/antagonists & inhibitors , Pyrazoles/chemistry , Pyrazoles/pharmacology , Binding Sites , Dihydroorotate Dehydrogenase , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Jurkat Cells , Models, Molecular , Oxidoreductases Acting on CH-CH Group Donors/chemistry , Protein Conformation , Structure-Activity RelationshipABSTRACT
A new generation of potent hDHODH inhibitors designed by a scaffold-hopping replacement of the quinolinecarboxylate moiety of brequinar, one of the most potent known hDHODH inhibitors, is presented here. Their general structure is characterized by a biphenyl moiety joined through an amide bridge with an acidic hydroxyazole scaffold (hydroxylated thiadiazole, pyrazole and triazole). Molecular modelling suggested that these structures should adopt a brequinar-like binding mode involving interactions with subsites 1, 2 and 4 of the hDHODH binding site. Initially, the inhibitory activity of the compounds was studied on recombinant hDHODH. The most potent compound of the series in the enzymatic assays was the thiadiazole analogue 4 (IC50 16 nM). The activity was found to be dependent on the fluoro substitution pattern at the biphenyl moiety as well as on the choice/substitution of the heterocyclic ring. Structure determination of hDHODH co-crystallized with one representative compound from each series (4, 5 and 6) confirmed the brequinar-like binding mode as suggested by modelling. The specificity of the observed effects of the compound series was tested in cell-based assays for antiproliferation activity using Jurkat cells and PHA-stimulated PBMC. These tests were also verified by addition of exogenous uridine to the culture medium. In particular, the triazole analogue 6 (IC50 against hDHODH: 45 nM) exerted potent in vitro antiproliferative and immunosuppressive activity without affecting cell survival.
Subject(s)
Azoles/chemistry , Enzyme Inhibitors/chemistry , Oxidoreductases Acting on CH-CH Group Donors/antagonists & inhibitors , Binding Sites , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Dihydroorotate Dehydrogenase , Drug Design , Enzyme Inhibitors/pharmacology , Humans , Hydroxylation , Immunosuppression Therapy , Jurkat Cells , Models, Molecular , Molecular Structure , Structure-Activity Relationship , X-Ray DiffractionABSTRACT
Adult-onset dominant leukodystrophies are a heterogeneous group of rare disorders, whose etiology, pathogenesis and molecular background are still unknown. We report the neuropathological and biochemical investigations of the brains and their myelin proteins components in 2 members of an Italian family affected by an adult-onset autosomal dominant leukoencephalopathy. Clinical signs included spastic paraparesis, pseudobulbar syndrome, action tremor of head and hands, and moderate memory impairment. No mental deterioration or neuropathy was present. Onset was subacute (range 42-53 years) and progression spanned 4 to 7 years. The neuropathological phenotype overlapped that of orthochromatic leukodystrophies. The biochemical analysis revealed an abnormal myelin-associated glycoprotein (MAG); the defect was localized at the C-terminal domain of the L-MAG isoform, resulting in a protein approximately 5 kDa shorter than the normal counterpart. No mutation in the MAG gene-coding regions was uncovered, and linkage analysis formally excluded the entire MAG locus. We show that the identified MAG protein alteration is probably due to an abnormal post-translational event. Considering MAG function in the maintenance of myelin, the abnormal protein may have a role in the pathogenesis of this disease. This is the first report of a possible pathogenetic role of MAG in a hereditary disease affecting the central white matter.
Subject(s)
Brain/pathology , Leukodystrophy, Globoid Cell/genetics , Leukodystrophy, Globoid Cell/pathology , Myelin-Associated Glycoprotein/metabolism , Protein Processing, Post-Translational , Adult , Age of Onset , Blotting, Western , Brain/ultrastructure , Electrophoresis, Polyacrylamide Gel , Female , Humans , Leukodystrophy, Globoid Cell/physiopathology , Magnetic Resonance Imaging , Male , Microscopy, Electron, Transmission , Middle Aged , Myelin-Associated Glycoprotein/chemistry , Myelin-Associated Glycoprotein/genetics , Pedigree , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
BACKGROUND: In HIV-infected patients some clinical and immunological benefits of antiretroviral therapy, which frequently include a combination of HIV protease inhibitors (PIs) and reverse transcriptase inhibitors (RTIs), cannot be solely explained by the drugs' action on viral enzymes. Proteasomes constitute the central protease of the ubiquitin ATP-dependent pathway involved in many cellular processes, as well as in HIV maturation and aggressiveness. OBJECTIVE: To explore whether the PIs nelfinavir and saquinavir and the RTIs abacavir, nevirapine, delavirdine, stavudine and didanosine affect proteasome function in vitro and in vivo. METHODS: Peptidase activity of purified human 26S and 20S proteasomes was assayed with and without the drugs at different concentrations. Intracellular proteasome proteolytic activity was evaluated by searching for ubiquitin-tagged proteins in HL60 cells incubated with and without the drugs. RESULTS: At therapeutic dosages, nelfinavir and saquinavir inhibited proteasome peptidase activity and caused intracellular accumulation of polyubiquitinated proteins, a hallmark of proteasome proteolytic inhibition in vivo; the RTIs failed to evoke either effect. CONCLUSION: Proteasomes are targeted by the two PIs but not the RTIs. Therefore, in HIV-infected patients the beneficial effect of a therapy including one of the two PIs should partly rely on inhibition of host proteasome function.
Subject(s)
HIV Protease Inhibitors/pharmacology , Nelfinavir/pharmacology , Proteasome Endopeptidase Complex/drug effects , Reverse Transcriptase Inhibitors/pharmacology , Saquinavir/pharmacology , HL-60 Cells , Humans , Proteasome Endopeptidase Complex/metabolism , Proteins/metabolism , Ubiquitin/metabolismABSTRACT
Proteasomes are multisubunit proteases involved in many cellular processes, including tumorigenesis and immune surveillance. In their catalytic core, the 20S proteasome, the beta1, beta2 and beta5 subunits show peptidylglutamyl peptide hydrolyzing (PGPH), trypsin-like and chymotrypsin-like activities, respectively. By IFN-gamma and TNFalpha stimulus, these subunits are replaced by their counterparts LMP2, MECL-1 and LMP7, defined inducible subunits, thus originating the immunoproteasome, and expression of the proteasome activator PA28 is enhanced. These modifications strengthen MHC-class I restricted peptide generation. The 20S proteasome has been detected immunohistochemically in formalin-fixed samples purified from fresh surgical specimens of 18 tumors (G20S) and from 8 samples of normal peritumoral tissue. The G20S, LMP2, MECL-1 and LMP7 increased in only 12 cases, along with unvaried trypsin-like and decreased PGPH and chymotrypsin-like activities; PA28 was unvaried in all 18 samples. The immunoproteasome alterations may represent an anomalous immunological attitude of glioblastomas.
Subject(s)
Central Nervous System Neoplasms/enzymology , Glioblastoma/enzymology , Proteasome Endopeptidase Complex/metabolism , Central Nervous System Neoplasms/pathology , Cysteine Endopeptidases/metabolism , Glioblastoma/pathology , Humans , Immunohistochemistry , Lymphocytes/enzymology , Lymphocytes/pathology , Macrophages/enzymology , Macrophages/pathology , Microglia/enzymology , Microglia/pathology , Multienzyme Complexes/metabolism , Peptide Hydrolases/metabolism , Proteasome Endopeptidase Complex/isolation & purificationABSTRACT
Imatinib mesylate is a tyrosine kinase inhibitor with selectivity for abelson tyrosine-protein kinase 1 (c-Abl), breakpoint cluster region (Bcr)-Abl fusion protein (Bcr-Abl), mast/stem cell growth factor receptor Kit (c-Kit), and platelet-derived growth factor receptor (PDGFR). Previous studies demonstrated that imatinib in the low micromolar range exerted antiproliferative effects on neuroblastoma cell lines. However, although neuroblastoma cells express c-Kit and PDGFR, the imatinib concentrations required to achieve significant growth inhibitory effects (≥ 10 µM) are substantially higher than those required for inhibition of ligand-induced phosphorylation of wild type c-Kit and PDGFR (≤ 1 µM), suggesting that additional mechanisms are responsible for the antitumor activity of imatinib on these cells. In this study, we show that treatment of neuroblastoma cell lines with 1-15 µM imatinib resulted in a dose dependent inhibition of 5-bromo-2'-deoxyuridine (BrdU) incorporation into newly synthesized DNA. The antiproliferative effect of imatinib was dependent on the upregulation of the cyclin-dependent kinase (CDK) inhibitor p27(KIP1) in the nuclear compartment as a result of increased p27(KIP1) protein stability. We demonstrate that the mechanism of p27(KIP1) stabilization relied on inhibition of p27(KIP1) phosphorylation on tyrosine residues by c-Abl. We provide evidence that in neuroblastoma cell lines a significant fraction of cellular c-Abl is phosphorylated on Tyr-245, consistent with an open and active conformation. Notably, exposure to imatinib did not affect Tyr-245 phosphorylation. Given the low affinity of active c-Abl for imatinib, these data provide a molecular explanation for the relatively high imatinib concentrations required to inhibit neuroblastoma cell proliferation.
Subject(s)
Antineoplastic Agents/pharmacology , Benzamides/pharmacology , Cyclin-Dependent Kinase Inhibitor p27/biosynthesis , Genes, abl/physiology , Neuroblastoma/metabolism , Piperazines/pharmacology , Pyrimidines/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/physiology , Dose-Response Relationship, Drug , Genes, abl/drug effects , Humans , Imatinib Mesylate , Up-Regulation/drug effects , Up-Regulation/physiologyABSTRACT
Previously, myelin from cerebral white matter (CWM) of two subjects of a family with orthochromatic adult-onset autosomal-dominant leukodystrophy (ADLD) was disclosed to exhibit defective large isoform of myelin-associated glycoprotein (L-MAG) and patchy distribution only in the elder subject. L-MAG and neural cell adhesion molecule (N-CAM) (N-CAM 180, 140, and 120) are structurally related and concur to myelin/axon interaction. In early developmental stages, in neurons and glia N-CAM is converted into polysialylated (PSA)-NCAM by two sialyltransferases sialyltransferase-X (STX) and polysialyltransferase-1 (PST). Notably, PSA-NCAM disrupts N-CAM adhesive properties and is nearly absent in the adult brain. Here, CWM extracts and myelin of the two subjects were searched for the expression pattern of the N-CAM isoforms and PSA-NCAM, and their CWM was evaluated for N-CAM, STX and PST gene copy number and gene expression as mRNA. Biochemically, we disclosed that in CWM extracts and myelin from both subjects, PSA-NCAM accumulates, N-CAM 180 considerably increases, N-CAM 140 is modestly modified and N-CAM 120 remarkably decreases; duplication of genes encoding N-CAM, STX and PST was not revealed, whereas PST mRNA was clearly increased. Immunohistochemically, in CWM of both subjects, we found an unusually diffuse accumulation of PSA-NCAM without inflammation markers. PSA-NCAM persistence, up-regulated PST mRNA and previously uncovered defective L-MAG may be early pathogenetic events in this ADLD form.
Subject(s)
Cerebrum/metabolism , Hereditary Central Nervous System Demyelinating Diseases/metabolism , Leukoencephalopathies/metabolism , Myelin-Associated Glycoprotein/metabolism , Neural Cell Adhesion Molecule L1/metabolism , Neural Cell Adhesion Molecules/metabolism , Sialic Acids/metabolism , Sialyltransferases/metabolism , Adult , Age of Onset , Blotting, Western , Family , Gene Dosage , Gene Expression Regulation , Hereditary Central Nervous System Demyelinating Diseases/genetics , Humans , Immunohistochemistry , Leukoencephalopathies/genetics , Middle Aged , Myelin Sheath/metabolism , Myelin-Associated Glycoprotein/genetics , Nerve Fibers, Myelinated/metabolism , Neural Cell Adhesion Molecule L1/genetics , Neural Cell Adhesion Molecules/genetics , Polymerase Chain Reaction , Protein Isoforms/metabolism , RNA, Messenger/metabolism , Sialic Acids/genetics , Sialyltransferases/geneticsABSTRACT
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder consisting of progressive loss of motor neurons. TDP-43 has been identified as a component of ubiquitin-immunoreactive inclusions of motor neurons in ALS. We focused on the diffuse cytoplasmic TDP-43 immunoreactivity in ALS neurons, and quantitatively assessed it in comparison with skein/round TDP-43 and ubiquitin immunostaining in motor neurons of 30 sporadic ALS cases. The percentage of spinal motor neurons with cytoplasmic TDP-43 immunoreactivity was higher than that of ubiquitin-immunoreactive ones. The percentage of TDP-43-positive motor neurons was independent of neuron counts in anterior horns, while the percentage of ubiquitinated neurons was inversely correlated. Aiming to define the cytosolic localization of TDP-43, the immunoblot analysis of spinal cord and frontal cortex showed that full-length TDP-43, the 45 kDa form and ubiquitinated TDP-43 are found in the soluble inclusion-free fraction. The present data suggest that delocalization, accumulation and ubiquitination of TDP-43 in the cytoplasm of motor neurons are early dysfunctions in the cascade of the events leading to motor neuron degeneration in ALS, preceding the formation of insoluble inclusion bodies. Being cytoplasmic accumulation an ongoing event during the course of the illness, a therapeutic approach to this incurable disease can be envisaged.
Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Cytoplasm/metabolism , DNA-Binding Proteins/metabolism , Motor Neurons/metabolism , Spinal Cord/metabolism , Adult , Aged , Amyotrophic Lateral Sclerosis/pathology , Cell Count , Disease Progression , Female , Frontal Lobe/metabolism , Hippocampus/metabolism , Humans , Male , Middle Aged , Motor Neurons/pathology , Neurons/metabolism , Neurons/pathology , Spinal Cord/pathology , Temporal Lobe/metabolism , Time Factors , Ubiquitin/metabolism , UbiquitinationABSTRACT
Sphingolipids are polar membrane lipids present as minor components in eukaryotic cell membranes. Sphingolipids are highly enriched in nervous cells, where they exert important biological functions. They deeply affect the structural and geometrical properties and the lateral order of cellular membranes, modulate the function of several membrane-associated proteins, and give rise to important intra- and extracellular lipid mediators. Sphingolipid metabolism is regulated along the differentiation and development of the nervous system, and the expression of a peculiar spatially and temporarily regulated sphingolipid pattern is essential for the maintenance of the functional integrity of the nervous system: sphingolipids in the nervous system participate to several signaling pathways controlling neuronal survival, migration, and differentiation, responsiveness to trophic factors, synaptic stability and synaptic transmission, and neuron-glia interactions, including the formation and stability of central and peripheral myelin. In several neurodegenerative diseases, sphingolipid metabolism is deeply deregulated, leading to the expression of abnormal sphingolipid patterns and altered membrane organization that participate to several events related to the pathogenesis of these diseases. The most impressive consequence of this deregulation is represented by anomalous sphingolipid-protein interactions that are at least, in part, responsible for the misfolding events that cause the fibrillogenic and amyloidogenic processing of disease-specific protein isoforms, such as amyloid beta peptide in Alzheimer's disease, huntingtin in Huntington's disease, alpha-synuclein in Parkinson's disease, and prions in transmissible encephalopathies. Targeting sphingolipid metabolism represents today an underexploited but realistic opportunity to design novel therapeutic strategies for the intervention in these diseases.