RESUMEN
In many cancers, high proliferation rates correlate with elevation of rRNA and tRNA levels, and nucleolar hypertrophy. However, the underlying mechanisms linking increased nucleolar transcription and tumorigenesis are only minimally understood. Here we show that IMP dehydrogenase-2 (IMPDH2), the rate-limiting enzyme for de novo guanine nucleotide biosynthesis, is overexpressed in the highly lethal brain cancer glioblastoma. This leads to increased rRNA and tRNA synthesis, stabilization of the nucleolar GTP-binding protein nucleostemin, and enlarged, malformed nucleoli. Pharmacological or genetic inactivation of IMPDH2 in glioblastoma reverses these effects and inhibits cell proliferation, whereas untransformed glia cells are unaffected by similar IMPDH2 perturbations. Impairment of IMPDH2 activity triggers nucleolar stress and growth arrest of glioblastoma cells even in the absence of functional p53. Our results reveal that upregulation of IMPDH2 is a prerequisite for the occurance of aberrant nucleolar function and increased anabolic processes in glioblastoma, which constitutes a primary event in gliomagenesis.
Asunto(s)
Carcinogénesis/metabolismo , Glioblastoma/metabolismo , IMP Deshidrogenasa/metabolismo , Línea Celular Tumoral , Nucléolo Celular/metabolismo , Proliferación Celular/fisiología , Transformación Celular Neoplásica/metabolismo , Humanos , IMP Deshidrogenasa/genética , ARN Ribosómico/metabolismoRESUMEN
PURPOSE: In neurofibromatosis type 1 (NF1) and in highly aggressive malignant peripheral nerve sheath tumors (MPNSTs), constitutively active RAS-GTP and increased MAPK signaling are important in tumorigenesis. Dual specificity phosphatases (DUSPs) are negative regulators of MAPK signaling that dephosphorylate p38, JNK, and ERK in different settings. Although often acting as tumor suppressors, DUSPs may also act as oncogenes, helping tumor cells adapt to high levels of MAPK signaling. We hypothesized that inhibiting DUSPs might be selectively toxic to cells from NF1-driven tumors. EXPERIMENTAL DESIGN: We examined DUSP gene and protein expression in neurofibroma and MPNSTs. We used small hairpin RNA (shRNA) to knock down DUSP1 and DUSP6 to evaluate cell growth, downstream MAPK signaling, and mechanisms of action. We evaluated the DUSP inhibitor, (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI), in MPNST cell lines and in cell-line and patient-derived MPNST xenografts. RESULTS: DUSP1 and DUSP6 are expressed in NF1-deleted tumors. Knockdown of DUSP1 and DUSP6, alone or in combination, reduced MPNST cell growth and led to ERK and JNK hyperactivation increasing downstream TP53 and p-ATM. The DUSP inhibitor, BCI, diminished the survival of NF1-deleted Schwann cells and MPNST cell lines through activation of JNK. In vivo, treatment of an established cell-line xenograft or a novel patient-derived xenograft (PDX) of MPNSTs with BCI increased ERK and JNK activation, caused tumor necrosis and fibrosis, and reduced tumor volume in one model. CONCLUSIONS: Targeting DUSP1 and DUSP6 genetically or with BCI effectively inhibits MPNST cell growth and promotes cell death, in vitro and in xenograft models. The data support further investigation of DUSP inhibition in MPNSTs.
Asunto(s)
Antineoplásicos/farmacología , Fosfatasa 1 de Especificidad Dual/antagonistas & inhibidores , Fosfatasa 6 de Especificidad Dual/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Neoplasias de la Vaina del Nervio/metabolismo , Neoplasias de la Vaina del Nervio/patología , Inhibidores de Proteínas Quinasas/farmacología , Animales , Línea Celular Tumoral , Variaciones en el Número de Copia de ADN , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Neurofibromatosis 1/genética , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Activation of redox-sensitive transcription factors like nuclear factor-E2-related factor 2 (Nrf2) can enhance the transcription of cytoprotective genes during oxidative stress. We investigated whether Nrf2 is activated by methamphetamine (METH) thereby altering neurotoxicity in Nrf2 +/+ and -/- adult mouse brain. A single dose of METH can induce the mRNA levels of Nrf2-regulated antioxidant and cytoprotective proteins in mouse brain. Multiple-day dosing with METH enhanced DNA oxidation and decreased tyrosine hydroxylase and dopamine transporter staining in the striatum, indicating dopaminergic nerve terminal toxicity, which was more severe in -/- mice, as were deficits in motor coordination and olfactory discrimination. These Nrf2-dependent effects were independent of changes in METH metabolism or the induction of hyperthermia. Similarly, METH increased striatal glial fibrillary acidic protein, indicating neurotoxicity. METH neurotoxicity was also observed in the glial cells and in the GABAergic system of the olfactory bulbs and was enhanced in -/- mice, whereas dopaminergic parameters were unaffected. With one-day dosing of METH, there were no differences between +/+ and -/- mice in either basal or METH-enhanced DNA oxidation and neurotoxicity markers. Nrf2-mediated pathways accordingly may protect against the neurodegenerative effects and functional deficits initiated by METH and perhaps other reactive oxygen species-enhancing neurotoxicants, when there is time for transcriptional activation and protein induction. In human users of METH, this mechanism may be essential when differences in drug abuse patterns may alter the induction and duration of Nrf2 activation thereby modulating susceptibility to the neurotoxic effects of METH.
Asunto(s)
Conducta Animal/efectos de los fármacos , Estimulantes del Sistema Nervioso Central/toxicidad , Metanfetamina/toxicidad , Factor 2 Relacionado con NF-E2/fisiología , Síndromes de Neurotoxicidad/etiología , Estrés Oxidativo/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Modelos Animales de Enfermedad , Dopamina/metabolismo , Fiebre/inducido químicamente , Fiebre/metabolismo , Fiebre/patología , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Técnicas para Inmunoenzimas , Ratones , Ratones Noqueados , Síndromes de Neurotoxicidad/metabolismo , Síndromes de Neurotoxicidad/patología , ARN Mensajero/genética , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor that mediates protective responses to oxidative stress, but its developmental role is unknown. Herein, we treated pregnant Nrf2-deficient knockout mice with methamphetamine (METH) (5-40 mg/kg ip), which increases fetal reactive oxygen species (ROS) and oxidatively damaged DNA in fetal brain tissue. METH-exposed Nrf2(-/-) fetuses were unable to increase mRNA levels of ROS-protective heme oxygenase-1, NAD(P)H:quinone oxidoreductase, or oxoguanine glycosylase 1, unlike wild-type controls, and exhibited enhanced DNA oxidation, fetal resorption, edema, and reduced fetal weight, with greater toxicity in female Nrf2(-/-) fetuses. Postnatal neurodevelopmental deficits in activity and olfactory function were exacerbated, with gender-dependent differences, and the olfactory bulb GABAergic marker GAD-65 was decreased in Nrf2(-/-) offspring exposed in utero to METH. In utero METH-initiated olfactory deficits may be a sensitive postnatal functional test for long-term neurotoxicity, and indicated a broad fetal role for Nrf2. The results show that fetal Nrf2 deficiency enhances METH-initiated oxidative DNA damage and toxicity, suggesting that Nrf2 activation of cytoprotective proteins mitigates the effects of ROS and their oxidative damage to cellular macromolecules, thereby protecting the developing fetus from adverse structural and postnatal neurodevelopmental consequences.
Asunto(s)
Estimulantes del Sistema Nervioso Central/toxicidad , Metanfetamina/toxicidad , Factor 2 Relacionado con NF-E2/metabolismo , Neurogénesis/fisiología , Efectos Tardíos de la Exposición Prenatal/metabolismo , Animales , Encéfalo/efectos de los fármacos , Encéfalo/embriología , Encéfalo/metabolismo , Femenino , Feto , Inmunohistoquímica , Ratones , Ratones Noqueados , Neurogénesis/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Embarazo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Neurotoxicity of the amphetamine analogs methamphetamine (METH) and 3,4-methylenedioxyamphetamine (MDA) (the active metabolite of ecstasy) may involve their prostaglandin H synthase (PHS)-dependent bioactivation to free radical intermediates that generate reactive oxygen species and oxidatively damage cellular macromolecules. We used Chinese hamster ovary-K1 (CHO-K1) cell lines either untransfected or stably expressing human PHS-1 (hPHS-1) or hPHS-2 to investigate hPHS isozyme-dependent oxidative damage and cytotoxicity. Both METH and MDA (250-1000 µM) caused concentration-independent cytotoxicity in hPHS-1 cells, suggesting maximal bioactivation at the lowest concentration. In hPHS-2 cells, with half the activity of hPHS-1 cells, METH (250-1000 µM) cytotoxicity was less than that for hPHS-1 cells but was concentration dependent and increased by exogenous arachidonic acid (AA), which increased hPHS activity. Whereas 10 µM MDA and METH were not cytotoxic, at 100 µM both analogs caused AA-dependent and concentration-dependent increases in cytotoxicity and DNA oxidation in both hPHS-1/2 cells. The hPHS-2 isozyme appeared to provide more efficacious bioactivation of these amphetamine analogs. Acetylsalicylic acid, an irreversible inhibitor of both hPHS-1 and hPHS-2, blocked cytotoxicity and DNA oxidation in both cell lines and untransfected CHO-K1 cells lacking PHS activity were similarly resistant. Accordingly, isozyme-dependent hPHS-catalyzed bioactivation of METH and MDA can cause oxidative macromolecular damage and cytotoxicity, which may contribute to their neurotoxicity.
Asunto(s)
3,4-Metilenodioxianfetamina/toxicidad , Ciclooxigenasa 1/metabolismo , Ciclooxigenasa 2/metabolismo , ADN/metabolismo , Metanfetamina/toxicidad , 8-Hidroxi-2'-Desoxicoguanosina , Animales , Aspirina/farmacología , Células CHO , Supervivencia Celular/efectos de los fármacos , Cricetinae , Cricetulus , Ciclooxigenasa 1/genética , Ciclooxigenasa 2/genética , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Activación Enzimática , Humanos , Oxidación-Reducción , TransfecciónRESUMEN
Prostaglandin H synthase (PHS)-2 (COX-2) is implicated in the neurodegeneration of Alzheimer and Parkinson diseases. Multiple mechanisms may be involved, including PHS-catalyzed bioactivation of neurotransmitters, precursors, and metabolites to neurotoxic free radical intermediates. Herein, in vitro studies with the purified PHS-1 (COX-1) isoform and in vivo studies of aging PHS-1 knockout mice were used to evaluate the potential neurodegenerative role of PHS-1-catalyzed bioactivation of endogenous neurotransmitters to free radical intermediates that enhance reactive oxygen species formation and oxidative DNA damage. The brains of 2-year-old wild-type (+/+) PHS-1 normal and heterozygous (+/-) and homozygous (-/-) PHS-1 knockout mice were analyzed for 8-oxo-2'-deoxyguanosine formation, characterized by high-performance liquid chromatography with electrochemical detection and by immunohistochemistry. Compared to aging PHS-1(+/+) normal mice, aging PHS-1(-/-) knockout mice had less oxidative DNA damage in the cortex, hippocampus, cerebellum, and brain stem. This PHS-1-dependent oxidative damage was not observed in young mice. In vitro incubation of purified PHS-1 and 2'-deoxyguanosine with dopamine, L-DOPA, and epinephrine, but not glutamate or norepinephrine, enhanced oxidative DNA damage. These results suggest that PHS-1-dependent accumulation of oxidatively damaged macromolecules including DNA may contribute to the mechanisms and risk factors of aging-related neurodegeneration.
Asunto(s)
Ciclooxigenasa 1/genética , Daño del ADN , 8-Hidroxi-2'-Desoxicoguanosina , Factores de Edad , Animales , Química Encefálica , Desoxiguanosina/análogos & derivados , Desoxiguanosina/biosíntesis , Desoxiguanosina/química , Dopamina/química , Epinefrina/química , Femenino , Radicales Libres/metabolismo , Eliminación de Gen , Ácido Glutámico/química , Levodopa/química , Masculino , Ratones , Ratones Noqueados , Norepinefrina/química , Oxidantes/química , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The dopamine (DA) precursor l-dihydroxyphenylalanine (L-DOPA) and metabolites dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 3-methoxytyramine may serve as substrates for prostaglandin H synthase (PHS)-catalyzed bioactivation to free radical intermediates. We used CHO-K1 cells expressing human (h) PHS-1 or hPHS-2 to investigate hPHS isozyme-dependent oxidative damage and cytotoxicity. hPHS-1- and hPHS-2-expressing cells incubated with DA, L-DOPA, DOPAC, or HVA exhibited increased cytotoxicity compared to untransfected cells, and cytotoxicity was increased further by exogenous arachidonic acid (AA), which increased hPHS activity. Preincubation with catalase, which detoxifies reactive oxygen species, or acetylsalicylic acid, an inhibitor of hPHS-1 and -2, reduced the cytotoxicity caused by DA, L-DOPA, DOPAC, and HVA in hPHS-1 and -2 cells both with and without AA. Protein oxidation was increased in hPHS-1 and -2 cells exposed to DA or L-DOPA and further increased by AA addition. DNA oxidation was enhanced earlier and at lower substrate concentrations than protein oxidation in both hPHS-1 and -2 cells by DA, L-DOPA, DOPAC, and HVA and further enhanced by AA addition. hPHS-2 cells seemed more susceptible than hPHS-1 cells, whereas untransfected CHO-K1 cells were less susceptible. Thus, isozyme-specific, hPHS-dependent oxidative damage and cytotoxicity caused by neurotransmitters, their precursors, and their metabolites may contribute to neurodegeneration associated with aging.
Asunto(s)
Ciclooxigenasa 1/metabolismo , Ciclooxigenasa 2/metabolismo , Daño del ADN , Dopamina/química , Dopamina/farmacología , 8-Hidroxi-2'-Desoxicoguanosina , Animales , Células CHO , Catalasa/metabolismo , Cricetinae , Cricetulus , Ciclooxigenasa 1/genética , Ciclooxigenasa 2/genética , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Dinoprostona/metabolismo , Humanos , L-Lactato Deshidrogenasa/metabolismo , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The role of prostaglandin H synthase-1 (PHS-1) and a related model enzyme, horseradish peroxidase (HRP), in catalyzing the bioactivation of dopamine (DA) and epinephrine and their precursors and metabolites to potential neurodegenerative free radical intermediates was examined. To determine the potential contribution of PHS-dependent reactive oxygen species (ROS) formation, the neurotransmitter DA or its precursor and metabolites were incubated in vitro with purified ovine PHS-1 and calf thymus DNA. DA, its L-dihydroxyphenylalanine (L-DOPA), precursor, and its dihydroxyphenylacetic acid (DOPAC) metabolite were excellent PHS-1 substrates, resulting in PHS-1-dependent ROS formation that initiated oxidative DNA damage, selectively quantified as 8-oxo-2'-deoxyguanosine. Most substrates generated isotropic electron spin resonance (ESR) spectra with a resolved hyperfine structure attributable to ortho-semiquinone free radical intermediates upon autoxidation at pH 6, with up to a 18-fold increase via HRP-catalyzed oxidation. Remarkably, HRP-mediated oxidation of DOPAC and dihydroxymandelic acid (DHMA) produced asymmetric ESR spectra characteristic of an immobilized radical, possibly due to free radical intermediates and melanin or melanin-like polymers. These results show that the precursors and metabolites of endogenous neurotransmitters, while inactive in receptor binding assays, may actually play an important role in free radical formation. Additionally, ROS generated by PHS-catalyzed bioactivation produce oxidative DNA damage in the central nervous system, which may initiate neurodegeneration associated with aging.
Asunto(s)
Ciclooxigenasa 1/metabolismo , Daño del ADN , Dopamina/metabolismo , Espectroscopía de Resonancia por Spin del Electrón/métodos , Epinefrina/metabolismo , 8-Hidroxi-2'-Desoxicoguanosina , Catálisis , Desoxiguanosina/análogos & derivados , Desoxiguanosina/análisis , Dopamina/análisis , Radicales Libres/metabolismo , Peroxidasa de Rábano Silvestre/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Espectrofotometría UltravioletaRESUMEN
Reactive oxygen species (ROS) are implicated in amphetamine-initiated neurodegeneration, but the mechanism is unclear. Here, we show that amphetamines are bioactivated by CNS prostaglandin H synthase (PHS) to free radical intermediates that cause ROS formation and neurodegenerative oxidative DNA damage. In vitro incubations of purified PHS-1 with 3,4-methylenedioxyamphetamine (MDA) and methamphetamine (METH) demonstrated PHS-catalyzed time- and concentration-dependent formation of an amphetamine carbon- and/or nitrogen-centered free radical intermediate, and stereoselective oxidative DNA damage, evidenced by 8-oxo-2'-deoxyguanosine (8-oxo-dG) formation. Similarly in vivo, MDA and METH caused dose- and time-dependent DNA oxidation in multiple brain regions, remarkably dependent on the regional PHS levels, including the striatum and substantia nigra, wherein neurodegeneration of dopaminergic nerve terminals was evidenced by decreased immunohistochemical staining of tyrosine hydroxylase. Motor impairment using the rotarod test was evident within 3 wk after the last drug dose, and persisted for at least 6 months. Pretreatment with the PHS inhibitor acetylsalicylic acid blocked MDA-initiated DNA oxidation and protected against functional motor impairment for at least 1.5 months after drug treatment. This is the first direct evidence for PHS-catalyzed bioactivation of amphetamines causing temporal and regional differences in CNS oxidative DNA damage directly related to structural and functional neurodegenerative consequences.