RESUMEN
The involvement of the nicotinamide adenine dinucleotide (NAD(+)) salvage pathway in cancer cell survival is poorly understood. Here we show that the NAD(+) salvage pathway modulates cancer cell survival through the rarely mutated tumour suppressor p73. Our data show that pharmacological inhibition or knockdown of nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme in the NAD(+) salvage pathway, enhances autophagy and decreases survival of cancer cells in a p53-independent manner. Such NAMPT inhibition stabilizes p73 independently of p53 through increased acetylation and decreased ubiquitination, resulting in enhanced autophagy and cell death. These effects of NAMPT inhibition can be effectively reversed using nicotinamide mononucleotide (NMN), the enzymatic product of NAMPT. Similarly, knockdown of p73 also decreases NAMPT inhibition-induced autophagy and cell death, whereas overexpression of p73 alone enhances these effects. We show that the breast cancer cell lines (MCF-7, MDA-MB-231 and MDA-MB-468) harbour significantly higher levels of NAMPT and lower levels of p73 than does the normal cell line (MCF-10A), and that NAMPT inhibition is cytotoxic exclusively to the cancer cells. Furthermore, data from 176 breast cancer patients demonstrate that higher levels of NAMPT and lower levels of p73 correlate with poorer patient survival, and that high-grade tumours have significantly higher NAMPT/p73 mRNA ratios. Therefore, the inverse relationship between NAMPT and p73 demonstrable in vitro is also reflected from the clinical data. Taken together, our studies reveal a new NAMPT-p73 nexus that likely has important implications for cancer diagnosis, prognosis and treatment.
Asunto(s)
Autofagia , Citocinas/metabolismo , NAD/metabolismo , Neoplasias/metabolismo , Nicotinamida Fosforribosiltransferasa/metabolismo , Proteína Tumoral p73/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Supervivencia Celular , Citocinas/genética , Humanos , Células Jurkat , Células MCF-7 , NAD/genética , Neoplasias/genética , Neoplasias/mortalidad , Neoplasias/patología , Nicotinamida Fosforribosiltransferasa/genética , Proteína Tumoral p73/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
BACKGROUND: Sarcoidosis and Sjögren's syndrome are two different diseases; however, when affecting the salivary glands, both diseases exhibit similar clinical signs and symptoms, which often complicates the diagnosis. The purpose of this study was to investigate the possibility of using salivary electrophoresis to differentiate between the two diseases. METHODS: Saliva was collected from patients with sarcoidosis and patients with Sjögren's syndrome. Salivary flow rate, total protein, and electrophoretic profiles were examined. RESULTS: Mean salivary flow rate was 0.41 ± 0.07 ml/min/gland vs. 0.43 ± 0.07 ml/min/gland; total salivary protein was 130.0 ± 29.2 mg% vs. 104.0 ± 8.8 mg% for sarcoidosis vs. Sjögren's syndrome, respectively. No differences were observed in salivary flow rate, total salivary protein, or electrophoretic profile between patients with sarcoidosis and patients with Sjögren's syndrome (P = 0.768, 0.718, and 1.000, respectively). CONCLUSIONS: Salivary protein electrophoresis does not appear to be useful to differentiate between sarcoidosis and Sjögren's syndrome.
Asunto(s)
Saliva/química , Enfermedades de las Glándulas Salivales/diagnóstico , Sarcoidosis/diagnóstico , Síndrome de Sjögren/diagnóstico , Adulto , Anciano , Estudios de Casos y Controles , Tos/diagnóstico , Diagnóstico Diferencial , Disnea/diagnóstico , Electroforesis en Gel Bidimensional/métodos , Femenino , Humanos , Concentración de Iones de Hidrógeno , Focalización Isoeléctrica/métodos , Masculino , Persona de Mediana Edad , Enfermedades Nasales/diagnóstico , Saliva/metabolismo , Proteínas y Péptidos Salivales/análisis , Tasa de Secreción/fisiología , Sialadenitis/diagnóstico , Trastornos del Gusto/diagnóstico , Xeroftalmia/diagnóstico , Xerostomía/diagnósticoRESUMEN
BACKGROUND: Post dural puncture headache can be a debilitating complication of subarachnoid anaesthesia for a new mother. Successful management of post dural puncture headache requires adherence to clear policies and protocols with close follow up of patients by an experienced obstetric anaesthetist. OBJECTIVES: To examine the cumulative incidence and severity of post dural puncture headache in obstetric patients who consented for spinal anaesthesia for Caesarean section. DESIGN: Prospective cohort study. SUBJECTS: All women who underwent Caesarean sections under spinal anaesthesia and fulfilled the inclusion criteria. SETTING: Aga Khan University Hospital, a teaching and referral hospital in Nairobi, Kenya. RESULTS: The overall cumulative incidence of post dural puncture headache was found to be 20.35 % but was significantly higher in patients in whom the quincke type of needle was used than in those whose spinal anaesthetics were administered using the pencil point needle (24.2% and 4.5% respectively: p=0.042). CONCLUSIONS: The incidence of post dural puncture headache can be significantly reduced in the obstetric population at the Aga Khan University Hospital if the pencil point spinal needle was to be routinely used. We recommend that the Quincke needles should not be used in the obstetric population at the Aga Khan University Hospital.
Asunto(s)
Anestesia Raquidea/efectos adversos , Cesárea , Cefalea Pospunción de la Duramadre/epidemiología , Trastornos Puerperales/epidemiología , Adulto , Estudios de Cohortes , Femenino , Humanos , Incidencia , Kenia , Cefalea Pospunción de la Duramadre/diagnóstico , Cefalea Pospunción de la Duramadre/terapia , Embarazo , Trastornos Puerperales/diagnóstico , Trastornos Puerperales/terapia , Adulto JovenRESUMEN
The tight regulation of protein kinase C (PKC) activity is crucial for maintaining normal cell proliferation. Excessive PKC activity leads to uncontrolled growth and malignant transformation. It has been reported that the activity of PKC is higher in astroglial cell lines than in normal astrocytes. Previously, we demonstrated that PKC epsilon is overexpressed in astroglial cell lines and in samples from primary high-grade astroglial brain tumors. Because there are no PKC epsilon isozyme-specific inhibitors, we chose a genetic approach to confirm that PKC epsilon is a potential target for inhibiting astroglial cell proliferation. We regulated the expression of a dominant negative PKC epsilon mutant (PKC epsilon 1-401 encoding amino acid 1-401) in U-373MG human astrocytoma cells using a tetracycline-regulated expression vector and established stable clones. Induction of expression of the dominant negative PKC epsilon mutant by the addition of doxycycline, a tetracycline derivative, completely blocked proliferation of U-373MG cells in proliferation and clonogenic assays. Although the induction of the dominant negative PKC epsilon mutant did not markedly affect mitogen-induced tyrosine phosphorylation of the mitogen-activated protein (MAP) kinases, it inhibited the induction of c-Fos protein expression by substance P (SP) and fetal bovine serum (FBS). These results clearly show that the expression of dominant negative PKC epsilon leads to the inhibition of U-373MG cellular proliferation demonstrating that this isozyme may be a potential therapeutic target for astroglial brain tumors.
Asunto(s)
Astrocitoma/enzimología , Astrocitoma/genética , Regulación Enzimológica de la Expresión Génica/fisiología , Inhibidores de Crecimiento/genética , Isoenzimas/genética , Mutación/genética , Proteína Quinasa C/genética , Animales , Astrocitoma/patología , Bovinos , División Celular/genética , Inducción Enzimática/genética , Sangre Fetal/fisiología , Genes Dominantes/fisiología , Inhibidores de Crecimiento/biosíntesis , Inhibidores de Crecimiento/fisiología , Humanos , Isoenzimas/biosíntesis , Isoenzimas/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Proteína Quinasa C/biosíntesis , Proteína Quinasa C/fisiología , Proteína Quinasa C-epsilon , Proteínas Proto-Oncogénicas c-fos/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Proteínas Proto-Oncogénicas c-fos/fisiología , Sustancia P/fisiología , Células Tumorales CultivadasRESUMEN
Prognosis for astroglial brain tumors that are not amenable to surgical resection remains poor. Consequently, a need to identify new cellular targets and chemotherapeutics for the treatment of astroglial tumors remains. Important reports indicate that human astroglial cell lines express higher protein kinase C (PKC) activity in comparison to normal astrocytes. PKC designates a family of kinases that regulate many cellular functions including cell growth and differentiation. The tight regulation of PKC activity is crucial for maintaining normal cellular proliferation since excessive activity leads to uncontrolled growth and cellular transformation. PKCepsilon, one of the 11 known PKC isozymes, has been shown to function as an oncogene in rodent fibroblasts by enhancing c-Raf-1 kinase activity leading to the stimulation of mitogen-activated protein (MAP) kinase pathway. We recently demonstrated that the ability of substance P (SP) neuropeptide to activate MAP kinase pathway in U-373MG astrocytoma cells correlates with its ability to selectively translocate PKCepsilon from cytosolic to membrane fraction, and that PKC inhibitors (e.g. CGP 41251) inhibit the activation of this pathway by SP or the PKC activator 12-O-tetradecanoyl phorbol 13-acetate (TPA). In this study, we demonstrated that PKCepsilon is overexpressed in many astroglial cell lines (n=27 lines), thus providing new evidence as to the possible involvement of this isozyme in the pathology of astroglial tumors. Consistently, we demonstrated that PKCepsilon is overexpressed in primary pediatric anaplastic astrocytoma (grade III) tumor samples as well as in cell lines derived from them, and that glioblastoma multiforme (grade IV) and gliosarcoma tumor samples, but not pilocytic astrocytomas (grade I), also express high levels of PKCepsilon. Therefore, the reported increase in PKC activity in brain tumor derived cell lines may be, in part, attributed to the overexpression of PKCepsilon and possibly other PKC isozymes. Consequently, we propose that the use of PKCepsilon selective inhibitors may be beneficial in the treatment of astroglial brain tumors.
Asunto(s)
Astrocitos/metabolismo , Astrocitoma/metabolismo , Neoplasias Encefálicas/metabolismo , Regulación Enzimológica de la Expresión Génica/fisiología , Regulación Neoplásica de la Expresión Génica/fisiología , Isoenzimas/genética , Proteína Quinasa C/genética , Glioblastoma/metabolismo , Gliosarcoma/metabolismo , Humanos , Proteína Quinasa C-epsilonRESUMEN
Protein kinase C (PKC) designates a family of kinases that regulate many essential functions including cell growth and differentiation. The tight regulation of PKC activity is crucial for maintaining normal cellular proliferation and excessive activity leads to abnormal or uncontrolled cell growth. Recent reports indicate that malignant glioma cell lines express 100 to 1000-fold higher PKC activity when compared to non-neoplastic astrocytes. This high activity correlates well with the proliferation of tumor cells in vitro. We recently reported on the anti-proliferative properties of selective PKC inhibitors on the growth of U-373MG human astrocytoma cell line, and their ability to block mitogen-activated protein (MAP) kinase pathway activated by substance P (SP) neuropeptide receptor signaling via a PKC-dependent mechanism. Therefore, inhibiting PKC activity by selective PKC inhibitors may present a promising approach for improving astroglial brain tumor therapy. For this purpose, we constructed a high throughput model cell system to evaluate the efficacy of PKC inhibitors. This system is based on the measurement of light production in U-373MG cells stably transfected with the luciferase reporter gene whose expression depends on the transcriptional activation of GAL4-Elk1 fusion protein by enzyme components of the MAP kinase pathway and the upstream activation of PKC (PKC activation-->MAP kinases-->GAL4-Elk1 phosphorylation-->luciferase expression-->luciferase activity). In brief, we have demonstrated that the PKC activator 12-O-tetradecanoyl phorbol 13-acetate (TPA)-induced luciferase activity in this cell system is mediated via the MAP kinase pathway and can be blocked in the presence of MEK1 selective inhibitors (PD 098059 or U0126). We also demonstrated that TPA-induced luciferase activity in U-373MG stable clones can be blocked by PKC inhibitors (CGP 41251, Go 6976, and GF 109203X) in a concentration dependent manner. In contrast, epidermal growth factor (EGF)-induced luciferase activity, which is independent of PKC activation (Ras-->Raf-1-->MEK1-->MAP kinases-->GAL4-Elk1 phosphorylation-->luciferase expression-->luciferase activity) can only be blocked using a selective EGF receptor inhibitor (AG 1478). In conclusion, we have constructed a model cell system for the high throughput screening and identification of PKC inhibitors potentially active against astrocytoma cells in culture.
Asunto(s)
Antineoplásicos/farmacología , Astrocitoma/tratamiento farmacológico , Proteínas de Unión al ADN , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/farmacología , Canales de Potasio/análisis , Proteína Quinasa C/antagonistas & inhibidores , Proteínas Proto-Oncogénicas , Factores de Transcripción , Antineoplásicos/uso terapéutico , Astrocitoma/genética , Astrocitoma/patología , Carbazoles/farmacología , Carbazoles/uso terapéutico , División Celular/efectos de los fármacos , Inhibidores Enzimáticos/uso terapéutico , Humanos , Indoles/farmacología , Indoles/uso terapéutico , Canales de Potasio/genética , Estaurosporina/análogos & derivados , Estaurosporina/farmacología , Estaurosporina/uso terapéutico , Activación Transcripcional , Células Tumorales Cultivadas , Proteína Elk-1 con Dominio etsRESUMEN
Prognosis for astroglial brain tumors that are not amenable to surgical resection remains poor and even successful treatment with current chemoradiotherapy is associated with debilitating sequelae. Consequently, a need to identify novel therapeutics for the treatment of brain tumors remains. Regulation of protein kinase C (PKC) whose activity regulates many cellular functions is crucial for maintaining normal cellular proliferation. Recent reports indicate that malignant glioma cell lines express 100 to 1000-fold higher PKC activity when compared to non-neoplastic astrocytes. In this study we used a novel approach for the evaluation of known PKC inhibitors (CGP 41251, Go 6976, and tamoxifen) as chemotherapeutic agents for the inhibition of growth of an astroglial derived cell line. For this purpose, we constructed a model cell system based on the measurement of light production in cells transfected with the luciferase reporter gene whose expression was quantitated by a highly sensitive, rapid, and easy to perform assay. We isolated U-373MG/MEK1C clone whose highly increased luciferase activity was independent of external stimuli and directly proportional to cell number, and therefore, was used as a measure of cell proliferation to quantitate the effects of several PKC inhibitors on growth of astrocytoma cells in vitro. In conclusion, we have constructed a novel cell system that can be utilized for high throughput screening and identification of potential anti-cancer drugs active against astrocytoma cells in culture.
Asunto(s)
Antineoplásicos/farmacología , Astrocitoma/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteína Quinasa C/antagonistas & inhibidores , Astrocitoma/genética , Astrocitoma/patología , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Clonales , Células Eucariotas/citología , Células Eucariotas/efectos de los fármacos , Células Eucariotas/enzimología , Humanos , Luciferasas/efectos de los fármacos , Luciferasas/metabolismo , MAP Quinasa Quinasa 1 , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/efectos de los fármacos , Proteínas Tirosina Quinasas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transfección/genética , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoRESUMEN
Functional bombesin receptors were identified in most human glioblastoma cell lines examined (approximately 85% of lines). Bombesin stimulated the release of intracellular Ca2+ in human adult (U-373MG, D-247MG, U-118MG, U-251MG, D-245MG, U-105MG, D-54MG, A-172MG, and D-270MG lines) and pediatric (SJ-S6 and SJ-G2 lines) glioblastoma cell lines. Stimulation of the glioblastoma cell line U-373MG with bombesin or gastrin-releasing peptide (GRP) induced mitogenesis, measured by [3H]thymidine incorporation into DNA, and stimulated the tyrosine phosphorylation of the mitogen-activated protein (MAP) kinases (Erk1 and Erk2). The stimulation of the MAP kinase phosphorylation in U-373MG cells was time- and peptide concentration-dependent. Both bombesin and GRP showed similar potencies in stimulation of intracellular Ca2+ release and activation of the MAP kinase pathway in U-373MG cells, whereas neuromedin B (NMB) peptide was less potent. Bombesin and GRP induced the release of cytosolic Ca2+ in a concentration-dependent manner. Because bombesin and GRP were more potent than NMB peptide in increasing the cytosolic Ca2+ levels in U-373MG cells, we concluded that the BB2 subtype (also known as GRP-preferring receptor subtype) of the bombesin receptor is expressed in this cell line. The bombesin receptor antagonist ([Leu13-psi(CH2NH)Leu14]bombesin) blocked bombesin induced Ca2+ release and attenuated MAP kinase activation in U-373MG cells demonstrating that bombesin is acting through a receptor-dependent mechanism. This study indicates that functional bombesin receptors are widely expressed in human glioblastoma cell lines.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Glioblastoma/metabolismo , Proteínas Quinasas Activadas por Mitógenos , Receptores de Bombesina/metabolismo , Adulto , Bombesina/análogos & derivados , Bombesina/farmacología , Calcio/metabolismo , División Celular , Niño , Activación Enzimática/efectos de los fármacos , Péptido Liberador de Gastrina , Glioblastoma/patología , Humanos , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Neuroquinina B/análogos & derivados , Neuroquinina B/farmacología , Péptidos/farmacología , Fosforilación , Receptores de Bombesina/antagonistas & inhibidores , Receptores de Bombesina/efectos de los fármacos , Células Tumorales Cultivadas , Tirosina/metabolismoRESUMEN
The substance P (SP) receptor (NK-1 subtype) is widely expressed in primary human astrocytomas and glioblastomas and many brain tumor-derived cell lines. SP receptor activation stimulates the mitogen-activated protein (MAP) kinase pathway and the expression of immediate-early genes (e.g., c-Fos and c-Myc), resulting in an increase in DNA synthesis in human astrocytoma U-373 MG cells. In this study, we investigated the role of protein kinase C (PKC) in SP receptor activation of the MAP kinase pathway. SP peptide, epidermal growth factor, and the PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) induced the tyrosine phosphorylation of the Erk1 and Erk2 MAP kinases in a concentration-dependent manner in U-373 MG cells. Pretreatment of the cells with PKC inhibitors, CGP 41251 or tamoxifen, inhibited tyrosine phosphorylation of Erk1 and Erk2 MAP kinases induced by low concentrations of SP or TPA and significantly attenuated phosphorylation at high concentrations of SP or TPA. The inhibitory effect exhibited by tamoxifen on SP-induced MAP kinase activation is similar to that exhibited by the selective PKC inhibitor CGP 41251, suggesting that the PKC enzyme is the in situ target for both inhibitors. Furthermore, SP-induced c-Fos phosphoprotein expression is inhibited by CGP 41251 or tamoxifen with similar efficacy. Importantly, neither CGP 41251 nor tamoxifen has any detectable effect on the MAP kinase activation by epidermal growth factor, consistent with the ability of this growth factor to activate the MAP kinase pathway by a PKC-independent mechanism. Prolonged treatment with TPA resulted in down-regulation of PKC and selective inhibition of TPA- and SP-induced Erk1 and Erk2 tyrosine phosphorylation in U-373 MG cells. Consistent with the in situ results, CGP 41251 and tamoxifen significantly inhibited endogenous PKC enzymatic activity from U-373 MG cells in vitro. In contrast to CGP 41251 and tamoxifen, Gö 6976, a highly selective inhibitor for PKC alpha and PKC beta 1 isozymes, did not inhibit SP- or TPA-induced tyrosine phosphorylation of Erk1 and Erk2 MAP kinases; rather, it inhibited a signaling pathway leading to the phosphorylation of cAMP-responsive element binding protein in U-373 MG cells. To investigate whether selective PKC isozyme(s) are involved in the activation of the MAP kinase pathway by SP, we determined the expression of PKC isozymes in U-373 MG cells. We found that U-373 MG cells express nine different PKC isozymes (alpha, beta I, beta II, epsilon, delta, eta, zeta, iota, and mu) and that stimulation with SP results in significant and selective translocation of PKC epsilon isozyme from cytosolic to membrane fraction. This establishes a correlation between the ability of SP to activate the MAP kinase pathway and its ability to translocate PKC epsilon. In conclusion, the results presented in this study demonstrate that SP receptor activation of PKC, possibly PKC epsilon, leads to the activation of the MAP kinase pathway, and that this pathway can be inhibited by known PKC inhibitors.
Asunto(s)
Astrocitoma/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Inhibidores Enzimáticos/farmacología , Proteínas Quinasas Activadas por Mitógenos , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Estaurosporina/análogos & derivados , Sustancia P/farmacología , Tamoxifeno/farmacología , Carbazoles/farmacología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Activación Enzimática/efectos de los fármacos , Humanos , Indoles/farmacología , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Estaurosporina/farmacología , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales Cultivadas , Tirosina/metabolismoRESUMEN
The mitogenic substance P receptor (NK-1 subtype) is expressed in many primary human tumors with the highest frequency of expression appearing in astrocytomas and glioblastomas (75% and 100%, respectively). Recently, we showed that substance P neuropeptide induces DNA synthesis in the human astrocytoma U-373MG cells by activating the mitogen-activated protein (MAP) kinase pathway leading to the induction of c-Fos and c-Myc expression. The induction of these immediate early genes is necessary for the progression of cells form G1 to S phase of the cell cycle. In this study, we demonstrate that U-373MG cells are highly sensitive to the growth-inhibitory action of rapamycin at nanomolar concentrations (IC50 <1 ng/ml). We also show that SP peptide stimulates protein synthesis in the U-373MG cell line by activating a rapamycin-sensitive signaling pathway. Further, we demonstrate that SP is potent in stimulating PHAS-I protein (also known as 4E-BP1) phosphorylation and p70 S6 kinase (p70(S6K)) phosphorylation and enzymatic activity, and that this stimulation is inhibited by subnanomolar concentrations of rapamycin. In contrast, rapamycin was not at all effective in repressing SP-induced activation of MAP kinase pathway, c-Fos phosphoprotein expression, and DNA synthesis in U-373MG astrocytoma cells.
RESUMEN
The neuropeptide substance P (SP) regulates many biological processes through binding to and activating the SP receptor (NK-1 subtype). Activation of the SP receptor induces mitogenesis in several cell types. In this study, we characterized the mitogenic response induced by SP peptide in the U-373MG astrocytoma cell line and showed that activation of the SP receptor induces [3H]thymidine incorporation into DNA. We also found that SP potently induces c-myc mRNA and protein in the U-373MG cells. Tyrphostin A25, which blocks activity of tyrosine kinases, significantly inhibited SP-induced mitogenesis, suggesting that the mitogenic response induced by SP peptide involves phosphorylation by tyrosine kinases. Furthermore, stimulation of the SP receptor activates tyrosine phosphorylation and enzymatic activity of extracellular signal-regulated kinases (Erk1 and Erk2), also called the mitogen-activated protein kinases (MAPKs). This result suggests that MAPKs participate in the SP peptide-induced signaling pathway. The addition of CP 96,345 ([(2S,3S)-cis-2-(diphenylmethyl)-N-[(2-methoxyphenyl)-methyl]-1 -azabicyclo[2.2.2]octan-3-amine]; an NK-1 receptor antagonist) or PD 098059 (MEK1 inhibitor) inhibited both DNA synthesis and activation of the MAPK pathway, substantiating that SP stimulates mitogenesis by activating the MAPK pathway through receptors of the NK-1 subtype. Our results demonstrate that SP peptide is a strong mitogen in the U-373MG astrocytoma cell line and establish a clear correlation between SP-induced mitogenesis and activation of MAPK signaling pathway.
Asunto(s)
Astrocitoma/patología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Sustancia P/farmacología , Secuencia de Aminoácidos , Animales , Astrocitoma/metabolismo , Compuestos de Bifenilo/farmacología , ADN/biosíntesis , Activación Enzimática , Flavonoides/farmacología , Genes myc , Humanos , Datos de Secuencia Molecular , Fosforilación , ARN Mensajero/análisis , Conejos , Receptores de Neuroquinina-1/fisiologíaRESUMEN
The two-component regulatory system consisting of OmpR and EnvZ controls the differential expression of major outer membrane porin proteins OmpF and OmpC of Escherichia coli K-12. We have isolated and characterized two mutations in rpoA, the gene encoding the alpha subunit of RNA polymerase, that decrease the expression of OmpF. These mutations have a number of properties that distinguish them from previously isolated rpoA mutations that affect porin expression. The rpoA203 mutation decreases the expression of porin genes ompF and ompC and also decreases the expression of the malE and phoA genes. In contrast, rpoA207 decreases the expression of ompF but does not affect ompC, malE, or phoA transcription. Our results suggest that mutations at various positions in the alpha subunit may affect the OmpR-dependent transcription of ompF and ompC differently and may be useful for analyzing the mechanism underlying their differential expression in response to medium osmolarity.