RESUMEN
BACKGROUND: Dominant negative regulation of critical cell cycle molecules could perturb survival of cancer cells and help develop novel therapies. METHODS: To perturb the activity of c-Myc, which regulates G0/G1 transitions, we overexpressed Mad1 protein with an adenoviral vector, AdMad. Studies were conducted with established cell lines, including HepG2, HuH-7 and PLC/PRF/5 liver cancer cells, RAT-1A embryonic fibroblasts and U373MG astrocytoma cells. RESULTS: After AdMad-treatment, transduced cells exhibited decreased proliferation rates in culture conditions. RAT-1A embryonic fibroblasts and U373MG astrocytoma cells showed accumulations in G0/G1, whereas HepG2 and HuH-7 cells accumulated in G0/G1, and additionally in G2/M, albeit to a lesser extent. An in vitro assay using hepatocyte growth factor to stimulate proliferation in HuH-7 cells showed blunting of growth factor responsiveness, along with inhibition of cell cycle progression in AdMad-treated cells. No cytotoxicity was observed in AdMad-treated cells in culture, although cells lost clonogenic capacity in soft agar. In vivo assays using HepG2 cell tumors in immunodeficient mice showed that overexpression of AdMad prevented tumorigenesis. CONCLUSIONS: These studies indicate roles of Mad in G2/M, as well as the potential of manipulating cell cycle controls for treating liver cancer.
Asunto(s)
Carcinoma Hepatocelular/patología , Proteínas de Unión al ADN/metabolismo , Técnicas de Transferencia de Gen , Neoplasias Hepáticas/patología , Proteínas Represoras , Factores de Transcripción/metabolismo , Adenoviridae/genética , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Carcinoma Hepatocelular/inmunología , División Celular , Proteínas de Unión al ADN/genética , Vectores Genéticos , Humanos , Interfase , Neoplasias Hepáticas/inmunología , Ratones , Ratones SCID , Factores de Transcripción/genética , Células Tumorales CultivadasRESUMEN
In this report, we describe a novel lytic mechanism exploited by antimicrotubule drugs (AMDs) such as Taxol which are frequently used to treat multiple human cancers including breast and ovarian cancers. In cells lacking the G1-arresting capacity due to the defect in retinoblastoma or p53 gene function, AMDs trigger hyperploid progression and death. The hyperploid progression occurs via continued cell-cycle progression without cell division. Blocking hyperploid progression through hydroxyurea or ectopically expressed p27(Kip1), a G1-specific Cdk inhibitor, abrogates AMD cytotoxicity. Thus, AMDs induce lethality in G1-checkpoint-defective cells by triggering hyperploid progression. The phenomenon is reminiscent of that observed previously with bub-1 yeast mutant. The potential significance of this finding lies in that hyperploid progression-mediated death may be exploited to develop a therapy with tumor-specificity at the genetic level. As a large fraction of human cancers are mutated in p53 gene, it may have a wide therapeutic applicability.
Asunto(s)
Astrocitoma/metabolismo , Astrocitoma/patología , Proteínas de Ciclo Celular , Nocodazol/farmacología , Paclitaxel/farmacología , Proteínas Supresoras de Tumor , Vincristina/farmacología , Ciclo Celular/efectos de los fármacos , Muerte Celular , División Celular/efectos de los fármacos , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Fibroblastos/efectos de los fármacos , Citometría de Flujo , Humanos , Hidroxiurea/farmacología , Cariotipificación , Proteínas Asociadas a Microtúbulos/farmacología , Fosforilación , Poliploidía , Proteína de Retinoblastoma/metabolismo , Timidina/metabolismo , Células Tumorales CultivadasRESUMEN
The principle hurdles for gene therapy are selectivity and efficacy. Toward that end, we constructed an adenovirus gene delivery system to enable robust, glial-specific, and repressible ectopic expression. A replication-incompetent (E1-deleted) adenovirus 5 vector was modified by the addition of three tandem repeats of a 300-bp fragment enhancer region of the glial fibrillary acidic protein gene coupled to a minimal promoter sequence from human cytomegalovirus to drive a tetracycline-controlled transactivator. Using beta-galactosidase as a reporter gene, we demonstrated high level expression in cells of glial origin (including cell lines derived from glioblastoma multiforme) but no detectable expression in nonglial cells (neuroblastoma or fibroblasts). Furthermore, expression was tightly regulated by anhydrous tetracycline. To our knowledge, this is the first gene therapy delivery system that is glial specific and which also allows for repression of ectopic gene expression.
Asunto(s)
Adenoviridae , Neoplasias Encefálicas/terapia , Virus Defectuosos , Terapia Genética/métodos , Vectores Genéticos/uso terapéutico , Proteína Ácida Fibrilar de la Glía/genética , Glioma/terapia , Antibacterianos/farmacología , Neoplasias Encefálicas/metabolismo , Genes Reporteros , Vectores Genéticos/genética , Glioma/metabolismo , Humanos , Sensibilidad y Especificidad , Tetraciclina/farmacología , Células Tumorales Cultivadas , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
Abnormal proliferation of vascular smooth muscle cells (VSMCs) contributes to intimal hyperplasia during atherosclerosis and restenosis, but the endogenous cell cycle regulatory factors underlying VSMC growth in response to arterial injury are not well understood. In the present study, we report that downregulation of cyclin-dependent kinase 2 (cdk2) activity in serum-deprived VSMCs was associated with the formation of complexes between cdk2 and its inhibitory protein p27(KIP1) (p27). Ectopic overexpression of p27 in serum-stimulated VSMCs resulted in the inhibition of cdk2 activity and repression of cyclin A promoter activity. Collectively, these findings indicate that p27 may contribute to VSMC growth arrest in vitro. Using the rat carotid model of balloon angioplasty, a marked upregulation of p27 was observed in injured arteries. High levels of p27 expression in the media and neointima correlated with downregulation of cdk2 activity at 2 wk after angioplasty, and adenovirus-mediated overexpression of p27 in balloon-injured arteries attenuated neointimal lesion formation. Thus, the inhibition of cdk2 function and repression of cyclin A gene transcription through the induction of the endogenous p27 protein provides a mechanism for the inhibition of VSMC growth at late time points after angioplasty.
Asunto(s)
Quinasas CDC2-CDC28 , Arterias Carótidas/metabolismo , Proteínas de Ciclo Celular , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Ciclinas/biosíntesis , Proteínas Asociadas a Microtúbulos/fisiología , Músculo Liso Vascular/metabolismo , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Supresoras de Tumor , Túnica Íntima/fisiología , Adenoviridae , Angioplastia , Animales , Traumatismos de las Arterias Carótidas , Células Cultivadas , Medio de Cultivo Libre de Suero , Quinasa 2 Dependiente de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Regulación de la Expresión Génica , Vectores Genéticos , Luciferasas/biosíntesis , Proteínas Asociadas a Microtúbulos/biosíntesis , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , Factores de Tiempo , Transfección , Túnica Íntima/lesionesRESUMEN
Although genetic alterations of chromosome band 9p21-22 occur frequently in head and neck squamous cell carcinoma (HNSCC) cell lines, alterations of the cyclin-dependent kinase inhibitor p16INK4a located in this region are less common in corresponding primary tumors. To further investigate genetic alterations at 9p21-22 and p16INK4a in primary HNSCC, a paired set of 21 tumors and blood specimens that were shown previously to exhibit allelic loss at 3p and elsewhere, were tested for LOH at 9p21-22 using eight different highly polymorphic marker. Sixteen of the samples (81%) exhibited LOH for at least one marker. Frequent LOH was found surrounding p16INK4a and at three additional non-contiguous regions of 9p21-22. No homozygous deletions were identified. SSCP screening and direct sequence analysis led to the identification of mutations the p16INK4a gene in two tumors. p16INK4a was not hypermethylated in any of the samples studied. Furthermore, there was no correlation between LOH at 9p21-22 and the RB1 tumor suppressor gene. These findings indicate that in the set of tumors that we tested, LOH at 9p21-22 is common in primary HNSCC but that genetic alterations of p16INK4a located in this region are unusual. Additional tumor suppressor genes at 9p21-22 may therefore be involved in the pathogenesis of this tumor.
Asunto(s)
Carcinoma de Células Escamosas/genética , Cromosomas Humanos Par 9 , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Neoplasias de Cabeza y Cuello/genética , Pérdida de Heterocigocidad , Mapeo Cromosómico , Metilación de ADN , Humanos , Repeticiones de Microsatélite , Polimorfismo Conformacional Retorcido-Simple , Proteína de Retinoblastoma/genéticaRESUMEN
p21WAF1/CIP1 is a downstream effector of the p53 tumor suppressor gene and a universal cyclin-dependent kinase (CDK) inhibitor. To determine the ability of p21WAF1/CIP1 to function as a tumor suppressor, we constructed a replication-defective adenovirus vector containing p21WAF1/CIP1 (Adp21WAF1/CIP1) to effect ectopic overexpression in a p53-defective human astrocytoma cell line, U-373MG. We observed a marked decrease in CDC2 and CDK2 kinase activity associated with a corresponding decrease in the amount of CDC2 but not CDK2 protein; a decreased growth potential of Adp21WAF1/CIP1-infected cells demonstrated by diminished [3H]thymidine incorporation, increased cell doubling time and G1-arrested cell cycle; an association between Adp21WAF1/CIP1-infected cells and inhibition of aneuploid cell accumulation; and an alteration of the malignant phenotype of cells was evidenced by the loss of anchorage-independent growth in soft agar and the failure to induce tumorigenesis in both peripheral and intracerebral xenograft models, including the prevention of tumor formation Adp21WAF1/CIP1 infection 2 days post tumor cell implantation. Adp21WAF1/CIP1. Adp21WAF1/CIP1 appears to be a strong candidate for gene therapy studies based on these studies indicating that Adp21WAF1/CIP1 inhibits proliferation, tumorigenicity and aneuploidy in human brain tumor cells.
Asunto(s)
Aneuploidia , Astrocitoma/metabolismo , Astrocitoma/terapia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/terapia , Ciclinas/metabolismo , Animales , Apoptosis , Astrocitoma/genética , Neoplasias Encefálicas/genética , Proteínas de Ciclo Celular/metabolismo , División Celular/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/fisiología , Virus Defectuosos , Fase G1/fisiología , Genes p53/fisiología , Terapia Genética , Vectores Genéticos , Humanos , Ratones , Ratones Desnudos , Fenotipo , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
To investigate how overexpression of p27KIP1, a downstream effector of TGF-beta and a universal cyclin-dependent kinase (CDK) inhibitor could influence the malignant phenotype of malignant human brain tumor cells, an adenovirus vector system was used to transfer the human p27KIP1 gene (Adp27KIP1) into the human astrocytoma cell line, U-373MG. Inhibition of CDK activity in Adp27KIP1-infected cells was indicated by inhibition of [3H]thymidine incorporation, an increase in cell doubling time and by cell cycle arrest in G1. Notably, ectopic overexpression of p27KIP1 was associated with a marked decrease in the accumulation of aneuploid cells. Diminished malignant potential of Adp27KIP1-infected cells was manifested by the loss of anchorage-independent growth in soft agar and by the inability to induce tumorgenesis in a xenograft model. These studies suggest that p27KIP1 is a tumor suppressor gene and supports the use of Adp27KIP1 for gene therapy of human brain tumors.
Asunto(s)
Aneuploidia , Astrocitoma/patología , Neoplasias Encefálicas/patología , Proteínas de Ciclo Celular , Inhibidores Enzimáticos , Proteínas Asociadas a Microtúbulos/biosíntesis , Proteínas Supresoras de Tumor , Animales , Astrocitoma/genética , Western Blotting , Neoplasias Encefálicas/genética , Ciclo Celular , División Celular , Línea Celular , Supervivencia Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , ADN de Neoplasias/biosíntesis , Expresión Génica , Humanos , Ratones , Ratones Desnudos , Proteínas Asociadas a Microtúbulos/análisis , Protamina Quinasa/metabolismo , Proteínas Recombinantes/biosíntesis , Timidina/metabolismo , Transfección , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
Ependymomas, which comprise 5% of central nervous system tumors, have not been extensively characterized genetically. The p53 tumor suppressor gene is frequently mutated in human cancer, and is important in the pathogenesis of other central nervous system (CNS) tumors. Chromosomal DNA corresponding to the p53 tumor suppressor gene was amplified by the polymerase chain reaction (PCR) from 31 archival ependymoma specimens. DNA was screened for the presence of p53 mutations by single strand conformational polymorphism (SSCP) analysis; samples with altered mobility were further tested for the presence of mutation by direct DNA sequence analysis. Of the 31 ependymomas tested, one contained a detectable DNA sequence change in the p53 gene. Sequencing revealed a silent mutation in exon 6, at codon 213, which represents a known p53 sequence polymorphism. These finding suggest that in contrast to many other human cancers, p53 mutation is not important in the pathogenesis or progression of ependymomas.
Asunto(s)
Neoplasias Encefálicas/genética , Ependimoma/genética , Genes p53 , Mutación Puntual , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-SimpleRESUMEN
Previous molecular genetic studies revealed that allelic loss of chromosome arm 3p is a frequent event in upper aerodigestive tract squamous cell carcinoma (UADT SCC). Recently, the Von-Hippel Lindau (VHL) tumor suppressor gene was identified at chromosome band 3p25-26. To determine if the VHL locus is altered in these tumors, a paired series of 26 tumors and blood from patients with UADT SCC that were previously shown to exhibit allelic loss of 3p were tested for LOH surrounding the VHL locus using four different polymorphic markers. All of the samples (100%) exhibited LOH for at least 1 marker. However, no LOH was detected using a polymorphism within exon 1 of the VHL gene which was informative for 18 of the 26 cases. Furthermore, mutations of the VHL gene could not be identified by single-strand conformation polymorphism, dideoxyfingerprint or direct DNA sequence analysis. In addition, the VHL gene was not inactivated by hypermethylation in any of the 26 tumor samples studied. These findings demonstrate that allelic loss of chromosome arm 3p in UADT SCC involves regions surrounding the VHL locus but does not include the VHL gene. The VHL gene, therefore, does not appear to be involved in the pathogenesis of UADT SCC.
Asunto(s)
Carcinoma de Células Escamosas/genética , Deleción Cromosómica , Cromosomas Humanos Par 3 , Genes Supresores de Tumor , Neoplasias de Cabeza y Cuello/genética , Enfermedad de von Hippel-Lindau/genética , Humanos , Metilación , Mutación , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
We recently reported that cyclic AMP (cAMP) specifically inhibits lipopolysaccharide (LPS)-induced interleukin 1 beta (IL-1 beta) transcription initiation in astrocytic cells but enhances the LPS induction of IL-1 beta in monocytic cells. The purpose of this study was to determine how cAMP differentially regulates LPS-induced IL-1 beta transcription in these two cell types. Two essential components of the mitogen-activated protein (MAP) kinase signal-transduction pathway, extracellular-signal-regulated kinase (ERK2; p41 mapk) and Raf-1, have been shown to be targets of LPS stimulation in other cell types, and therefore may be linked to the regulation of IL-1 beta transcription. In the human astrocytic cell line, U-373MG, LPS was found to strongly activate (and cAMP to inhibit) both ERK2 and Raf-1. In the human monocytic cell line, THP-1, LPS minimally activated ERK2 and did not activate Raf-1. These findings suggest that, in astrocytic cells, elevated intracellular cAMP levels may negatively regulate LPS activation of IL-1 beta via the MAP kinase signalling pathway. In contrast, this pathway is not significantly activated by LPS in monocytic cells, thus inhibition by elevated intracellular cAMP levels would not affect IL-1 beta transcription.
Asunto(s)
Astrocitos/efectos de los fármacos , Lipopolisacáridos/farmacología , Monocitos/efectos de los fármacos , Proteínas Quinasas/biosíntesis , Astrocitos/enzimología , Línea Celular , Células Cultivadas , Inducción Enzimática , Humanos , Monocitos/enzimología , Células Tumorales CultivadasRESUMEN
In bacterial sepsis and meningitis, large concentrations of interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) correlate directly with morbidity and mortality. This laboratory has reported previously that elevated temperature in the physiologic range is associated with down regulation of IL-1 beta and TNF alpha expression in cultured astroglia after lipopolysaccharide (LPS) stimulation. To further investigate the role of elevated temperature in the CNS inflammatory response, the effects of LPS and elevated temperature on the expression of genes that participate in the inflammatory response were determined in cultured transformed human fetal astrocytes and in an astrocytoma cell line. The effect of physiologic temperature elevation on cytokine concentrations in cerebrospinal fluid (CSF) was also investigated in a rabbit meningitis model. The findings indicate that astrocytes express a wide variety of cytokines, growth factors, growth factor receptors, and other genes that could play important roles in CNS inflammation. Furthermore, temperature elevation in the febrile range can lead to alterations in the patterns of expression of many genes involved in the inflammatory response of these cells.
Asunto(s)
Astrocitos/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Inflamación/genética , Lipopolisacáridos/farmacología , Temperatura , Animales , Astrocitos/metabolismo , Astrocitoma/patología , Encéfalo/citología , Encéfalo/embriología , Neoplasias Encefálicas/patología , Células Cultivadas , Proteínas del Líquido Cefalorraquídeo/análisis , Citocinas/líquido cefalorraquídeo , Fiebre/líquido cefalorraquídeo , Fiebre/etiología , Humanos , Meningitis/líquido cefalorraquídeo , Meningitis/inducido químicamente , Conejos , Células Tumorales CultivadasRESUMEN
To investigate how overexpression of MAD, an antagonist of MYC oncogenes influences the malignant phenotype of human cancer cells, an adenovirus vector system was used to transfer the human MAD gene (AdMAD) into human astrocytoma cells. Decreased growth potential of AdMAD-infected cells was evidenced by a decrease in [3H]thymidine incorporation, an increase in cell doubling time and alteration of cell-cycle distribution. Diminished malignant potential of AdMAD-infected cells was manifested by their loss of anchorage-independent growth in soft agar and by their inability, in general, to induce tumorigenesis in a xenograft animal model. These studies indicate that adenovirus constructs encoding MAD dramatically inhibit the proliferation and tumorigenicity of human astrocytoma cells and support the use of MAD for gene therapy of human tumours.
Asunto(s)
Astrocitoma/patología , Neoplasias Encefálicas/patología , Proteínas de Unión al ADN/fisiología , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Proteínas Represoras , Factores de Transcripción , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Linfoma de Burkitt/patología , Carcinoma/patología , Carcinoma de Células Escamosas/patología , Adhesión Celular , Ciclo Celular , División Celular , Replicación del ADN , ADN de Neoplasias/biosíntesis , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Humanos , Sustancias Macromoleculares , Ratones , Ratones Desnudos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Trasplante de Neoplasias , Neoplasias Ováricas/patología , Fenotipo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Células Tumorales Cultivadas/trasplanteRESUMEN
Dexamethasone inhibits lipopolysaccharide-induced synthesis of the cytokine, interleukin-1 beta, in cerebrospinal fluid of patients with bacterial meningitis. Along with monocytes, astrocytes are capable of producing lipopolysaccharide-induced interleukin-1 beta in the central nervous system. The objective of this study was to investigate the induction of interleukin-1 beta mRNA by lipopolysaccharide, and the inhibition of this process by dexamethasone, in human astrocytes using the astrocytoma cell line U-373MG as a model system. Dexamethasone-mediated inhibition of induction of interleukin-1 beta mRNA by lipopolysaccharide required a functional glucocorticoid receptor. In contrast to monocytes, lipopolysaccharide induction of interleukin-1 beta mRNA in U-373MG cells required de novo protein synthesis. Dexamethasone also had no effect on lipopolysaccharide-induced interleukin-1 beta transcriptional initiation in U-373MG cells. U-373MG cells were similar to monocytes, however, with respect to the ability of dexamethasone to decrease interleukin-1 beta mRNA half-life. These findings demonstrate that the mode of lipopolysaccharide induction of interleukin-1 beta mRNA, and inhibition of this process by dexamethasone, can vary in different cell types.
Asunto(s)
Astrocitos/efectos de los fármacos , Dexametasona/farmacología , Interleucina-1/antagonistas & inhibidores , Interleucina-1/genética , Lipopolisacáridos/farmacología , ARN Mensajero/efectos de los fármacos , Astrocitos/metabolismo , Astrocitoma/inmunología , Cicloheximida/farmacología , Humanos , Interleucina-1/biosíntesis , Mifepristona/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Transcripción Genética/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
A 15-month-old female presented with eczema, thrombocytopenia, recurrent infections and failure to thrive. She had low serum IgM and IgG subclasses and an abnormal lymphocyte proliferative response to periodate in vitro. Molecular X chromosome inactivation analysis, using the polymorphic HUMARA DNA probe, showed that the infant has random X chromosome inactivation. We conclude that she has an atypical form of Wiskott-Aldrich syndrome which may be inherited in an autosomal recessive manner.
Asunto(s)
Compensación de Dosificación (Genética) , Síndrome de Wiskott-Aldrich/genética , División Celular/inmunología , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Lactante , Linfocitos/inmunología , Síndrome de Wiskott-Aldrich/inmunologíaRESUMEN
BACKGROUND: In human brain tumors, sensitivity to procarbazine as measured by sensitivity in a xenograft tumor model correlated inversely with amounts of the DNA repair enzyme O6-alkylguanine DNA alkyltransferase (AT). METHODS: To test the hypothesis that mutations of the p53 tumor suppressor gene in human tumors also can correlate with the response to chemotherapy, p53 mutations2 were identified in primary human malignant brain tumors and cell lines in which AT activity and procarbazine sensitivity in a xenograft model was ascertained. RESULTS: Mutations were identified in 7 of 21 (33%) specimens tested. Specimens containing p53 mutations tended to exhibit an increased growth delay in procarbazine-treated xenografts and lower amounts of AT. CONCLUSIONS: p53 mutations in brain tumors may contribute to procarbazine sensitivity by failing to induce arrest at the G1/S cell-cycle checkpoint, thereby preventing the repair of procarbazine-induced genetic alterations.
Asunto(s)
Neoplasias Encefálicas/enzimología , Neoplasias Encefálicas/genética , Genes p53/genética , Metiltransferasas/metabolismo , Procarbazina/uso terapéutico , Animales , Secuencia de Bases , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Ciclo Celular , Análisis Mutacional de ADN , Reparación del ADN , Humanos , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Mutación , Trasplante de Neoplasias , O(6)-Metilguanina-ADN Metiltransferasa , Reacción en Cadena de la Polimerasa , Células Tumorales CultivadasRESUMEN
The response to LPS includes synthesis by monocytes of the inflammatory mediator IL-1 beta. Although the intracellular signaling pathways activated by LPS that lead to IL-1 beta production have been studied extensively in monocytes, these pathways have not been investigated in astrocytes, an important source of IL-1 beta in the central nervous system. cAMP has been implicated in LPS signaling as a positive regulator of IL-1 beta mRNA accumulation in monocytes. In this study, we demonstrate that in human astrocytes (both fetal and the astrocytoma cell line, U-373 MG), agents that elevate intracellular cAMP decrease LPS-induced IL-1 beta mRNA accumulation. Elevated intracellular cAMP does not affect IL-1 beta mRNA stability, but inhibits LPS-induced transcription initiation of IL-1 beta in U-373 MG cells. Elevated intracellular cAMP may be a negative feedback regulatory mechanism to inhibit IL-1 beta production employed by astrocytes that (unlike monocytic cells) lack a glycosyl-phosphatidylinositol (GPI)-anchored form of the LPS receptor, CD14. Whether cAMP inhibits an LPS-inducible signaling pathway or negatively affects cAMP-dependent transcription factors remains to be determined.
Asunto(s)
Astrocitos/inmunología , AMP Cíclico/fisiología , Interleucina-1/biosíntesis , Lipopolisacáridos/antagonistas & inhibidores , Transcripción Genética/fisiología , Astrocitos/efectos de los fármacos , Northern Blotting , Línea Celular , Regulación de la Expresión Génica , Humanos , Interleucina-1/genética , Lipopolisacáridos/farmacología , ARN Mensajero/metabolismo , Transcripción Genética/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
BACKGROUND: Little is known about the molecular genetic events that contribute to the pathogenesis of squamous cell carcinoma of the upper aerodigestive tract. Previous molecular genetic studies have been limited to the identification of mutations of the p53 (also known as TP53) tumor suppressor gene, activation of a limited set of oncogenes, allelic loss at 3p and other locations, and occasional association with human papillomavirus infection. PURPOSE: Our purpose was to screen tumor tissue and blood from patients with squamous cell carcinoma of the upper aerodigestive tract for loss of heterozygosity at polymorphic loci corresponding to each of the autosomal chromosomes and to identify the locations of additional putative tumor suppressor genes, other than RB (also known as RB1) and p53, that may contribute to the pathogenesis of this disease. METHODS: Tumor tissue and blood were obtained from 68 consecutive patients with squamous cell carcinoma of the upper aerodigestive tract. In all cases, tumor tissue was obtained from the center of the surgical specimen. The relative absence of non-neoplastic tissue was confirmed by frozen-section histologic examination of immediately adjacent tissue. Initially, 30 paired tissue and blood samples were tested for loss of heterozygosity by polymerase chain reaction (PCR) to amplify 43 different highly polymorphic sequences containing small oligonucleotide repeats. After PCR amplification, with unique oligonucleotides flanking the repeat, visualization and sizing of the alleles on DNA sequencing gels were performed. Specific loss of heterozygosity was distinguished from random genetic loss due to generalized chromosomal instability if it occurred in more than 20% of specimens tested for a particular marker. RESULTS: Significant loss of heterozygosity (> 20%) occurred at alleles at chromosome bands 3p21 (32%), 3p25-26 (56%), 8pter-21.1 (31%), 13q14 (27%), and 17p12 (45%). Loss of heterozygosity at more than two loci was significant with a poor prognosis (P = .039). CONCLUSIONS: These findings demonstrate that squamous cell carcinoma of the upper aerodigestive tract exhibits genetic alterations at multiple loci and that allelic loss at more than two locations is indicative of a poor prognosis (the likelihood of the patient dying of disease). IMPLICATIONS: While tumor suppressor genes at 3p (VHL), 13q (RB), and 17p (p53) have been identified, altered genes at other loci on 3p and on 8p have not yet been characterized. Furthermore, the genotype at these loci for squamous cell carcinoma of the upper aerodigestive tract has prognostic importance and may identify the patients who should receive the most aggressive treatment.
Asunto(s)
Alelos , Carcinoma de Células Escamosas/genética , Deleción Cromosómica , Cromosomas Humanos , Genes Supresores de Tumor/genética , Neoplasias de Cabeza y Cuello/genética , Adulto , Anciano , Anciano de 80 o más Años , Cromosomas Humanos Par 13 , Cromosomas Humanos Par 17 , Cromosomas Humanos Par 3 , Cromosomas Humanos Par 8 , Femenino , Heterocigoto , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , PronósticoRESUMEN
Loss of function of the p53 tumor suppressor gene by point mutation is the most commonly detected genetic alteration in human cancer. There is growing evidence that amplification and overexpression of the MDM2 gene are alternative mechanisms that also lead to functional inactivation of p53. While p53 mutations and MDM2 amplification have been reported to occur in rhabdomyosarcoma and osteogenic sarcoma, the incidence of MDM2 in other pediatric solid tumors is not known. We therefore tested a series of other pediatric solid tumors for MDM2 gene amplification. MDM2 amplification could not be detected in specimens from 40 Wilms' tumors, 15 neuroblastomas, 12 sarcomas, or 4 hepatoblastomas tested. To determine whether MDM2 amplification was an alternative mechanism of p53 inactivation in adult carcinomas that frequently possess p53 mutations, 68 samples of squamous cell carcinomas of the upper aerodigestive tract, 24% of which were previously shown to contain p53 mutations, were also tested for MDM2 amplification. MDM2 amplification did not occur in any of the tumor specimens tested. These findings suggest that MDM2 amplification may only occur in a limited subset of human tumors. Loss of function of p53 may be an essential event in human tumorigenesis. If so, then other mechanisms of p53 inactivation must occur in those tumors that exhibit neither p53 mutation nor MDM2 amplification.
Asunto(s)
Carcinoma de Células Escamosas/genética , Amplificación de Genes , Genes p53 , Proteínas de Neoplasias/genética , Neoplasias/genética , Proteínas Nucleares , Proteínas Proto-Oncogénicas , Adulto , Secuencia de Bases , Niño , Humanos , Datos de Secuencia Molecular , Mutación , Proteínas Proto-Oncogénicas c-mdm2RESUMEN
BACKGROUND: Alteration of the ras family of oncogenes and of the tumor suppressor genes p53 and RB are the most common genetic events in human tumors. Although there have been no reports of the prevalence of these alterations in Wilms tumors, overexpression of the N-myc and insulin-like growth factor-II (IGF-II) genes have been observed, and alteration of another tumor suppressor gene (WT1) has been demonstrated. METHODS: Forty-four Wilms tumor specimens were tested for the presence of N-, K-, and H-ras mutations in codons 12, 13, and 61 by single-strand conformation polymorphism (SSCP) analysis and direct DNA sequence analysis. Sixteen tumors were tested for abnormalities of WT1 by Southern and northern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR). N-myc, c-myc, WT1, and IGF-II mRNA expression was measured in 16 tumors by Northern blot analysis. Thirty-eight tumors were screened for p53 mutations by SSCP analysis and direct DNA sequence analysis. Nine tumors were analyzed for loss of heterozygosity (LOH) of RB. RESULTS: Although the authors confirmed that N-myc and IGF-II are overexpressed in Wilms tumors, no mutations of ras family, p53, or RB genes were identified, and no gross alterations of WT1 were detected by Southern or Northern blot analysis. CONCLUSIONS: These findings suggest that H-ras, K-ras, N-ras, p53, and RB are not involved in the pathogenesis of Wilms tumor.
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Proteínas de Unión al ADN/genética , Genes de Retinoblastoma , Genes p53 , Genes ras , Neoplasias Renales/genética , Tumor de Wilms/genética , Secuencia de Bases , Preescolar , Deleción Cromosómica , Femenino , Humanos , Lactante , Masculino , Datos de Secuencia Molecular , Mutación , Proteínas WT1RESUMEN
OBJECTIVE: The primary objective of this study was to determine the incidence of p53 and retinoblastoma tumor suppressor gene mutations and human papillomavirus infection in squamous cell carcinoma and adjacent normal mucosa of the upper aerodigestive tract. The secondary objective was to associate these findings with clinical and histopathologic features. DESIGN: Point mutations of p53 were identified by single-strand conformation polymorphism analysis and confirmed by direct DNA sequence analysis. Polymerase chain reaction-based methods were used to identify loss of heterozygosity of the retinoblastoma tumor suppressor gene and the presence of human papillomavirus sequences. SETTING: University-based tertiary care center. PATIENTS OR OTHER PARTICIPANTS: Forty-five consecutive cases of upper aerodigestive tract squamous cell carcinoma. RESULTS: Eleven point mutations of p53 were identified in tumor samples (24%). No functional p53 mutations were detected in adjacent normal tissue from eight of these individuals nor was there evidence of p53 alteration in normal tissue adjacent to 12 of 30 additional tumors tested that demonstrated conformational alterations by single-strand conformation polymorphism analysis. The p53 mutations were significantly associated with local invasion. Loss of heterozygosity (which has a 20% chance of random occurrence in tumors) was detected at the retinoblastoma locus in 15% of the tumors tested. Five of the specimens (11%) were positive for human papillomavirus sequences (two of which also contained p53 mutations). CONCLUSIONS: These findings suggest that p53 but not retinoblastoma or human papillomavirus is an important prognostic factor and is involved as a late event in the pathogenesis of upper aerodigestive tract squamous cell carcinoma.