RESUMEN
Many aspects of mammalian physiology and behavior are driven by an intrinsic timekeeping system that has an important role in synchronizing various biological processes within an organism and coordinating them with the environment. It is believed that deregulation of this coordination may cause the development of various pathologies. However, recent studies using mice deficient in individual components of the circadian system clearly demonstrated more complex interaction of the circadian system with various biological processes. The growing amount of evidence suggests that in addition to their roles in the core clock mechanism, some of the components of the molecular oscillator are involved in modulation of such diverse physiological processes as response to genotoxic stress, regulation of the cell cycle, aging, and carcinogenesis. These new data provide a mechanistic link between deregulation of the circadian system and/or some of its core components and the development of various pathologies, suggesting novel strategies for the disease treatment and prevention.
Asunto(s)
Envejecimiento/fisiología , Ritmo Circadiano/fisiología , Neoplasias/etiología , Transactivadores/fisiología , Factores de Transcripción ARNTL , Envejecimiento/genética , Envejecimiento Prematuro/etiología , Envejecimiento Prematuro/genética , Envejecimiento Prematuro/fisiopatología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/deficiencia , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Proteínas CLOCK , Ritmo Circadiano/genética , Humanos , Ratones , Ratones Noqueados , Ratones Mutantes , Modelos Biológicos , Neoplasias/genética , Neoplasias/fisiopatología , Transactivadores/genética , Transcripción GenéticaRESUMEN
By screening a chemical library for the compounds protecting cells from adriamycin (Adr), a series of small molecules was isolated that interfered with the accumulation of Adr in mouse fibroblasts by enhancing efflux of the drug. Isolated compounds also stimulated efflux of Rhodamine 123 (Rho-123), another substrate of multidrug transporters. Stimulation of drug efflux was detectable in the cells expressing P-glycoprotein (P-gp), but not in their P-gp-negative variants, and was completely reversible by the P-gp inhibitors. A dramatic stimulation of P-gp activity against Adr and Rho-123 by the identified compounds was accompanied by suppression of P-gp-mediated efflux of other substrates, such as Taxol (paclitaxel) or Hoechst 33342, indicating that they act as modulators of substrate specificity of P-gp. Consistently, P-gp modulators dramatically altered the pattern of cross-resistance of P-gp-expressing cells to different P-gp substrates: an increase in resistance to Adr, daunorubicin, and etoposide was accompanied by cell sensitization to Vinca alkaloids, gramicidin D, and Taxol with no effect on cell sensitivity to colchicine, actinomycin D, puromycin, and colcemid, as well as to several non-P-gp substrates. The relative effect of P-gp modulators against different substrates varied among the isolated compounds that can be used as fine tools for analyzing mechanisms of drug selectivity of P-gp. These results raise the possibility of a rational control over cell sensitivity to drugs and toxins through modulation of P-gp activity by small molecules.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antibióticos Antineoplásicos/metabolismo , Doxorrubicina/metabolismo , Resistencia a Múltiples Medicamentos , Imidazoles/metabolismo , Tiazoles/metabolismo , Animales , Antibióticos Antineoplásicos/farmacología , Línea Celular , Doxorrubicina/farmacología , Evaluación Preclínica de Medicamentos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Imidazoles/química , Imidazoles/farmacología , Ratones , Estructura Molecular , Fenotipo , Rodamina 123/metabolismo , Rodamina 123/farmacología , Especificidad por Sustrato , Tiazoles/química , Tiazoles/farmacología , Proteína p53 Supresora de Tumor/metabolismoAsunto(s)
Regiones no Traducidas 3' , Transducción de Señal , Activador de Tejido Plasminógeno/genética , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , ADN , Humanos , Ratones , Datos de Secuencia Molecular , Plásmidos , ARN Mensajero/genética , Ratas , Eliminación de Secuencia , Homología de Secuencia de Ácido NucleicoRESUMEN
Chemotherapy and radiation therapy for cancer often have severe side effects that limit their efficacy. Because these effects are in part determined by p53-mediated apoptosis, temporary suppression of p53 has been suggested as a therapeutic strategy to prevent damage of normal tissues during treatment of p53-deficient tumors. To test this possibility, a small molecule was isolated for its ability to reversibly block p53-dependent transcriptional activation and apoptosis. This compound, pifithrin-alpha, protected mice from the lethal genotoxic stress associated with anticancer treatment without promoting the formation of tumors. Thus, inhibitors of p53 may be useful drugs for reducing the side effects of cancer therapy and other types of stress associated with p53 induction.
Asunto(s)
Antineoplásicos/efectos adversos , Apoptosis/efectos de los fármacos , Rayos gamma/efectos adversos , Neoplasias/terapia , Tolerancia a Radiación/efectos de los fármacos , Tiazoles/farmacología , Tolueno/análogos & derivados , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Benzotiazoles , División Celular/efectos de los fármacos , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , ADN/biosíntesis , Daño del ADN , Fase G2/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , Neoplasias/tratamiento farmacológico , Neoplasias/radioterapia , Factores de Tiempo , Tolueno/farmacología , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/fisiología , Rayos Ultravioleta/efectos adversosRESUMEN
Activation of p53 plays a central role in the cell's response to various stress signals. We investigated whether p53 is activated upon disruption of actin microfilaments, caused by cytochalasin D (CD). We show that treatment with CD leads to accumulation of p53 in the cells and activation of p53-dependent transcription. Treatment with CD led to arrest of G1-to-S transition in cells retaining wild-type p53, while cells with inactivated p53 showed partial rescue from it. CD also induces apoptosis in p53+/+, but not in p53-/- cells. The obtained data suggest that disruption of the actin microfilaments activates p53-dependent pathways.
Asunto(s)
Citoesqueleto de Actina/efectos de los fármacos , Citocalasina D/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Actinas/efectos de los fármacos , Animales , Apoptosis , División Celular , Supervivencia Celular , Células Cultivadas , Fibroblastos , Ratones , Ratas , Activación TranscripcionalRESUMEN
[3H]5'-O-Phosphonylmethylthymidine with a specific activity of 71 Ci/mmol was obtained by isotope exchange. Its incubation with a HeLa cell culture resulted in the formation of [3H]-labeled 5'-O-(beta-phosphoryl-alpha-phosphonylmethyl)thymidine, 5'-O-(beta,gamma-diphosphoryl-alpha-phosphonylmethyl)thymidine, and [3H]DNA. This proved the ability of 5'-O-phosphonylmethylthymidine to undergo phosphorylation and incorporation into the DNA of human cells.
Asunto(s)
ADN de Neoplasias/metabolismo , Compuestos Organofosforados/metabolismo , Timidina/análogos & derivados , Células HeLa , Humanos , Fosforilación , Timidina/metabolismo , TritioRESUMEN
p53 is a multifunctional tumor suppressor protein involved in the negative control of cell growth. Mutations in p53 cause alterations in cellular phenotype, including immortalization, neoplastic transformation, and resistance to DNA-damaging drugs. To help dissect distinct functions of p53, a set of genetic suppressor elements (GSEs) capable of inducing different p53-related phenotypes in rodent embryo fibroblasts was isolated from a retroviral library of random rat p53 cDNA fragments. All the GSEs were 100-300 nucleotides long and were in the sense orientation. They fell into four classes, corresponding to the transactivator (class I), DNA-binding (class II), and C-terminal (class III) domains of the protein and the 3'-untranslated region of the mRNA (class IV). GSEs in all four classes promoted immortalization of primary cells, but only members of classes I and III cooperated with activated ras to transform cells, and only members of class III conferred resistance to etoposide and strongly inhibited transcriptional transactivation by p53. These observations suggest that processes related to control of senescence, response to DNA damage, and transformation involve different functions of the p53 protein and furthermore indicate a regulatory role for the 3'-untranslated region of p53 mRNA.
Asunto(s)
Genes p53 , Proteína p53 Supresora de Tumor/biosíntesis , Animales , Secuencia de Bases , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , ADN Complementario , Proteínas de Unión al ADN/metabolismo , Resistencia a Medicamentos , Embrión de Mamíferos , Etopósido/toxicidad , Fibroblastos , Datos de Secuencia Molecular , Fenotipo , ARN Mensajero/biosíntesis , Ratas , Retroviridae , Supresión Genética , Transactivadores/metabolismoAsunto(s)
Proteína p53 Supresora de Tumor/genética , Secuencia de Bases , Cloranfenicol O-Acetiltransferasa/genética , Genes p53 , Datos de Secuencia Molecular , Mutación , Oligodesoxirribonucleótidos , Plásmidos , Regiones Promotoras Genéticas , Unión Proteica , Transactivadores/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The ability of ras oncogenes and mutant p53 to activate reporter gene expression from human and rodent mdr1 gene promoters was described, although functional significance of this finding was unclear. We analyzed the influence of various forms of recombinant human ras and p53 on the mdr1 gene expression and P-glycoprotein (Pgp) function in rodent immortalized fibroblasts. The ras genes, in addition to activation of exogenous human mdr1 gene promoter, caused an increase in (i) expression of endogenous mdr1 mRNA, (ii) Pgp activity as determined by flow cytometry analysis of Rhodamine 123 exclusion, and (iii) resistance of cells to the cytotoxic action of colchicine and some other drugs. To elucidate whether the same signalling pathway is responsible for multidrug resistance induced by various oncogenes and protein kinase C (PKC), we tested the effects of v-mos and the PKC agonist 12-O-tetradecanoylphorbol-13-acetate. Similarly to cells transformed by ras, a Rat1 subline transformed by the v-mos oncogene was characterized by decreased drug sensitivity. On the contrary, Rat1 cells treated with the protein kinase C agonist 12-O-tetradecanoylphorbol-13-acetate showed neither increased mdr1 mRNA expression nor stimulation of Pgp function. Introduction by retrovirus-mediated gene transfer of wild-type p53 into Rat1 cells or into murine p53-deficient 10(1) and 10(3) cells did not change the Pgp function significantly, whereas in Rat1 cells transformed by activated N-ras or v-mos, expression of wild-type p53 caused partial reversion of oncogene-induced drug resistance.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Resistencia a Múltiples Medicamentos/genética , Regulación de la Expresión Génica , Genes p53 , Genes ras , Regiones Promotoras Genéticas , Transfección , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Animales , Línea Celular Transformada , Supervivencia Celular/efectos de los fármacos , Colchicina/farmacología , Fibroblastos , Expresión Génica , Genes mos , Humanos , Ratones , Proteínas Oncogénicas v-mos/biosíntesis , Proteínas Proto-Oncogénicas p21(ras)/biosíntesis , Ratas , Proteínas Recombinantes/biosíntesis , Acetato de Tetradecanoilforbol/farmacología , Proteína p53 Supresora de Tumor/biosíntesisRESUMEN
Expression of urokinase in murine and rat cells was performed by two recombinant constructs, one containing cDNA and the other--hybrid (cDNA/genome) variant of human urokinase gene conserving 7 introns of 10, in the eukaryotic retrovirus vector pPS-3-neo. DNA of both constructs was introduced into packaging cell line psi 2 by a standard Ca-phosphate transfection technique. Infection of mouse and rat fibroblasts BALB/c 3T3 and Rat I with virus particles, produced by transfected psi 2 cells, led to an integration into the host genome of one or two recombinant proviral copies. Stable expression and secretion into the culture medium of glycosylated high molecular weight human urokinase was observed for both cell types. For the hybrid gene construct, precise excision of intervening sequences was shown during transferring of genetic material from packaging to recipient cells.