RESUMEN
An enzymatic assay system is described that allows quantitative localization within different cellular structures of recombinant proteins. The system is based on alpha-complementation of beta-galactosidase. The large omega-fragment of beta-galactosidase is expressed in predefined cellular structures with the aid of attached protein localization signals. The obtained reporter cell lines are used for the introduction of a second construct that expresses a protein of study fused with a shorter alpha-fragment of beta-galactosidase. Physical proximity of the two recombinant proteins carrying beta-galactosidase fragments results in reconstitution of an active enzyme, and the activity can be measured in a plate reader. The recombinant constructs are based on lentiviral vectors, which allows rapid and efficient introduction of recombinant proteins into cells by infection with stocks of lentiviral particles. The efficiency of the system is demonstrated with transcriptional factor FOXO3A, which is shuttling between cytoplasm and nuclei in model colon carcinoma cell line RKO.
Asunto(s)
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Factores de Transcripción Forkhead/metabolismo , Vectores Genéticos , Lentivirus , Proteínas Recombinantes/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Línea Celular Tumoral , Núcleo Celular/genética , Citoplasma/genética , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/genética , Humanos , Proteínas Recombinantes/genéticaRESUMEN
Inactivation of tumor suppressor p53 accompanies the majority of malignant diseases in humans. Restoration of p53 functions in tumor results in death of cancer cells, which can be used in cancer therapy. In cervical cancer a product of E6 gene of the human papilloma virus promotes accelerated degradation of p53 in proteasome system. Therefore, one of the approaches to reactivation of p53 in cervical carcinoma cells could be the use of small molecules that inhibit functions of viral proteins. By using as a test system human cervical carcinoma cells (HeLa cell line bearing human papilloma virus type 18, HPV-18) with introduced reporter construct that expresses beta-galactosidase under control of a p53-dependent promoter we carried out screening of a library of small molecules to select small molecules capable of reactivating transcriptional activity of p53. We then characterized the effects of two most active compounds in cell lines that differ in the status of p53-dependent signaling pathway. Both of the compounds caused specific activation of p53 in the cell lines expressing HPV-18, to a lesser extent--HPV-16, and do not cause any effect in control p53 negative cells, or in the cells with undisrupted p53 pathway. Activation of p53 in cervical carcinoma cells was accompanied by the induction of the p53-dependent gene CDKN1 (p21), by inhibition of proliferation, and by the induction of apoptosis. Both of the compounds were capable of deep inhibition of transcription from the HPV genome, which apparently was the cause for p53 reactivation in response to decreased expression of the E6 protein. The observed low toxicity for normal cells allows considering these chemical compounds as prototypes for future anticancer drugs.
Asunto(s)
Antineoplásicos/farmacología , Proteínas de Unión al ADN/metabolismo , Papillomavirus Humano 18/efectos de los fármacos , Proteínas Oncogénicas Virales/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Antineoplásicos/química , Apoptosis , Benzodioxoles/química , Benzodioxoles/farmacología , Benzopiranos/química , Benzopiranos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Genes Reporteros , Células HeLa , Papillomavirus Humano 16/efectos de los fármacos , Papillomavirus Humano 16/metabolismo , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/metabolismo , Humanos , Regiones Promotoras Genéticas , Piranos/química , Piranos/farmacología , Quinolinas/química , Quinolinas/farmacología , Transcripción Genética , Neoplasias del Cuello Uterino , beta-Galactosidasa/metabolismoRESUMEN
A study was made of the effect of activated oncogene N-RAS on the function of tumor suppressor p53 and the proliferating ability of rat embryo fibroblasts REF52. The proliferation rate and the portion of S-phase cells increased in the first three days of N-RAS expression. After 5-7 days, the p53 function was enhanced, as manifest in increased p53 lifespan and nuclear content and induced transcription of p53-responsive genes. In particular, Cdk2 p21WAF1/CIP1, an inhibitor of cyclin-dependent kinase 2, was produced to a higher level and arrested the cell cycle in G1. Cells with abrogated or dramatically inhibited N-RAS expression were generated at this stage. Having a selective advantage, these cells gradually displaced N-RAS-expressing cells arrested in G1, so that one month after oncogene induction the culture mostly consisted of morphologically normal, actively proliferating Res-negative cells. Neither cell cycle arrest nor reversion to the normal phenotype were observed in N-RAS expressing cells devoid of the p53 function. Thus, p53 prevented stable N-RAS-induced transformation of REF52 cells, arresting the cell cycle and expediting revertant selection.
Asunto(s)
Transformación Celular Neoplásica/genética , Fibroblastos/fisiología , Proteína p53 Supresora de Tumor/fisiología , Proteínas ras/genética , Animales , Ciclo Celular/genética , División Celular/genética , Células Cultivadas , Fibroblastos/patología , Regulación de la Expresión Génica , Genes ras , Ratas , Proteínas ras/metabolismoRESUMEN
The effect of modifications of p53 expression on the incidence of numerical and structural chromosome aberrations was studied. Infection of LIM1215 cells containing two alleles of the wild-type p53 gene (P53wt) with the recombinant viruses that expressed mutant cDNAs coding for human p53 (His273, Trp248, and His175) resulted in appearance of hyperdiploid cells in populations and an increased proportion of metaphases with chromosome breakage. Expression of the exogenous p53wt or vectors HSG/neo and pPS/neo, which did not contain the p53 cDNA, did not induce numerical or structural chromosome aberrations. Treatment of cells with caffeine decreased the p53wt content and increased the proportion of metaphases with chromosome breaks; however, it did not induce hyperdiploidy in the majority of cell lines. Only in the subline that expressed the exogenous p53Trp248 did caffeine treatment increase the proportion of hyperdiploid variants, which was correlated with the hyperexpression of the product of the mutant allele. The increase in the frequency of chromosome breaks probably resulted from p53wt inactivation, whereas changes in chromosome number might be induced by some additional activities of p53 determined by mutations. Possible mechanisms for inducing heteroploidy by mutant p53 variants, including the role of endoreduplication in inducing hyper- and polyploidy, are discussed.
Asunto(s)
Aberraciones Cromosómicas , Genes p53 , Poliploidía , Proteína p53 Supresora de Tumor/genética , Línea Celular , Expresión Génica , Humanos , Cariotipificación , Mutación , TransfecciónRESUMEN
Previously, we showed that, development of multidrug resistance (MDR) in mouse P388 leukemia cells, is often associated with the appearance of newly-formed chromosome-like structures that contain amplified copies of the mdrl gene. In the present study, we compared amplicon content in P388 sublines showing different types of these structures. A strong correlation between the formation of specific acentric markers consisting of two identical arms and the absence of the sorcin gene co-amplification was found. In all the sublines containing other types of chromosome-like structures, the sorcin gene is co-amplified.
Asunto(s)
Proteínas de Unión al Calcio/genética , Resistencia a Múltiples Medicamentos/genética , Resistencia a Antineoplásicos/genética , Amplificación de Genes , Proteínas de Neoplasias/genética , Animales , Cromosomas , Marcadores Genéticos , Ratones , Células Tumorales CultivadasRESUMEN
The effect of the expression of the exogenous human mutant p53 (Arg-->His in codon 273) on the amplification rate of the gene dhfr in permissive Rat-1 and LIM1215 cells was studied. It was shown that injection of a retroviral construction with p53His273 resulted in the accumulation of methotrexate-resistant variants with an increased number of dhfr copies in populations of recipient cells. Luria-Delbruck fluctuation analysis revealed a four- to six-fold increase in the rate of appearance of new methotrexate-resistant cells. Chromosomal analysis demonstrated an extrachromosomal location of amplified DNA in cells containing p53His273, as was the case for control sublines. The data obtained indicate that modifications of p53 may induce gene amplification not only via removing the proliferation block of cells with amplified genes in selective medium, but also via some other mechanisms, that seem to increase the chromosomal recombination rate.