Asunto(s)

Resistencia a Antineoplásicos , Leucemia Mieloide Aguda , Humanos , Resistencia a Antineoplásicos/genética , Proteínas Nucleares/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Proteína Forkhead Box M1/genética

Asunto(s)

Resistencia a Antineoplásicos , Proteína Forkhead Box M1/fisiología , Leucemia Mieloide Aguda/fisiopatología , Proteínas Nucleares/fisiología , Línea Celular Tumoral , Proteína Forkhead Box M1/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Nucleofosmina , Unión Proteica

Asunto(s)

Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Leucemia/patología , Nucleósidos/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Pirimidinas/farmacología , Compuestos de Bifenilo/farmacología , Inhibidores de Caspasas , Caspasas/metabolismo , Sinergismo Farmacológico , Humanos , Immunoblotting , Leucemia/tratamiento farmacológico , Leucemia/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Nitrofenoles/farmacología , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Sulfonamidas/farmacología , Células Tumorales Cultivadas

RESUMEN

Tumor suppressor p53 is stabilized in response to gamma-irradiation or treatment with DNA-damaging agents, and as a result p53 transcriptionally activates its targets leading to cell-cycle arrest or apoptosis. P-TEFb (positive transcription elongation factor b) inhibitors such as flavopiridol or 4-amino-6-hydrazino-7-b-d-ribofuranosyl-7H-pyrrolo[2,3-d]-pyrimidine-5-carboxamide (ARC) upregulate p53 protein levels, but inhibit the expression of its targets p21 and hdm2. DNA-damaging agents, doxorubicin and cisplatin are being used in combination with P-TEFb inhibitor flavopiridol in clinical trials for the treatment of some cancer patients. In this study, we found that P-TEFb inhibitors block the phosphorylation of p53 induced by doxorubicin. Furthermore, treatment of cells with P-TEFb inhibitors together with doxorubicin inhibits doxorubicin-induced binding of p53 to DNA and p53 transcriptional activity. These data suggest that P-TEFb inhibitors may antagonize the activation of p53 by DNA-damaging agents in tumors with wild-type p53.

Asunto(s)

Antineoplásicos/farmacología , Daño del ADN/efectos de los fármacos , ADN de Neoplasias/efectos de los fármacos , Genes p53/efectos de los fármacos , Factor B de Elongación Transcripcional Positiva/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/metabolismo , Antineoplásicos/antagonistas & inhibidores , Antineoplásicos/metabolismo , Flavonoides/farmacología , Células HCT116 , Humanos , Nucleósidos/farmacología , Fosforilación/efectos de los fármacos , Piperidinas/farmacología , Pirimidinas/farmacología , Proteína p53 Supresora de Tumor/genética

RESUMEN

We have previously described the identification of a nucleoside analog transcriptional inhibitor ARC (4-amino-6-hydrazino-7-beta-D-ribofuranosyl-7H-pyrrolo[2,3-d]-pyrimidine-5-carboxamide) that was able to induce apoptosis in cancer cell lines of different origin. Here, we report the characterization of ARC on a panel of neuroblastoma cell lines. We found that these cell lines were more than 10-fold sensitive to ARC than to the well-known nucleoside analog DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole), and that ARC-induced apoptosis proceeds through mitochondrial injury. Also, we observed that ARC-mediated cell death was accompanied by caspase-3 cleavage and repression of antiapoptotic proteins such as Mcl-1 and survivin. Conversely, we found that overexpression of Mcl-1-protected neuroblastoma cell line NB-1691 from ARC-induced apoptosis. Furthermore, we found that while ARC inhibited the phosphorylation of Akt Ser-473 in multiple cancer cell lines, forced expression of myristoylated Akt promoted resistance to ARC-induced apoptosis in neuroblastoma cells. In addition, we observed that ARC was able to downregulate the protein levels of N-myc, a commonly amplified oncogene in neuroblastomas, and Akt protected N-myc from ARC-induced downregulation. These data suggest that ARC may antagonize different antiapoptotic pathways and induce apoptosis in neuroblastoma cells via multiple mechanisms. Overall, ARC could represent an attractive candidate for anticancer drug development against neuroblastomas.

Asunto(s)

Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neuroblastoma/patología , Nucleósidos/farmacología , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Proteínas Proto-Oncogénicas c-myc/efectos de los fármacos , Pirimidinas/farmacología , Caspasa 3/metabolismo , Humanos , Proteínas Inhibidoras de la Apoptosis , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/efectos de los fármacos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas de Neoplasias/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Survivin , Células Tumorales Cultivadas

RESUMEN

Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) pose major public health concerns worldwide. HCV is clearly associated with the occurrence of hepatocellular carcinoma, and recently HIV infection has also been linked to the development of a multitude of cancers. Previously, we identified a novel nucleoside analog transcriptional inhibitor ARC (4-amino-6-hydrazino-7-beta-D-ribofuranosyl-7H-pyrrolo[2,3-d]-pyrimidine-5-carboxamide) that exhibited proapoptotic and antiangiogenic properties in vitro. Here, we evaluated the effect of ARC on HIV-1 transcription and HCV replication. Using reporter assays, we found that ARC inhibited HIV-1 Tat-based transactivation in different cell systems. Also, using hepatoma cells that harbor subgenomic and full-length replicons of HCV, we found that ARC inhibited HCV replication. Together, our data indicate that ARC could be a promising candidate for the development of antiviral therapeutics against HIV and HCV.

Asunto(s)

Antivirales/farmacología , VIH-1/efectos de los fármacos , Hepacivirus/efectos de los fármacos , Nucleósidos/farmacología , Pirimidinas/farmacología , Transcripción Genética/efectos de los fármacos , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Genes Reporteros , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

Asunto(s)

Metilación de ADN , Genes myc , Transcripción Genética , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas p21(ras)/genética

RESUMEN

The p53 tumor-suppressor is a transcription factor that is stabilized in response to cellular stress leading to growth arrest or apoptosis. p21(WAF1/CIP1) is a major transcriptional target of p53 and it plays a critical role in p53-dependent cell cycle arrest. In this study, we identified multiple alternate human p21 transcripts that have their transcriptional start sites in the direct proximity of the distal p53 response element. These transcripts are upregulated as a result of DNA damage-induced p53 activation. Furthermore, the basal expression of these alternate transcripts is strongly regulated by p53 and they are undetectable in p53-knocked down cells. This is in contrast to classical p21 transcripts, which have reduced, albeit detectable expression levels in the absence of p53. The existence of the alternate transcripts underscores the complexity of the human p21 genomic locus and opens up new avenues for further investigation.

Asunto(s)

Empalme Alternativo/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Transcripción Genética/fisiología , Proteína p53 Supresora de Tumor/genética , Secuencia de Bases , Línea Celular , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

The cyclin-dependent kinase inhibitor p21((WAF1/CIP1)) inhibits proliferation both in vitro and in vivo, and overexpression of p21 in normal and tumor cell lines results in cell cycle arrest. In contrast, ectopic expression of Myc alleviates G(1) cell cycle arrest. Recent studies showed that Myc can repress p21 transcription, thereby overriding a p21-mediated cell cycle checkpoint. We found that activation of a Myc-estrogen receptor fusion protein by 4-hydroxytamoxifen in mouse cells resulted in suppression of endogenous p21 transcription. This effect was observed in the absence of de novo protein synthesis and was independent of histone deacetylase activity. In transient transfection studies, Myc effectively repressed p21 promoter constructs containing only 119 bp of sequence upstream of the transcription start site. This region contains multiple Sp1-binding sites and a potential initiator element, but no canonical Myc DNA-binding sites. Deletion of the potential initiator element does not affect repression of the p21 promoter by c-Myc. Coimmunoprecipitation and glutathione S-transferase pull-down experiments demonstrate that c-Myc may form complexes with Sp1/Sp3. We found that the central region of c-Myc interacts with the zinc finger domain of Sp1. Because Sp1 is required for p21 transcription, it is possible that Myc may down-regulate p21 transcription, at least in part, by sequestering Sp1. Repression of the p21 promoter may contribute to the ability of c-Myc to promote cell proliferation.

Asunto(s)

Ciclinas/genética , Proteínas de Unión al ADN/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-myc/fisiología , Factor de Transcripción Sp1/metabolismo , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Línea Celular Transformada , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Cartilla de ADN , Humanos , Ratones , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción Sp3

RESUMEN

The CDK inhibitor p21WAF1/CIP1 is a negative regulator of the cell cycle, and its expression is induced during terminal differentiation in vitro and in vivo. Expression of p21 is controlled at the transcriptional level by both p53-dependent and -independent mechanisms. Our previous studies established that p21 is expressed in the Caco-2 adenocarcinoma cell line, and its expression is induced by a p53-independent mechanism during differentiation of these cells. Here we have found that transcription of p21 in Caco-2 cells is controlled primarily by the transcription factors Sp1 and Sp3 through two Sp1 binding sites, Sp1-1 and Sp1-2, located between -119 and -114 bp and between -109 and -104 bp of the p21 promoter, respectively. Sp1 and Sp3 binding to the p21 promoter increased during Caco-2 cell differentiation, while the absolute level of Sp1 did not change and the absolute level of Sp3 increased approximately twofold. Transfection experiments in the SL2 Drosophila cell line that lacks endogenous Sp3 activity demonstrated that Sp1 transactivates the p21 promoter primarily through the Sp1-2 site, while Sp3 acts through the Sp1-1 site. In these cells Sp3 is a stronger transactivator of the p21 promoter than Sp1. Our data suggest that induction of p21 transcription during Caco-2 differentiation is modulated by Sp1/Sp3 interactions with the p21 promoter.

Asunto(s)

Adenocarcinoma/genética , Neoplasias del Colon/genética , Ciclinas/genética , Proteínas de Unión al ADN/metabolismo , Factor de Transcripción Sp1/metabolismo , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Células CACO-2 , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Drosophila/citología , Humanos , Regiones Promotoras Genéticas , Unión Proteica , Factor de Transcripción Sp3 , Transcripción Genética

RESUMEN

We recently reported that E2F1 could transactivate the p21 promoter via cis-acting elements between -119 to +16 bp of the p21 gene. Here we show that activated V12-H-Ras can induce the p21 promoter through the same region of the p21 promoter by a p53-independent mechanism in NIH3T3 cells. In contrast, activated Ras was not able to induce the p21 promoter in E2F1-/- fibroblasts, suggesting that E2F1 is required for induction of the p21 promoter by activated Ras. Cotransfection of increasing concentrations of dominant negative E2F1 alone, or with dominant negative DP1 into NIH3T3 cells suppressed induction of the p21 promoter by activated Ras. These data suggest that p53-independent induction of the p21 promoter by activated Ras is mediated at least in part by E2F1. Oncogene (2000) 19, 961 - 964.

Asunto(s)

Proteínas Portadoras , Proteínas de Ciclo Celular , Ciclinas/genética , Ciclinas/fisiología , Proteínas de Unión al ADN , Regulación de la Expresión Génica/genética , Factores de Transcripción/fisiología , Transcripción Genética/genética , Proteínas ras/fisiología , Células 3T3 , Animales , Línea Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Ratones , Proteína 1 de Unión a Retinoblastoma , Factor de Transcripción DP1 , Factores de Transcripción/genética , Proteínas ras/genética

Asunto(s)

Ciclinas/genética , Regulación de la Expresión Génica , Animales , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Genes ras , Humanos , Transcripción Genética , Proteína p53 Supresora de Tumor/metabolismo

RESUMEN

The Cdk inhibitor p21(WAF1/CIP1) is a negative regulator of the cell cycle, although its expression is induced by a number of mitogens that promote cell proliferation. We have found that E2F1 and E2F3, transcription factors that activate genes required for cell cycle progression, are strong activators of the p21 promoter. In contrast, HBP1 (HMG-box protein-1), a novel retinoblastoma protein-binding protein, can repress the p21 promoter and inhibit induction of p21 expression by E2F. Both E2Fs and HBP1 regulate p21 transcription through cis-acting elements located between nucleotides -119 to +16 of the p21 promoter and the DNA binding domains of each of these proteins are required for activity. Sequences between -119 and -60 basepairs containing four Sp1 consensus elements and two noncanonical E2F binding sites are of major importance for E2F activation, although E2F1 and E2F3 differ in the extent of their ability to activate expression when this segment is deleted. The opposing effects of E2Fs and HBP1 on p21 promoter activity suggest that interplay between these factors may determine the level of p21 transcription in vivo.

Asunto(s)

Proteínas Portadoras , Ciclinas/genética , Ciclinas/metabolismo , Proteínas de Unión al ADN , Proteínas Represoras/metabolismo , Proteína de Retinoblastoma/metabolismo , Transcripción Genética , Secuencia de Bases , Sitios de Unión , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Factor de Transcripción E2F2 , Factor de Transcripción E2F3 , Regulación de la Expresión Génica , Secuencias Hélice-Asa-Hélice/genética , Proteínas del Grupo de Alta Movilidad/genética , Proteínas del Grupo de Alta Movilidad/metabolismo , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Proteínas Represoras/genética , Proteína 1 de Unión a Retinoblastoma , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción DP1 , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas

Asunto(s)

División Celular/fisiología , Ciclinas/fisiología , Animales , Apoptosis , Ciclo Celular/fisiología , Diferenciación Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Regulación del Desarrollo de la Expresión Génica , Humanos , Neoplasias/fisiopatología

RESUMEN

The cyclin-dependent kinase, proliferating cell nuclear antigen, and stress-activated protein kinase/c-jun NH2 terminal kinase inhibitor p21WAF1/CIP1 can induce G1 arrest, and its expression coincides with the cessation of replication in many systems. We examined expression of p21 during the early stages of carbon tetrachloride intoxication in the mouse liver and observed a dramatic increase in p21 RNA levels between 4 and 8 h after administration. p21 expression, visualized by in situ hybridization, is induced in pericentral hepatocytes before carbon tetrachloride-induced necrosis. Examination of c-fos and c-myc expression patterns confirm that these immediate-early genes are induced in similar regions of the mouse liver. p21 induction is not dependent on p53; we observed similar levels and localization of p21 in wild-type and p53 null animals. Immunohistochemical localization of p21 and CCAAT/enhancer-binding protein expression shows that p21 protein accumulation is limited to a subset of CCAAT/enhancer-binding protein-positive hepatocytes. A second peak of periportal and intermediate zone-specific p21 gene expression, appearing 1-2 days after injection, is also p53 independent and may represent cell cycle checkpoints or postmitotic growth arrest. Sporadic p21 expression was also detected in pairs of hepatocytes distributed throughout the liver acini in healthy animals. Together, these data suggest several roles for p21 in the liver in response to toxicity, regeneration, and growth inhibition.

Asunto(s)

Tetracloruro de Carbono/toxicidad , Ciclinas/biosíntesis , Ciclinas/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Proteína p53 Supresora de Tumor/fisiología , Animales , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Regulación de la Expresión Génica/efectos de los fármacos , Hígado/citología , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Noqueados

RESUMEN

Progression through the cell cycle is regulated by cyclins and cyclin-dependent kinases (Cdks). The cyclin kinase inhibitor p21 (also known as WAF1, CIP1, SDI1, and MDA-6) can induce G1 arrest and block entry into S phase by inactivating Cdks or by inhibiting activity of proliferating cell nuclear antigen (PCNA). In normal cells, p21 exists in quaternary complexes with cyclin, Cdk, and PCNA. Transcription of the p21 gene is activated by p53-dependent and -independent mechanisms. Mice deficient in p21 exhibit no apparent phenotype, although p21 function has been demonstrated to be necessary for p53-mediated G1 arrest following irradiation of p21-deficient mouse embryonic fibroblasts. Thus, the function of p21 under normal circumstances appears to be redundant. p21 is expressed in terminally differentiating cells of a variety of tissues in a p53-independent manner. Overexpression of p21 results in G1 arrest and has been shown to suppress effectively tumor growth in vitro and in vivo. We review the recent literature describing the functional characterization of p21. This protein plays a key role in regulating the cell cycle and may have potential gene therapy applications.

Asunto(s)

Ciclo Celular/fisiología , Ciclinas/fisiología , Inhibidores Enzimáticos , Animales , Ciclo Celular/efectos de los fármacos , Diferenciación Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/antagonistas & inhibidores , Ciclinas/farmacología , Daño del ADN , Humanos , Neoplasias/genética , Neoplasias/patología

RESUMEN

We examined the relationship between expression of the p21 (WAF1/CIP1) inhibitor of cyclin-dependent kinases, cessation of proliferation, and terminal differentiation in the epithelia of the gastrointestinal tract. Using in situ hybridization, we performed a detailed study of patterns of p21 mRNA expression in different regions of the stomach, along the length of the intestine, and in tongue, cervix, and hair follicle. We detected strong hybridization only in cells that had ceased proliferation and begun the process of terminal differentiation. Induction of p21 transcription may serve as a useful marker for dissection of differentiation programs in these diverse epithelia. To determine the relative levels of p21 expressed in various regions of the gastrointestinal tract from the esophagus to the colon, we used quantitative RT-PCR with endogenous and exogenous sequences as internal standards. The highest levels of p21 expression were detected in the distal small intestine. To further investigate the role that cell cycle regulation may play during differentiation of intestinal epithelial cells, we examined the expression of p53, p21, cyclin D1, cyclin E, and E2F1 in the Caco-2 colon carcinoma cell line, which differentiates spontaneously after reaching confluence. p21 and p53 mRNA and protein levels increase as Caco-2 cells differentiate. In both undifferentiated and differentiated Caco-2 cells, p53 protein was not inducible by DNA damaging agents, suggesting the absence of functionally wildtype protein. Caco-2 cells should provide a useful model system for studying regulation of p21 and determining if it plays a role during intestinal epithelial cell differentiation.

Asunto(s)

Células CACO-2/fisiología , Ciclinas/genética , Inhibidores Enzimáticos/metabolismo , Interfase/fisiología , Western Blotting , Células CACO-2/citología , Ciclo Celular/genética , Diferenciación Celular/genética , Diferenciación Celular/fisiología , División Celular/fisiología , Cuello del Útero/citología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Células Epiteliales , Epitelio/fisiología , Femenino , Regulación de la Expresión Génica/fisiología , Folículo Piloso/citología , Humanos , Hibridación in Situ , Intestino Delgado/química , Intestino Delgado/citología , ARN Mensajero/análisis , Lengua/citología

RESUMEN

The S mu-C mu intron of the IgH chain locus is conserved in rodents, but its biological function is unknown. It has been shown that switch recombination breakpoints are concentrated within the repetitive sequences in the S mu region in mitogen-activated normal B cells. In Ig-secreting hybridomas these breakpoints occur most frequently at the most 5' end, and immediately upstream of the S mu DNA. The S mu-C mu intron appears remarkably protected from recombination. Because the nucleoprotein complexes that drive transcription and recombination may overlap, the transcriptional characteristics of this fragment were studied. The cis-acting regulatory elements in the S mu-C mu intron were identified by ligating the entire intron, or a series of subfragments to the TK promoter and bacterial chloramphenicol acetyltransferase gene. Expression of these constructs was tested in activated B cells and the nonlymphoid cell lines HeLa and HepG2. The complete S mu-C mu intron (1 kb) had a negative effect on TK promoter activity in activated B cells only when placed upstream of the promoter, in both orientations. Segmentation of the S mu-C mu intron has revealed that this region contains multiple negative elements active in B cells. A subfragment located at the 3' end of the S mu-C mu intron contains a B-cell-specific negative element, while the subfragment located at the 5'end has cell-type-independent repressing activity.

Asunto(s)

Linfocitos B/metabolismo , Regulación de la Expresión Génica , Región de Cambio de la Inmunoglobulina , Cadenas mu de Inmunoglobulina/genética , Intrones , Animales , Células Cultivadas , Expresión Génica , Reordenamiento Génico de Cadena Pesada de Linfocito B , Ratones , Ratones Endogámicos BALB C , Secuencias Reguladoras de Ácidos Nucleicos , Transcripción Genética

RESUMEN

The data on the effects of antisense RNA in plants is reviewed. Results of expression of the genes for selective markers, antisense reporter genes, functioning and viral genes are analyzed. The molecular mechanisms for inhibiting effects of antisense RNA and the potential use of the phenomenon in the plants biotechnology are discussed. The formation of long duplexes between the antisense RNA and messenger RNA are supposed to be irrelevant to suppression of gene expression in plants by the antisense RNA.

Asunto(s)

Ingeniería Genética , Plantas/genética , ARN sin Sentido/genética

RESUMEN

Isolation of genomic clones containing the human insulin gene by screening the human genomic library for this gene using the cDNA rat insulin probe is reported. The analysis of promoter-enhancer region and exons sequences has revealed their identity to analogous sequences determined earlier.

Asunto(s)

Clonación Molecular , Insulina/genética , Humanos