RESUMEN
Regulation of gene expression at the level of transcriptional elongation has been shown to be important in stem cells and tumour cells, but its role in the whole animal is only now being fully explored. Neural crest cells (NCCs) are a multipotent population of cells that migrate during early development from the dorsal neural tube throughout the embryo where they differentiate into a variety of cell types including pigment cells, cranio-facial skeleton and sensory neurons. Specification of NCCs is both spatially and temporally regulated during embryonic development. Here we show that components of the transcriptional elongation regulatory machinery, CDK9 and CYCLINT1 of the P-TEFb complex, are required to regulate neural crest specification. In particular, we show that expression of the proto-oncogene c-Myc and c-Myc responsive genes are affected. Our data suggest that P-TEFb is crucial to drive expression of c-Myc, which acts as a 'gate-keeper' for the correct temporal and spatial development of the neural crest.
Asunto(s)
Ciclina T/genética , Quinasa 9 Dependiente de la Ciclina/genética , Regulación del Desarrollo de la Expresión Génica , Genes myc , Cresta Neural/embriología , Factor B de Elongación Transcripcional Positiva/genética , Elongación de la Transcripción Genética , Proteínas de Xenopus/genética , Xenopus laevis/embriología , Animales , Ciclina T/deficiencia , Quinasa 9 Dependiente de la Ciclina/deficiencia , Isoxazoles/farmacología , Leflunamida , Morfolinos/farmacología , Factor B de Elongación Transcripcional Positiva/deficiencia , Proteínas Proto-Oncogénicas c-myc/biosíntesis , ARN Polimerasa II/metabolismo , Factores de Transcripción SOXE/biosíntesis , Factores de Transcripción SOXE/genética , Elongación de la Transcripción Genética/efectos de los fármacos , Transcripción Genética , Proteínas de Xenopus/deficiencia , Xenopus laevis/genéticaRESUMEN
It has been demonstrated that miR-874 plays important roles in many types of cancers. Nevertheless, its biological function in breast cancer remains largely unknown. In this study, we found that the expression level of miR-874 is down-regulated in breast cancer in comparison with the adjacent normal tissues. The overexpression of miR-874 is able to inhibit cell proliferation and induce cell apoptosis and cell cycle arrest in MCF7 and MDA-MB-231 cells. Using a bioinformatics method, we further show that CDK9 is a direct target of miR-874 and that its protein level is negatively regulated by miR-874. Therefore, the data reported in this manuscript demonstrate that miR-874 is an important regulator in breast cancer and imply that the miR-874/CDK9 axis has potential as a therapeutic target for breast cancer.
Asunto(s)
Apoptosis/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Quinasa 9 Dependiente de la Ciclina/genética , MicroARNs/genética , Adulto , Anciano , Secuencia de Bases , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , Quinasa 9 Dependiente de la Ciclina/deficiencia , Regulación hacia Abajo/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Persona de Mediana EdadRESUMEN
Previously we determined that S81 is the highest stoichiometric phosphorylation on the androgen receptor (AR) in response to hormone. To explore the role of this phosphorylation on growth, we stably expressed wild-type and S81A mutant AR in LHS and LAPC4 cells. The cells with increased wild-type AR expression grow faster compared with parental cells and S81A mutant-expressing cells, indicating that loss of S81 phosphorylation limits cell growth. To explore how S81 regulates cell growth, we tested whether S81 phosphorylation regulates AR transcriptional activity. LHS cells stably expressing wild-type and S81A mutant AR showed differences in the regulation of endogenous AR target genes, suggesting that S81 phosphorylation regulates promoter selectivity. We next sought to identify the S81 kinase using ion trap mass spectrometry to analyze AR-associated proteins in immunoprecipitates from cells. We observed cyclin-dependent kinase (CDK)9 association with the AR. CDK9 phosphorylates the AR on S81 in vitro. Phosphorylation is specific to S81 because CDK9 did not phosphorylate the AR on other serine phosphorylation sites. Overexpression of CDK9 with its cognate cyclin, Cyclin T, increased S81 phosphorylation levels in cells. Small interfering RNA knockdown of CDK9 protein levels decreased hormone-induced S81 phosphorylation. Additionally, treatment of LNCaP cells with the CDK9 inhibitors, 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole and Flavopiridol, reduced S81 phosphorylation further, suggesting that CDK9 regulates S81 phosphorylation. Pharmacological inhibition of CDK9 also resulted in decreased AR transcription in LNCaP cells. Collectively these results suggest that CDK9 phosphorylation of AR S81 is an important step in regulating AR transcriptional activity and prostate cancer cell growth.
Asunto(s)
Quinasa 9 Dependiente de la Ciclina/metabolismo , Receptores Androgénicos/metabolismo , Serina/metabolismo , Antagonistas de Receptores Androgénicos/metabolismo , Andrógenos/metabolismo , Animales , Células COS , Procesos de Crecimiento Celular/genética , Células Cultivadas , Chlorocebus aethiops , Ciclina T/biosíntesis , Ciclina T/genética , Ciclina T/metabolismo , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 9 Dependiente de la Ciclina/deficiencia , Quinasa 9 Dependiente de la Ciclina/genética , Diclororribofuranosil Benzoimidazol/farmacología , Flavonoides/farmacología , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Fosforilación , Piperidinas/farmacología , Regiones Promotoras Genéticas , ARN Interferente Pequeño/genética , Receptores Androgénicos/genética , Serina/genética , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , TransfecciónRESUMEN
Preclinical studies were performed of a novel selective imidazopyridine cyclin-dependent kinase (cdk) inhibitor, AZ703. In vitro kinase assays showed that IC50 values for AZ703 against purified cyclin E/cdk2 and cyclin B/cdk1 were 34 and 29 nmol/L, respectively. In contrast, the IC50 against cdk4 was 10 micromol/L. AZ703 also inhibited cdk7 and cdk9 with IC50 values of 2.1 micromol/L and 521 nmol/L, respectively. Treatment of U2OS, NCI-H1299, and A549 cells for 24 hours resulted in growth arrest involving multiple cell cycle phases. At low drug concentrations (< 2 micromol/L), G2 arrest predominated, whereas at higher concentrations (> or = 2 micromol/L), S-G2 arrest was observed. When cells were synchronized in G1 by starvation and released into AZ703, a block in G1 occurred that was not evident in exponentially growing cells. Cell cycle arrest was associated with reduced phosphorylation of the retinoblastoma protein and p27(Kip1) at cdk2 phospho-sites. Following longer exposures, apoptosis was evident. Cells were further sensitized to AZ703 following recruitment to S phase by synchronization. Consistent with the inhibition of cdks during S and G2 that modulate the activity and stability of E2F-1, AZ703 treatment induced E2F-1 expression. In U2OS and NCI-H1299 cells engineered to inducibly express the dominant-negative mutant E2F-1 (1-374), expression of the mutant decreased AZ703-mediated apoptosis, indicating dependence on E2F-1 transcriptional targets. AZ703-induced apoptosis in NCI-H1299 cells was enhanced by small interfering RNA-mediated depletion of cdk9, which caused reduced levels of Mcl-1 and XIAP, suggesting that cdk2, cdk1, and cdk9 represent a rational subset of family members for drug targeting.