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/genéticaRESUMEN
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 CultivadasRESUMEN
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 InversaRESUMEN
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.