RESUMO
Apoptosis is a fundamental host defence mechanism against invading microbes. Inactivation of NF-kappaB attenuates encephalomyocarditis virus (EMCV) virulence by triggering rapid apoptosis of infected cells, thereby pre-emptively limiting viral replication. Recent evidence has shown that hypoxia-inducible factor (HIF) increases NF-kappaB-mediated anti-apoptotic response in clear-cell renal cell carcinoma (CCRCC) that commonly exhibit hyperactivation of HIF due to the loss of its principal negative regulator, von Hippel-Lindau (VHL) tumour suppressor protein. Here, we show that EMCV challenge induces a strong NF-kappaB-dependent gene expression profile concomitant with a lack of interferon-mediated anti-viral response in VHL-null CCRCC, and that multiple established CCRCC cell lines, as well as early-passage primary CCRCC cultured cells, are acutely susceptible to EMCV replication and virulence. Functional restoration of VHL or molecular suppression of HIF or NF-kappaB dramatically reverses CCRCC cellular susceptibility to EMCV-induced killing. Notably, intratumoural EMCV treatment of CCRCC in a murine xenograft model rapidly regresses tumour growth. These findings provide compelling pre-clinical evidence for the usage of EMCV in the treatment of CCRCC and potentially other tumours with elevated HIF/NF-kappaB-survival signature.
Assuntos
Carcinoma de Células Renais/terapia , Vírus da Encefalomiocardite/fisiologia , Neoplasias Renais/terapia , Vírus Oncolíticos/fisiologia , Animais , Apoptose , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Vírus da Encefalomiocardite/genética , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Camundongos , Camundongos SCID , NF-kappa B/metabolismo , Vírus Oncolíticos/genética , Interferência de RNA , RNA Interferente Pequeno , Transdução de Sinais , Transplante Heterólogo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
Hypoxia-inducible factor (HIF) is a central regulator of cellular responses to hypoxia, and under normal oxygen tension the catalytic alpha subunit of HIF is targeted for ubiquitin-mediated destruction via the VHL-containing E3 ubiquitin ligase complex. Principally known for its association with oncogenesis, HIF has been documented to have a role in the antibacterial response. Interferons, cytokines with antiviral functions, have been shown to upregulate the expression of HIF-1alpha, but the significance of HIF in the antiviral response has not been established. Here, using renal carcinoma cells devoid of VHL or reconstituted with functional wild-type VHL or VHL mutants with various abilities to negatively regulate HIF as an ideal model system of HIF activity, we show that elevated HIF activity confers dramatically enhanced resistance to vesicular stomatitis virus (VSV)-mediated cytotoxicity. Inhibition of HIF activity using a small-molecule inhibitor, chetomin, enhanced cellular sensitivity to VSV, while treatment with hypoxia mimetic CoCl2 promoted resistance. Similarly, targeting HIF-2alpha by RNA interference also enhanced susceptibility to VSV. Expression profiling studies show that upon VSV infection, the induction of genes with known antiviral activity, such as that encoding beta interferon (IFN-beta), is significantly enhanced by HIF. These results reveal a previously unrecognized role of HIF in the antiviral response by promoting the expression of the IFN-beta gene and other genes with antiviral activity upon viral infection.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Vírus da Estomatite Vesicular Indiana/imunologia , Vírus da Estomatite Vesicular Indiana/patogenicidade , Proteína Supressora de Tumor Von Hippel-Lindau/fisiologia , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular , DNA Complementar/genética , Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Interferons/genética , Camundongos , Mutação , Interferência de RNA , Proteína Supressora de Tumor Von Hippel-Lindau/genéticaRESUMO
Real-time quantitative PCR (qPCR) is a powerful tool for quantifying specific DNA target sequences. Although determination of relative quantity is widely accepted as a reliable means of measuring differences between samples, there are advantages to being able to determine the absolute copy numbers of a given target. One approach to absolute quantification relies on construction of an accurate standard curve using appropriate external standards of known concentration. We have validated the use of tissue genomic DNA as a universal external standard to facilitate quantification of any target sequence contained in the genome of a given species, addressing several key technical issues regarding its use. This approach was applied to validate mRNA expression of gene candidates identified from microarray data and to determine gene copies in transgenic mice. A simple method that can assist achieving absolute quantification of gene expression would broadly enhance the uses of real-time qPCR and in particular, augment the evaluation of global gene expression studies.
