RESUMO
UNLABELLED: Infections with high-risk human papillomaviruses (hrHPV) contribute to cervical carcinoma. The cdk inhibitor and tumor suppressor p16INK4A is consistently upregulated in cervical carcinoma cells for reasons that are poorly understood. We report here that downregulation of p16INK4A gene expression in three different cervical carcinoma cell lines reduced expression of the E7 oncogene, suggesting a positive feedback loop involving E7 and p16INK4A. p16INK4A depletion induced cellular senescence in HeLa but not CaSki and MS-751 cervical carcinoma cells. IMPORTANCE: This study demonstrates that the cdk inhibitor p16INK4A, frequently used as surrogate marker for transforming infections by human papillomaviruses of the high-risk group, is required for high-level expression of the E7 oncoproteins of HPV-16, HPV-18, and HPV-45 in cervical carcinoma cells. It is also demonstrated that depletion of p16INK4A induces senescence in HeLa but not CaSki or MS-751 cervical carcinoma cells.
Assuntos
Proliferação de Células , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Células Epiteliais/fisiologia , Proteínas E7 de Papillomavirus/biossíntese , Linhagem Celular Tumoral , Feminino , Técnicas de Silenciamento de Genes , HumanosRESUMO
ROS (reactive oxygen species) generated by NADPH oxidases play an important role in cellular signal transduction regulating cell proliferation, survival and differentiation. Nox4 (NADPH oxidase 4) induces cellular senescence in human endothelial cells; however, intracellular targets for Nox4 remained elusive. In the present study, we show that Nox4 induces mitochondrial dysfunction in human endothelial cells. Nox4 depletion induced alterations in mitochondrial morphology, stabilized mitochondrial membrane potential and decreased production of H(2)O(2) in mitochondria. High-resolution respirometry in permeabilized cells combined with native PAGE demonstrated that Nox4 specifically inhibits the activity of mitochondrial electron transport chain complex I, and this was associated with a decreased concentration of complex I subunits. These data suggest a new pathway by which sustained Nox4 activity decreases mitochondrial function.
Assuntos
Complexo I de Transporte de Elétrons/antagonistas & inibidores , NADPH Oxidases/química , NADPH Oxidases/fisiologia , Complexo I de Transporte de Elétrons/química , Células Endoteliais/metabolismo , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana , Humanos , Potencial da Membrana Mitocondrial , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , NADPH Oxidase 4 , NADPH Oxidases/deficiência , Fosforilação Oxidativa , Consumo de Oxigênio , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/química , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/química , Transdução de Sinais/fisiologiaRESUMO
Persistent infections by high-risk human papillomaviruses (HPVs) are the main etiological factor for cervical cancer, and expression of HPV E7 oncoproteins was suggested to be a potential marker for tumor progression. The objective of this study was to generate new reagents for the detection of the HPV18 E7 oncoprotein in cervical smears. Rabbit monoclonal antibodies against recombinant E7 protein of HPV type 18 (HPV18) were generated and characterized using Western blotting, epitope mapping, indirect immunofluorescence, and immunohistochemistry. One clone specifically recognizing HPV18 E7 was used for the development of a sandwich enzyme-linked immunosorbent assay (ELISA). The assay was validated using recombinant E7 proteins of various HPV types and lysates from E7-positive cervical carcinoma cells. A total of 14 HPV18 DNA-positive cervical swab specimens and 24 HPV DNA-negative-control specimens were used for the determination of E7 protein levels by the newly established sandwich ELISA. On the basis of the average absorbance values obtained from all 24 negative controls, a cutoff above which a clinical sample can be judged E7 positive was established. Significant E7 signals 6- to 30-fold over background were found in 7 out of 14 abnormal HPV18 DNA-positive cervical smear specimens. This feasibility study demonstrates for the first time that HPV18 E7 oncoprotein can be detected in cervical smears.
Assuntos
Detecção Precoce de Câncer/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Papillomavirus Humano 18/isolamento & purificação , Proteínas E7 de Papillomavirus/análise , Infecções por Papillomavirus/diagnóstico , Vagina/virologia , Virologia/métodos , Anticorpos Monoclonais , Estudos de Viabilidade , Feminino , Papillomavirus Humano 18/imunologia , Humanos , Proteínas E7 de Papillomavirus/imunologia , Esfregaço VaginalRESUMO
The free radical theory of aging proposes that ROS (reactive oxygen species) are major driving forces of aging, and are also critically involved in cellular senescence. Besides the mitochondrial respiratory chain, alternative sources of ROS have been described that might contribute to cellular senescence. Noxs (NADPH oxidases) are well-known sources of superoxide, which contribute to the antimicrobial capabilities of macrophages, a process involving the prototypical member of the family referred to as Nox2. However, in recent years non-phagocytic homologues of Nox2 have been identified that are involved in processes other than the host defence. Superoxide anions produced by these enzymes are believed to play a major role in signalling by MAPKs (mitogen-activated protein kinases) and stress-activated kinases, but could also contribute to cellular senescence, which is known to involve oxygen radicals. In HUVECs (human umbilical vein endothelial cells), Nox4 is predominantly expressed, but its role in replicative senescence of HUVECs remains to be elucidated. Using shRNA (small-hairpin RNA)-mediated knockdown of Nox4, implicating lentiviral vectors, we addressed the question of whether lifelong depletion of Nox4 in HUVECs would influence the senescent phenotype. We found a significant extension of the replicative lifespan of HUVECs upon knockdown of Nox4. Surprisingly, mean telomere length was significantly reduced in Nox4-depleted cells. Nox4 depletion had no discernable influence on the activity of MAPKs and stress-activated kinases, but reduced the degree of oxidative DNA damage. These results suggest that Nox4 activity increases oxidative damage in HUVECs, leading to loss of replicative potential, which is at least partly independent of telomere attrition.
Assuntos
Senescência Celular , Dano ao DNA , Células Endoteliais/enzimologia , Sistema de Sinalização das MAP Quinases , NADPH Oxidases/metabolismo , Telômero/metabolismo , Células Cultivadas , Técnicas de Silenciamento de Genes , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , NADPH Oxidase 2 , NADPH Oxidase 4 , NADPH Oxidases/genética , Oxirredução , Superóxidos/metabolismo , Telômero/genéticaRESUMO
According to the free radical theory of aging, reactive oxygen species (ROS) act as a driving force of the aging process, and it is generally believed that mitochondrial dysfunction is a major source of increased oxidative stress in tissues with high content of mitochondria, such as muscle or brain. However, recent experiments in mouse models of premature aging have questioned the role of mitochondrial ROS production in premature aging. To address the role of mitochondrial impairment and ROS production for aging in human muscles, we have analyzed mitochondrial properties in muscle fibres isolated from the vastus lateralis of young and elderly donors. Mitochondrial respiratory functions were addressed by high-resolution respirometry, and ROS production was analyzed by in situ staining with the redox-sensitive dye dihydroethidium. We found that aged human skeletal muscles contain fully functional mitochondria and that the level of ROS production is higher in young compared to aged muscle. Accordingly, we could not find any increase in oxidative modification of proteins in muscle from elderly donors. However, the accumulation of lipofuscin was identified as a robust marker of human muscle aging. The data support a model, where ROS-induced molecular damage is continuously removed, preventing the accumulation of dysfunctional mitochondria despite ongoing ROS production.
Assuntos
Envelhecimento/fisiologia , Lipofuscina/metabolismo , Mitocôndrias/fisiologia , Músculo Esquelético/metabolismo , Estresse Oxidativo/fisiologia , Adulto , Idoso , Biópsia , Respiração Celular , Feminino , Humanos , Masculino , Consumo de Oxigênio , Espécies Reativas de Oxigênio/metabolismoRESUMO
E7 is the major oncoprotein of high-risk human papillomaviruses (HPV) which causes cervical cancer. To date E7 oncoproteins have not been investigated in cervical adenocarcinoma. In this study we generated a rabbit monoclonal anti-HPV-16 E7 antibody, RabMab42-3, which recognizes a conformational epitope in the E7 carboxy-terminal zinc-finger resulting in a strong increase in the sensitivity for the detection of cell-associated HPV-16 E7 protein relative to conventional polyclonal anti-HPV-16 E7 antibodies. Using RabMab42-3, we show that the subcellular localization of endogenous HPV-16 E7 oncoprotein varies during the cell cycle in cervical cancer cells. Moreover, we demonstrate for the first time that the HPV-16 E7 oncoprotein is abundantly expressed in cervical adenocarcinoma in situ and adenocarcinoma, suggesting an important role of HPV-16 E7 for the development of these tumors. Our findings suggest that the HPV-16 E7 oncoprotein could be a useful marker for the detection of cervical adenocarcinoma and their precursors.
Assuntos
Adenocarcinoma/virologia , Papillomavirus Humano 16/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Frações Subcelulares/metabolismo , Neoplasias do Colo do Útero/virologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Linhagem Celular Tumoral , Feminino , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas E7 de Papillomavirus/química , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Coelhos , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologiaRESUMO
Cellular senescence is now recognized as an important mechanism of tumor suppression, and the accumulation of senescent cells may contribute to the aging of various human tissues. Alterations of the cellular energy metabolism are considered key events in tumorigenesis and are also known to play an important role for aging processes in lower eukaryotic model systems. In this study, we addressed senescence-associated changes in the energy metabolism of human endothelial cells, using the HUVEC model of in vitro senescence. We observed a drastic reduction in cellular ATP levels in senescent endothelial cells. Although consumption of glucose and production of lactate significantly increased in senescent cells, no correlation was found between both metabolite conversion rates, neither in young endothelial cells nor in the senescent cells, which indicates that glycolysis is not the main energy source in HUVEC. On the other hand, glutamine consumption was increased in senescent HUVEC and inhibition of glutaminolysis by DON, a specific inhibitor of glutaminase, led to a significant reduction in the proliferative capacity of both early passage and late passage cells. Moreover, inhibition of glutaminase activity induced a senescent-like phenotype in young HUVEC within two passages. Together, the data indicate that glutaminolysis is an important energy source in endothelial cells and that alterations in this pathway play a role in endothelial cell senescence.