RESUMO
The growth of a tumor depends to a certain extent on an increase in mitotic events. Key steps during mitosis are the regulated assembly of the spindle apparatus and the separation of the sister chromatids. The microtubule-associated protein Aurora kinase A phosphorylates DLGAP5 in order to correctly segregate the chromatids. Its activity and recruitment to the spindle apparatus is regulated by TPX2. KIF11 and CKAP5 control the correct arrangement of the microtubules and prevent their degradation. In the present study, we investigated the role of these five molecules in non-small cell lung cancer (NSCLC). We analyzed the expression of the five genes in a large cohort of NSCLC patients (n=362) by quantitative real-time PCR. Each of the genes was highly overexpressed in the tumor tissues compared to corresponding normal lung tissue. The correlation of the expression of the individual genes depended on the histology. An increased expression of AURKA, DLGAP5, TPX2, KIF11 and CKAP5 was associated with poor overall survival (P=0.001-0.065). AURKA was a significant prognostic marker using multivariate analyses (P=0.006). Immunofluorescence studies demonstrated that the five mitosis-associated proteins co-localized with the spindle apparatus during cell division. Taken together, our data demonstrate that the expression of the mitosis-associated genes AURKA, DLGAP5, TPX2, KIF11 and CKAP5 is associated with the prognosis of NSCLC patients.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Aurora Quinase A/biossíntese , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Proteínas de Ciclo Celular/biossíntese , Feminino , Imunofluorescência , Humanos , Estimativa de Kaplan-Meier , Cinesinas/biossíntese , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Masculino , Proteínas Associadas aos Microtúbulos/biossíntese , Pessoa de Meia-Idade , Mitose/genética , Proteínas de Neoplasias/biossíntese , Proteínas Nucleares/biossíntese , Prognóstico , Modelos de Riscos Proporcionais , Reação em Cadeia da Polimerase em Tempo Real , TranscriptomaRESUMO
Regulation of iron homeostasis and the inflammatory response are tightly linked to protect the host from infection. Here we investigate how imbalanced systemic iron homeostasis in a murine disease model of hereditary hemochromatosis (Hfe(-/-) mice) affects the inflammatory responses of the lung. We induced acute pulmonary inflammation in Hfe(-/-) and wild-type mice by intratracheal instillation of 20 µg of lipopolysaccharide (LPS) and analyzed local and systemic inflammatory responses and iron-related parameters. We show that in Hfe(-/-) mice neutrophil recruitment to the bronchoalveolar space is attenuated compared to wild-type mice although circulating neutrophil numbers in the bloodstream were elevated to similar levels in Hfe(-/-) and wild-type mice. The underlying molecular mechanisms are likely multifactorial and include elevated systemic iron levels, alveolar macrophage iron deficiency and/or hitherto unexplored functions of Hfe in resident pulmonary cell types. As a consequence, pulmonary cytokine expression is out of balance and neutrophils fail to be recruited efficiently to the bronchoalveolar compartment, a process required to protect the host from infections. In conclusion, our findings suggest a novel role for Hfe and/or imbalanced iron homeostasis in the regulation of the inflammatory response in the lung and hereditary hemochromatosis.
Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Infiltração de Neutrófilos/fisiologia , Pneumonia/metabolismo , Animais , Hemocromatose/genética , Hemocromatose/imunologia , Hemocromatose/metabolismo , Proteína da Hemocromatose , Antígenos de Histocompatibilidade Classe I/genética , Lipopolissacarídeos/toxicidade , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Infiltração de Neutrófilos/genética , Pneumonia/induzido quimicamente , Pneumonia/genéticaRESUMO
Biological experiments are most often performed with immortalized cell lines because they are readily available and can be expanded without limitation. However, cell lines may differ from the in vivo situation in important aspects. Here we introduce a straightforward methodology to compare cell lines to their cognate primary cells and to derive a comparative functional phenotype. We used SILAC (stable isotope labeling by amino acids in cell culture) for quantitative, mass spectrometry-based comparison of the hepatoma cell line Hepa1-6 with primary hepatocytes. The resulting quantitative proteome of 4,063 proteins had an asymmetric distribution, with many proteins down-regulated in the cell line. Bioinformatic analysis of the quantitative proteomics phenotypes revealed that Hepa1-6 cells were deficient in mitochondria, reflecting re-arrangement of metabolic pathways, drastically up-regulate cell cycle-associated functions and largely shut down drug metabolizing enzymes characteristic for the liver. This quantitative knowledge of changes provides an important basis to adapt cell lines to more closely resemble physiological conditions.