ABSTRACT
Alveolar epithelia play an essential role in maintaining the integrity and homeostasis of lungs, in which alveolar epithelial type II cells (AECII) are a cell type with stem cell potential for epithelial injury repair and regeneration. However, mechanisms behind the physiological and pathological roles of alveolar epithelia in human lungs remain largely unknown, partially owing to the difficulty of isolation and culture of primary human AECII cells. In the present study, we aimed to characterize alveolar epithelia generated from A549 lung adenocarcinoma cells that were cultured in an air-liquid interface (ALI) state. Morphological analysis demonstrated that A549 cells could reconstitute epithelial layers in ALI cultures as evaluated by histochemistry staining and electronic microscopy. Immunofluorescent staining further revealed an expression of alveolar epithelial type I cell (AECI) markers aquaporin-5 protein (AQP-5), and AECII cell marker surfactant protein C (SPC) in subpopulations of ALI cultured cells. Importantly, molecular analysis further revealed the expression of AQP-5, SPC, thyroid transcription factor-1, zonula occludens-1 and Mucin 5B in A549 ALI cultures as determined by both immunoblotting and quantitative RT-PCR assay. These results suggest that the ALI culture of A549 cells can partially mimic the property of alveolar epithelia, which may be a feasible and alternative model for investigating roles and mechanisms of alveolar epithelia in vitro.
Subject(s)
Humans , Culture Media, Conditioned , Cell Culture Techniques/methods , Alveolar Epithelial Cells/physiology , A549 Cells/physiology , Reference Values , Time Factors , Microscopy, Electron, Scanning , Immunoblotting , Cell Count , Reproducibility of Results , Analysis of Variance , Pulmonary Surfactant-Associated Protein C/analysis , Aquaporin 5/analysis , Mucin-5B/analysis , Real-Time Polymerase Chain Reaction , Zonula Occludens-1 Protein/analysis , Thyroid Nuclear Factor 1/analysisABSTRACT
CONTEXT: Survivin is an inhibitor of apoptosis that is selectively over-expressed in common human cancers, but not in normal tissues, and that correlates with aggressive disease and unfavorable outcomes. AIMS: To identify the role of survivin in bladder carcinogenesis and the correlation between survivin protein expression and the occurrence of spontaneous apoptosis, proliferative activity of cancer cells. SETTINGS AND DESIGN: Retrospective analysis. METHODS AND MATERIAL: Bladder transitional cell cancer (BTCC) tissue samples for 128 patients were investigated, with normal bladder tissues serving as controls. From these tumor samples, 42 (32.8%) were grade I, 59 (46.1%) were grade II, and 27 (21.1%) were grade III; 72 (56.2%) were superficial, 56 (43.8%) were invasive. The survivin protein level was quantified by Western blot analysis. The apoptotic index (AI) using in situ labeling apoptotic DNA fragment kit and the Ki-67 labeling index (Ki-67LI) with an anti-Ki-67 monoclonal antibody were analyzed in these tumors, respectively. STATISTICAL ANALYSIS USED: Differences in the S/beta ratio between tumor grade and stage were evaluated by using unpaired t-test and F-test. The relationships between the S/beta ratios and AIs, Ki-67LIs of tumors were evaluated by Pearson correlation coefficient. RESULTS: High survivin levels were detected by Western blot analysis in tumor tissue extracts. None of the expression of survivin protein was found in normal bladder tissues. Survivin levels were significantly different from different clinical stages and pathological grades of the tumors (P > 0.05, respectively). Spearman rank correlation test revealed a positively correlation between survivin protein level and the proliferative activity (P < 0.001) and failed to find significant correlation between AI and survivin protein level (P > 0.05). CONCLUSIONS: Survivin protein expression played an important role in the malignant progression of BTCC.