Transcription profiling by array of human lung cancer cells after treatment with tobacco smoke condensate
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ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Hussain M
PROVIDER: S-ECPF-GEOD-13309 | biostudies-other | 2009 Apr
REPOSITORIES: biostudies-other
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Project description:Exposure to tobacco smoke is the number one risk factor for lung cancer. Although the DNA damaging properties of tobacco smoke have been well documented, relatively few studies have examined its effect on DNA repair pathways. This is especially true for the nucleotide excision repair (NER) pathway which recognizes and removes many structurally diverse DNA lesions, including those introduced by chemical carcinogens present in tobacco smoke. The aim of the present study was to investigate the effect of tobacco smoke on NER in human lung cells. We studied the effect of cigarette smoke condensate (CSC), a surrogate for tobacco smoke, on the NER pathway in two different human lung cell lines; IMR-90 lung fibroblasts and BEAS-2B bronchial epithelial cells. To measure NER, we employed a slot-blot assay to quantify the introduction and removal of UV light-induced 6-4 photoproducts and cyclobutane pyrimidine dimers. We find a dose-dependent inhibition of 6-4 photoproduct repair in both cell lines treated with CSC. Additionally, the impact of CSC on the abundance of various NER proteins and their respective RNAs was investigated. The abundance of XPC protein, which is required for functional NER, is significantly reduced by treatment with CSC while the abundance of XPA protein, also required for NER, is unaffected. Both XPC and XPA RNA levels are modestly reduced by CSC treatment. Finally, treatment of cells with MG-132 abrogates the reduction in the abundance of XPC protein produced by treatment with CSC, suggesting that CSC enhances proteasome-dependent turnover of the protein that is mediated by ubiquitination. Together, these findings indicate that tobacco smoke can inhibit the same DNA repair pathway that is also essential for the removal of some of the carcinogenic DNA damage introduced by smoke itself, increasing the DNA damage burden of cells exposed to tobacco smoke.
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Project description:BackgroundUnlike normal cells, cancer cells frequently have multiple centrosomes that can cluster to form bipolar mitotic spindles and allow for successful cell division. Inhibiting centrosome clustering, therefore, holds therapeutic promise to promote cancer cell-specific cell death.MethodsWe used confocal microscopy, real-time PCR, siRNA knockdown, and western blot to analyze centrosome clustering and declustering using normal lung bronchial epithelial and nonsmall-cell lung cancer (NSCLC) cell lines. Also, we used Ingenuity Pathway Analysis software to identify novel pathways associated with centrosome clustering.ResultsIn this study, we found that exposure to cigarette smoke condensate induces centrosome amplification and clustering in human lung epithelial cells. We observed a similar increase in centrosome amplification and clustering in unexposed NSCLC cell lines which may suggest a common underlying mechanism for lung carcinogenesis. We identified a cyclin D2-mediated centrosome clustering pathway that involves a sonic hedgehog-forkhead box protein M1 axis which is critical for mitosis. We also observed that cyclin D2 knockdown induced multipolar mitotic spindles that could eventually lead to cell death.ConclusionsHere we report a novel role of cyclin D2 in the regulation of centrosome clustering, which could allow the identification of tumors sensitive to cyclin D2 inhibitors. Our data reveal a pathway that can be targeted to inhibit centrosome clustering by interfering with the expression of cyclin D2-associated genes.
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Project description:Limited information is available about epigenetic mechanisms by which cigarette smoke enhances the initiation and progression of lung cancer. To examine this issue, A549 and Calu-6 lung cancer cells were cultured in normal media with or without tobacco smoke condensate (TSC) under clinically relevant exposure conditions. Ten-day TSC exposure dramatically increased the tumorigenicity of lung cancer cells in nude mice. Microarray and quantitative reverse transcription-PCR (RT-PCR) experiments revealed that this phenomenon coincided with diminished expression of Dickkopf-1 (Dkk-1). Western blot, chromatin immunoprecipitation, methylation-specific PCR, and pyrosequencing experiments showed that repression of Dkk-1 coincided with decreased H4K16Ac, increased H3K27me3, and recruitment of SirT1, EZH2, SUZ12, and Bmi1 without DNA hypermethylation within the Dkk-1 promoter despite prolonged TSC exposures. Removal of TSC from culture media resulted in loss of promoter-associated polycomb repressor complexes and reexpression of Dkk-1. siRNA-mediated knockdown of EZH2 and SirT1 partially abrogated TSC-mediated inhibition of Dkk-1 expression. Western blot and quantitative RT-PCR array experiments showed that TSC exposure as well as knockdown of Dkk-1 activated Wnt signaling and significantly up-regulated Wnt5a in lung cancer cells. Knockdown of Dkk-1 recapitulated the dramatic protumorigenic effects of TSC exposure in Calu-6 cells. Despite the transient nature of Dkk-1 repression following TSC exposure in vitro, Dkk-1 remained silenced in tumor xenografts derived from TSC-treated Calu-6 cells. Collectively, these data provide evidence that cigarette smoke directly engages polycomb machinery to activate a signaling network implicated in maintenance of cancer stem cells.
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Project description:The goal was to assess global gene expression changes in primary human bronchial epithelial cells exposed to environmental tobacco smoke (ETS) condensate. ETS-C was standardized by HPLC analysis and two timepoints of exposure in two different donor bronchial epithelial cell populations were assessed. These findings demonstrate that even short exposure (4.5 h) to ETS is sufficient to induce a stress response, as reflected by decreased antioxidant levels, induced HSP family members, and modulation of the family of glutathione metabolism enzymes in primary human lung cells. Upon longer exposures (48 h) with ETS-condensate, bronchial epithelial cells arrest at the G2/M phase of the cell cycle. Taken together, these data support a stress-induced state in primary human bronchial epithelial cells that culminates in cell cycle arrest. Keywords: time course, comparative, stress response
2006-12-25 | GSE4234 | GEO
Project description:PURPOSE The development of reliable gene expression profiling technology is having an increasing impact on our understanding of lung cancer biology. The present study aims to determine whether the phenotypic heterogeneity and genetic diversity of lung cancer are correlated. PATIENTS AND METHODS In this study, microarray analysis was performed in a set of 91 non-small cell lung cancer (NSCLC) samples in order to: establish gene signatures in primary adenocarcinomas and squamous-cell carcinomas; determine differentially expressed gene sequences at different stages of the disease; and identify sequences with biological significance for tumor progression. After microarray analysis, the expression level of 92 selected genes was validated by qPCR in an independent set of 70 samples. RESULTS Gene sequences were differentially expressed as a function of tumor type, stage, and differentiation grade. High upregulation was observed for KRT15 and PKP1, which may be good markers to distinguish squamous cell carcinoma samples. High downregulation was observed for DSG3 in stage IA adenocarcinomas. CONCLUSION Expression signatures in NSCLC distinguish tumor type, stage, and differentiation grade. Keywords: Tumor vs control comparative genomics study 91 samples studied, 46 tumors and 45 controls. All samples are paired except three.
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