Accelerated repair and reduced mutagenicity of DNA damage induced by cigarette smoke in human bronchial cells transfected with E.coli formamidopyrimidine DNA glycosylase.

Cigarette smoke (CS) is associated to a number of pathologies including lung cancer. Its mutagenic and carcinogenic effects are partially linked to the presence of reactive oxygen species and polycyclic aromatic hydrocarbons (PAH) inducing DNA damage. The bacterial DNA repair enzyme formamidopyrimidine DNA glycosylase (FPG) repairs both oxidized bases and different types of bulky DNA adducts. We investigated in vitro whether FPG expression may enhance DNA repair of CS-damaged DNA and counteract the mutagenic effects of CS in human lung cells. NCI-H727 non small cell lung carcinoma cells were transfected with a plasmid vector expressing FPG fused to the Enhanced Green Fluorescent Protein (EGFP). Cells expressing the fusion protein EGFP-FPG displayed accelerated repair of adducts and DNA breaks induced by CS condensate. The mutant frequencies induced by low concentrations of CS condensate to the Na(+)K(+)-ATPase locus (oua(r)) were significantly reduced in cells expressing EGFP-FPG. Hence, expression of the bacterial DNA repair protein FPG stably protects human lung cells from the mutagenic effects of CS by improving cells' capacity to repair damaged DNA

Phenotype of FPG-expressing human bronchial carcinoid H727 cells.

<p>(A) Proliferative activity. Data are the means ± SEM of three independent experiments. CE was calculated by seeding 100–500 cells and counting developed colonies three weeks later. The population doubling time (PDT) was calculated according to Glaab and Tindall <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087984#pone.0087984-Glaab1" target="_blank">[16]</a>. (B) CSC toxicity was evaluated by the In Vitro Toxicology Assay Kit MTT-based. Data are the means ± SEM of at least three independent experiments. (C) CSC mutagenicity was determined at the <i>Na⁺/K⁺ ATPase</i> locus (oua^r) as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087984#s2" target="_blank">Materials and Methods</a>. Data are the means ± SEM of at least three independent experiments. (D) Cell cycle distribution was determined by cytofluorimetry. No significant variation of cell cycle distribution was induced by CSC treatments at either 62.5 or 125 µg/ml doses.</p

FPG expression in human bronchial carcinoid H727 cells and transfected derivatives.

<p>(A) Fluorescence analysis. Fluorescence of cells was observed under an inverted fluorescence microscope at 100X magnification and images were taken with a digital color camera. H727, untransfected cells; H1, cells transfected with the pEGFP-C1 vector; HF12, HF45, cells transfected with the pEGFP-C1-FPG vector. (B) HF12 cells analyzed by confocal microscopy at 600X magnification showing distribution of EGFP-FPG protein in both the cytoplasm and nucleus. (C) and (D) Immunodetection analyses. Experiments were performed as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087984#s2" target="_blank">Materials and Methods</a>. Molecular weight markers were in the far-right lane (M) and β-actin was immunodetected as a loading control. (C) Immunodetection of EGFP. Recombinant pure EGFP protein (rEGFP) was run as an internal standard. (D) Immunodetection of FPG. Recombinant pure FPG protein (rFPG) was run as an internal standard.</p

Conventional and nuclear medicine imaging in Ectopic Cushing's syndrome: A systematic review

Context: Ectopic Cushing's Syndrome (ECS) can be a diagnostic challenge with the hormonal source difficult to find. This study analyzes the accuracy of imaging studies in ECS localization. EvidenceAcquisition:SystematicreviewofmedicalliteratureforECScaseseriesprovidingindividualpatient data on at least one conventional imaging technique (computed tomography [CT]/magnetic resonance imaging) and one of the following: 111In-pentetreotide (OCT), 131I/123I-metaiodobenzylguanidine, 18Ffluoro-2-deoxyglucose-positron emission tomography (FDG-PET), 18F-fluorodopa-PET (F-DOPA-PET), 68Ga-DOTATATE-PET/CT or 68Ga-DOTATOC-PET/CT scan (68Gallium-SSTR-PET/CT). Evidence Summary: The analysis comprised 231 patients (females, 50.2%; age, 42.6±17 y). Overall, 52.4%(121/231)had"overt" ECS,18.6%had"occult" ECS,and29%had"covert" ECS. Tumors were located in the lung (55.3%), mediastinum-thymus (7.9%), pancreas (8.5%), adrenal glands (6.4%), gastrointestinal tract (5.4%), thyroid (3.7%), and other sites (12.8%), and primary tumors were mostly bronchial neuroendocrine tumors (NETs) (54.8%), pancreatic NETs (8%), mediastinum-thymusNETs (6.9%), gastrointestinal NETs (5.3%), pheochromocytoma (6.4%), neuroblastoma (3.2%), andmedullary thyroid carcinoma (3.2%).Tumorswerelocalized byCTin66.2%(137/207), magnetic resonance imaging in 51.5% (53/103), OCT in 48.9% (84/172), FDG-PET in 51.7% (46/89), F-DOPAPET in 57.1% (12/21), 131/123II-metaiodobenzylguanidine in 30.8% (4/13), and 68Gallium-SSTRPET/ CT in 81.8% (18/22) of cases. Molecular imaging discovered 79.1% (53/67) of tumors unidentified by conventional radiology, with OCT the most commonly used, revealing the tumor in 64%, followed by FDG-PET in 59.4%. F-DOPA-PET was used in only seven covert cases (sensitivity, 85.7%). Notably, 68Gallium-SSTR-PET/CT had 100% sensitivity among covert cases. Conclusions: Nuclear medicine improves the sensitivity of conventional radiology when tumor site identification is problematic. OCT offers a good availability/reliability ratio, and FDG-PET was proven useful. 68Gallium-SSTR-PET/CT use was infrequent, despite offering the highest sensitivity