Dataset Information

Combining Optical Reporter Proteins with Different Half-lives to Detect Temporal Evolution of Hypoxia and Reoxygenation in Tumors.

ABSTRACT: Here we have developed a hypoxia response element driven imaging strategy that combined the hypoxia-driven expression of two optical reporters with different half-lives to detect temporal changes in hypoxia and hypoxia inducible factor (HIF) activity. For this purpose, human prostate cancer PC3 cells were transfected with the luciferase gene fused with an oxygen-dependent degradation domain (ODD-luc) and a variant of the enhanced green fluorescent protein (EGFP). Both ODD-luciferase and EGFP were under the promotion of a poly-hypoxia-response element sequence (5xHRE). The cells constitutively expressed tdTomato red fluorescent protein. For validating the imaging strategy, cells were incubated under hypoxia (1% O2) for 48 hours and then reoxygenated. The luciferase activity of PC3-HRE-EGFP/HRE-ODD-luc/tdtomato cells detected by bioluminescent imaging rapidly decreased after reoxygenation, whereas EGFP levels in these cells remained stable for several hours. After in vitro validation, PC3-HRE-EGFP/HRE-ODD-luc/tdtomato tumors were implanted subcutaneously and orthotopically in nude male mice and imaged in vivo and ex vivo using optical imaging in proof-of-principle studies to demonstrate differences in optical patterns between EGFP expression and bioluminescence. This novel "timer" imaging strategy of combining the short-lived ODD-luciferase and the long-lived EGFP can provide a time frame of HRE activation in PC3 prostate cancer cells and will be useful to understand the temporal changes in hypoxia and HIF activity during cancer progression and following treatments including HIF targeting strategies.

SUBMITTER: Danhier P

PROVIDER: S-EPMC4688563 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Combining Optical Reporter Proteins with Different Half-lives to Detect Temporal Evolution of Hypoxia and Reoxygenation in Tumors.

Danhier Pierre P Krishnamachary Balaji B Bharti Santosh S Kakkad Samata S Mironchik Yelena Y Bhujwalla Zaver M ZM

Neoplasia (New York, N.Y.) 20151201 12

Here we have developed a hypoxia response element driven imaging strategy that combined the hypoxia-driven expression of two optical reporters with different half-lives to detect temporal changes in hypoxia and hypoxia inducible factor (HIF) activity. For this purpose, human prostate cancer PC3 cells were transfected with the luciferase gene fused with an oxygen-dependent degradation domain (ODD-luc) and a variant of the enhanced green fluorescent protein (EGFP). Both ODD-luciferase and EGFP wer ...[more]

PMID: 26696369

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Project description:BackgroundPolycyclic aromatic hydrocarbons (PAHs) accumulate in urban soils, and PAH contamination can change soil microbial community composition. Environmental microbiota is associated with human commensal microbiota, immune system and health. Therefore, studies investigating the degradation of PAHs, and the consequences of soil pollution on microbial communities in urban landscaping materials, are crucial.MethodsFour landscaping materials (organic matter 1, 2, 13 and 56%) were contaminated with PAHs commonly found at urban sites (phenanthrene, fluoranthene, pyrene, chrysene and benzo(b)fluoranthene) in PAH concentrations that reflect urban soils in Finland (2.4 µg g -1 soil dry weight). PAHs were analyzed initially and after 2, 4, 8 and 12 weeks by gas chromatography-mass spectrometry. Half-lives of PAHs were determined based on 12-weeks degradation. Bacterial communities were analyzed at 1 and 12 weeks after contamination using Illumina MiSeq 16S rRNA gene metabarcoding.ResultsHalf-lives ranged from 1.5 to 4.4 weeks for PAHs with relatively low molecular weights (phenanthrene, fluoranthene and pyrene) in landscaping materials containing 1-2% organic matter. In contrast, in materials containing 13% and 56% organic matter, the half-lives ranged from 2.5 to 52 weeks. Shorter half-lives of phenanthrene and fluoranthene were thus associated with low organic matter content. The half-life of pyrene was inversely related to the relative abundance of Beta-, Delta- and Gammaproteobacteria, and diversity of Bacteroidetes and Betaprotebacteria. Compounds with higher molecular weights followed compound-specific patterns. Benzo(b)fluoranthene was resistant to degradation and half-life of chrysene was shorter when the relative abundance of Betaproteobacteria was high. Temporal microbiota changes involved increase in the relative abundance of Deltaproteobacteria and decrease in genera Flavobacterium and Rhodanobacter. Exposure to PAHs seems to adjust microbial community composition, particularly within class Beta- and Deltaproteobacteria.ConclusionsIn this study, PAH degradation depended on the organic matter content and bacterial community composition of landscaping materials. Contamination seems to alter bacterial community composition in landscaping materials depending on material type. This alteration includes changes in bacterial phyla associated with human health and immune system. This may open new possibilities for managing urban environments by careful selection of landscaping materials, to benefit health and wellbeing.

Dataset Information

Combining Optical Reporter Proteins with Different Half-lives to Detect Temporal Evolution of Hypoxia and Reoxygenation in Tumors.

Publications

Combining Optical Reporter Proteins with Different Half-lives to Detect Temporal Evolution of Hypoxia and Reoxygenation in Tumors.

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