BackgroundP16 DNA methylation is well known to be the most frequent event in cancer development. It has been reported that genetic inactivation of P16 drives cancer growth and metastasis, however, whether P16 DNA methylation is truly a driver in cancer metastasis remains unknown.ResultsA P16-specific DNA methyltransferase (P16-dnmt) expression vector is designed using a P16 promoter-specific engineered zinc finger protein fused with the catalytic domain of dnmt3a. P16-dnmt transfection significantly decreases P16 promoter activity, induces complete methylation of P16 CpG islands, and inactivates P16 transcription in the HEK293T cell line. The P16-Dnmt coding fragment is integrated into an expression controllable vector and used to induce P16-specific DNA methylation in GES-1 and BGC823 cell lines. Transwell assays show enhanced migration and invasion of these cancer cells following P16-specific DNA methylation. Such effects are not observed in the P16 mutant A549 cell line. These results are confirmed using an experimental mouse pneumonic metastasis model. Moreover, enforced overexpression of P16 in these cells reverses the migration phenotype. Increased levels of RB phosphorylation and NFκB subunit P65 expression are also seen following P16-specific methylation and might further contribute to cancer metastasis.ConclusionP16 methylation could directly inactivate gene transcription and drive cancer metastasis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0819-6) contains supplementary material, which is available to authorized users.
The impact of pollution on soil microbial communities and subsequent bioremediation can be measured quantitatively in situ using direct, non-culture-dependent techniques. Such techniques have advantages over culture-based methods, which often account for less than 1% of the extant microbial community. In 1988, a JP-4 fuel spill contaminated the glacio-fluvial aquifer at Wurtsmith Air Force Base, Michigan, USA. In this study, lipid biomarker characterization of the bacterial and eukaryotic communities was combined with polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of the eubacterial community to evaluate correlation between contaminant (JP-4 fuel) concentration and community structure shifts. Vadose, capillary fringe and saturated zone samples were taken from cores within and up- and down-gradient from the contaminant plume. Lipid biomarker analysis indicated that samples from within the plume contained increased biomass, with large proportions of typically gram-negative bacteria. Outside the plume, lipid profiles indicated low-biomass microbial communities compared with those within the initial spill site. 16S rDNA sequences derived from DGGE profiles from within the initial spill site suggested dominance of the eubacterial community by a limited number of phylogenetically diverse organisms. Used in tandem with pollutant quantification, these molecular techniques should facilitate significant improvements over current assessment procedures for the determination of remediation end-points.
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