Chemo-Enzymatic Fluorescence Labeling Of Genomic DNA For Simultaneous Detection Of Global 5-Methylcytosine And 5-Hydroxymethylcytosine.

5-Methylcytosine and 5-hydroxymethylcytosine are epigenetic modifications involved in gene regulation and cancer. We present a new, simple, and high-throughput platform for multi-color epigenetic analysis. The novelty of our approach is the ability to multiplex methylation and de-methylation signals in the same assay. We utilize an engineered methyltransferase enzyme that recognizes and labels all unmodified CpG sites with a fluorescent cofactor. In combination with the already established labeling of the de-methylation mark 5-hydroxymethylcytosine via enzymatic glycosylation, we obtained a robust platform for simultaneous epigenetic analysis of these marks. We assessed the global epigenetic levels in multiple samples of colorectal cancer and observed a 3.5-fold reduction in 5hmC levels but no change in DNA methylation levels between sick and healthy individuals. We also measured epigenetic modifications in chronic lymphocytic leukemia and observed a decrease in both modification levels (5-hydroxymethylcytosine: whole blood 30 %; peripheral blood mononuclear cells (PBMCs) 40 %. 5-methylcytosine: whole blood 53 %; PBMCs 48 %). Our findings propose using a simple blood test as a viable method for analysis, simplifying sample handling in diagnostics. Importantly, our results highlight the assay's potential for epigenetic evaluation of clinical samples, benefiting research and patient management.

5-Hydroxymethylcytosine as a clinical biomarker: Fluorescence-based assay for high-throughput epigenetic quantification in human tissues.

Epigenetic transformations may provide early indicators for cancer and other disease. Specifically, the amount of genomic 5-hydroxymethylcytosine (5-hmC) was shown to be globally reduced in a wide range of cancers. The integration of this global biomarker into diagnostic workflows is hampered by the limitations of current 5-hmC quantification methods. Here we present and validate a fluorescence-based platform for high-throughput and cost-effective quantification of global genomic 5-hmC levels. We utilized the assay to characterize cancerous tissues based on their 5-hmC content, and observed a pronounced reduction in 5-hmC level in various cancer types. We present data for glioblastoma, colorectal cancer, multiple myeloma, chronic lymphocytic leukemia and pancreatic cancer, compared to corresponding controls. Potentially, the technique could also be used to follow response to treatment for personalized treatment selection. We present initial proof-of-concept data for treatment of familial adenomatous polyposis.

Rapid Quantification of Oxidation and UV Induced DNA Damage by Repair Assisted Damage Detection-(Rapid RADD).

Knowing the amount and type of DNA damage is of great significance for a broad range of clinical and research applications. However, existing methods are either lacking in their ability to distinguish between types of DNA damage or limited in their sensitivity and reproducibility. The method described herein enables rapid and robust quantification of type-specific single-strand DNA damage. The method is based on repair-assisted damage detection (RADD) by which fluorescent nucleotides are incorporated into DNA damage sites using type-specific repair enzymes. Up to 90 DNA samples are then deposited on a multiwell glass slide, and analyzed by a conventional slide scanner for quantification of DNA damage levels. Accurate and sensitive measurements of oxidative or UV-induced DNA damage levels and repair kinetics are presented for both in vitro and in vivo models.

Microfluidic DNA combing for parallel single-molecule analysis.

DNA combing is a widely used method for stretching and immobilising DNA molecules on a surface. Fluorescent labelling of genomic information enables high-resolution optical analysis of DNA at the single-molecule level. Despite its simplicity, the application of DNA combing in diagnostic workflows is still limited, mainly due to difficulties in analysing multiple small-volume DNA samples in parallel. Here, we report a simple and versatile microfluidic DNA combing technology (µDC), which allows manipulating, stretching and imaging of multiple, microliter scale DNA samples by employing a manifold of parallel microfluidic channels. Using DNA molecules with repetitive units as molecular rulers, we demonstrate that the µDC technology allows uniform stretching of DNA molecules. The stretching ratio remains consistent along individual molecules as well as between different molecules in the various channels, allowing simultaneous quantitative analysis of different samples loaded into parallel channels. Furthermore, we demonstrate the application of µDC to characterise UVB-induced DNA damage levels in human embryonic kidney cells and the spatial correlation between DNA damage sites. Our results point out the potential application of µDC for quantitative and comparative single-molecule studies of genomic features. The extremely simple design of µDC makes it suitable for integration into other microfluidic platforms to facilitate high-throughput DNA analysis in biological research and medical point-of-care applications.

The PIM-2 kinase is an essential component of the ultraviolet damage response that acts upstream to E2F-1 and ATM.

The oncogenic nature ascribed to the PIM-2 kinase relies mostly on phosphorylation of substrates that act as pro-survival/anti-apoptotic factors. Nevertheless, pro-survival effects can also result from activating DNA repair mechanisms following damage. In this study, we addressed the possibility that PIM-2 plays a role in the cellular response to UV damage, an issue that has never been addressed before. We found that in U2OS cells, PIM-2 expression and activity increased upon exposure to UVC radiation (2-50 mJ/cm(2)), and Pim-2-silenced cells were significantly more sensitive to UV radiation. Overexpression of PIM-2 accelerated removal of UV-induced DNA lesions over time, reduced γH2AX accumulation in damaged cells, and rendered these cells significantly more viable following UV radiation. The protective effect of PIM-2 was mediated by increased E2F-1 and activated ATM levels. Silencing E2F-1 reduced the protective effect of PIM-2, whereas inhibiting ATM activity abrogated this protective effect, irrespective of E2F-1 levels. The results obtained in this study place PIM-2 upstream to E2F-1 and ATM in the UV-induced DNA damage response.

Lighting up individual DNA damage sites by in vitro repair synthesis.

DNA damage and repair are linked to fundamental biological processes such as metabolism, disease, and aging. Single-strand lesions are the most abundant form of DNA damage; however, methods for characterizing these damage lesions are lacking. To avoid double-strand breaks and genomic instability, DNA damage is constantly repaired by efficient enzymatic machinery. We take advantage of this natural process and harness the repair capacity of a bacterial enzymatic cocktail to repair damaged DNA in vitro and incorporate fluorescent nucleotides into damage sites as part of the repair process. We use single-molecule imaging to detect individual damage sites in genomic DNA samples. When the labeled DNA is extended on a microscope slide, damage sites are visualized as fluorescent spots along the DNA contour, and the extent of damage is easily quantified. We demonstrate the ability to quantitatively follow the damage dose response to different damaging agents as well as repair dynamics in response to UV irradiation in several cell types. Finally, we show the modularity of this single-molecule approach by labeling DNA damage in conjunction with 5-hydroxymethylcytosine in genomic DNA extracted from mouse brain tissue.

Single-molecule quantification of 5-hydroxymethylcytosine for diagnosis of blood and colon cancers.

BACKGROUND: The DNA modification 5-hydroxymethylcytosine (5hmC) is now referred to as the sixth base of DNA with evidence of tissue-specific patterns and correlation with gene regulation and expression. This epigenetic mark was recently reported as a potential biomarker for multiple types of cancer, but its application in the clinic is limited by the utility of recent 5hmC quantification assays. We use a recently developed, ultra-sensitive, fluorescence-based single-molecule method for global quantification of 5hmC in genomic DNA. The high sensitivity of the method gives access to precise quantification of extremely low 5hmC levels common in many cancers. METHODS: We assessed 5hmC levels in DNA extracted from a set of colon and blood cancer samples and compared 5hmC levels with healthy controls, in a single-molecule approach. RESULTS: Using our method, we observed a significantly reduced level of 5hmC in blood and colon cancers and could distinguish between colon tumor and colon tissue adjacent to the tumor based on the global levels of this molecular biomarker. CONCLUSIONS: Single-molecule detection of 5hmC allows distinguishing between malignant and healthy tissue in clinically relevant and accessible tissue such as blood and colon. The presented method outperforms current commercially available quantification kits and may potentially be developed into a widely used, 5hmC quantification assay for research and clinical diagnostics. Furthermore, using this method, we confirm that 5hmC is a good molecular biomarker for diagnosing colon and various types of blood cancer.

Activation of cell cycle arrest and apoptosis by the proto-oncogene Pim-2.

Potent survival effects have been ascribed to the serine/threonine kinase proto-oncogene PIM-2. Elevated levels of PIM-2 are associated with various malignancies. In human cells, a single Pim-2 transcript gives rise mainly to two protein isoforms (34, 41 kDa) that share an identical catalytic site but differ at their N-terminus, due to in-frame alternative translation initiation sites. In this study we observed that the 34 kDa PIM-2 isoform has differential nuclear and cytoplasmic forms in all tested cell lines, suggesting a possible role for the balance between these forms for PIM-2's function. To further study the cellular role of the 34 kDa isoform of PIM-2, an N-terminally HA-tagged form of this isoform was transiently expressed in HeLa cells. Surprisingly, this resulted in increased level of G1 arrested cells, as well as of apoptotic cells. These effects could not be obtained by a Flag-tagged form of the 41 kDa isoform. The G1 arrest and apoptotic effects were associated with an increase in T14/Y15 phosphorylation of CDK2 and proteasom-dependent down-regulation of CDC25A, as well as with up-regulation of p57, E2F-1, and p73. No such effects were obtained upon over-expression of a kinase-dead form of the HA-tagged 34 kDa PIM-2. By either using a dominant negative form of p73, or by over-expressing the 34 kDa PIM-2 in p73-silenced cells, we demonstrated that these effects were p73-dependent. These results demonstrate that while PIM-2 can function as a potent survival factor, it can, under certain circumstances, exhibit pro-apoptotic effects as well.