Malignant brain tumors are among the most lethal of human tumors, with limited treatment options currently available. A complex array of recurrent genetic and epigenetic changes has been observed in gliomas that collectively result in derangements of common cell signaling pathways controlling cell survival, proliferation, and invasion. One important determinant of gene expression is DNA methylation status, and emerging studies have revealed the importance of a recently identified demethylation pathway involving 5-hydroxymethylcytosine (5hmC). Diminished levels of the modified base 5hmC is a uniform finding in glioma cell lines and patient samples, suggesting a common defect in epigenetic reprogramming. Within the tumor microenvironment, infiltrating immune cells increase oxidative DNA damage, likely promoting both genetic and epigenetic changes that occur during glioma evolution. In this environment, glioma cells are selected that utilize multiple metabolic changes, including changes in the metabolism of the amino acids glutamate, tryptophan, and arginine. Whereas altered metabolism can promote the destruction of normal tissues, glioma cells exploit these changes to promote tumor cell survival and to suppress adaptive immune responses. Further understanding of these metabolic changes could reveal new strategies that would selectively disadvantage tumor cells and redirect host antitumor responses toward eradication of these lethal tumors.

hSMUG1 (human single-stranded selective monofunctional uracil-DNA glyscosylase) is one of three glycosylases encoded within a small region of human chromosome 12. Those three glycosylases, UNG (uracil-DNA glycosylase), TDG (thymine-DNA glyscosylase), and hSMUG1, have in common the capacity to remove uracil from DNA. However, these glycosylases also repair other lesions and have distinct substrate preferences, indicating that they have potentially redundant but not overlapping physiological roles. The mechanisms by which these glycosylases locate and selectively remove target lesions are not well understood. In addition to uracil, hSMUG1 has been shown to remove some oxidized pyrimidines, suggesting a role in the repair of DNA oxidation damage. In this paper, we describe experiments in which a series of oligonucleotides containing purine and pyrimidine analogs have been used to probe mechanisms by which hSMUG1 distinguishes potential substrates. Our results indicate that the preference of hSMUG1 for mispaired uracil over uracil paired with adenine is best explained by the reduced stability of a duplex containing a mispair, consistent with previous reports with Escherichia coli mispaired uracil-DNA glycosylase. We have also extended the substrate range of hSMUG1 to include 5-carboxyuracil, the last in the series of damage products from thymine methyl group oxidation. The properties used by hSMUG1 to select damaged pyrimidines include the size and free energy of solvation of the 5-substituent but not electronic inductive properties. The observed distinct mechanisms of base selection demonstrated for members of the uracil glycosylase family help explain how considerable diversity in chemical lesion repair can be achieved.

Rapidly proliferating tumors are exposed to a hypoxic microenvironment because of their density, high metabolic consumption, and interruptions in blood flow because of immature angiogenesis. Cellular responses to hypoxia promote highly malignant and metastatic behavior, as well as a chemotherapy-resistant state. To better understand the complex relationships between hypoxic adaptations and cancer progression, we studied the dynamic proteome responses of glioblastoma cells exposed to hypoxia via an innovative approach: quantification of newly synthesized proteins using heavy stable-isotope arginine labeling combined with accurate assessment of cell replication by quantification of the light/heavy arginine ratio of peptides in histone H4. We found that hypoxia affects cancer cells in multiple intertwined ways: inflammation, typically with over-expressed glucose transporter (GLUT1), DUSP4/MKP2, and RelA proteins; a metabolic adaptation with overexpression of all glycolytic pathway enzymes for pyruvate/lactate synthesis; and the EMT (epithelial-mesenchymal transition) and cancer stem cell (CSC) renewal with characteristic morphological changes and mesenchymal/CSC protein expression profiles. For the first time, we identified the vitamin B transporter protein TCN2, which is essential for one-carbon metabolism, as being significantly downregulated. Further, we found, by knockdown and overexpression experiments, that TCN2 plays an important role in controlling cancer cell transformation toward the highly aggressive mesenchymal/CSC stage; low expression of TCN2 has an effect similar to hypoxia, whereas high expression of TCN2 can reverse it. We conclude that hypoxia induces sequential metabolic responses of one-carbon metabolism in tumor cells. Our mass spectrometry data are available via ProteomeXchange with identifiers PXD005487 (TMT-labeling) and PXD007280 (label-free).

Abnormal epigenetic reprogramming is one of the major causes leading to irregular gene expression and regulatory pathway perturbations, in the cells, resulting in unhealthy cell development or diseases. Accurate measurements of these changes of epigenetic modifications, especially the complex histone modifications, are very important, and the methods for these measurements are not trivial. By following our previous introduction of PRM to targeting histone modifications (Tang, H.; Fang, H.; Yin, E.; Brasier, A. R.; Sowers, L. C.; Zhang, K. Multiplexed parallel reaction monitoring targeting histone modifications on the QExactive mass spectrometer. Anal. Chem. 2014, 86 (11), 5526-34), herein we validated this method by varying the protein/trypsin ratios via serial dilutions. Our data demonstrated that PRM with SILAC histones as the internal standards allowed reproducible measurements of histone H3/H4 acetylation and methylation in the samples whose histone contents differ at least one-order of magnitude. The method was further validated by histones isolated from histone H3 K36 trimethyltransferase SETD2 knockout mouse embryonic fibroblasts (MEF) cells. Furthermore, histone acetylation and methylation in human neural stem cells (hNSC) treated with ascorbic acid phosphate (AAP) were measured by this method, revealing that H3 K36 trimethylation was significantly down-regulated by 6 days of treatment with vitamin C.