Significance: The detrimental effects of ionizing radiation (IR) involve a highly orchestrated series of events that are amplified by endogenous signaling and culminating in oxidative damage to DNA, lipids, proteins, and many metabolites. Despite the global impact of IR, the molecular mechanisms underlying tissue damage reveal that many biomolecules are chemoselectively modified by IR. Recent Advances: The development of high-throughput “omics” technologies for mapping DNA and protein modifications have revolutionized the study of IR effects on biological systems. Studies in cells, tissues, and biological fluids are used to identify molecular features or biomarkers of IR exposure and response and the molecular mechanisms that regulate their expression or synthesis. Critical Issues: In this review, chemical mechanisms are described for IR-induced modifications of biomolecules along with methods for their detection. Included with the detection methods are crucial experimental considerations and caveats for their use. Additional factors critical to the cellular response to radiation, including alterations in protein expression, metabolomics, and epigenetic factors, are also discussed. Future Directions: Throughout the review, the synergy of combined “omics” technologies such as genomics and epigenomics, proteomics, and metabolomics is highlighted. These are anticipated to lead to new hypotheses to understand IR effects on biological systems and improve IR-based therapies. Antioxid. Redox Signal. 21: 260–292.

Cellular exposure to reactive oxygen species induces rapid oxidation of DNA, proteins, lipids and other biomolecules. At the proteome level, cysteine thiol oxidation is a prominent post‐translational process that is implicated in normal physiology and numerous pathologies. Methods for investigating protein oxidation include direct labeling with selective chemical probes and indirect tag‐switch techniques. Common to both approaches is chemical blocking of free thiols using reactive electrophiles to prevent post‐lysis oxidation or other thiol‐mediated cross‐reactions. These reagents are used in large excess, and their reactivity with cysteine sulfenic acid, a critical oxoform in numerous proteins, has not been investigated. Here we report the reactivity of three thiol‐blocking electrophiles, iodoacetamide, N‐ethylmaleimide and methyl methanethiosulfonate, with protein sulfenic acid and dimedone, the structural core of many sulfenic acid probes. We demonstrate that covalent cysteine ‐SOR (product) species are partially or fully susceptible to reduction by dithiothreitol, tris(2‐carboxyethyl)phosphine and ascorbate, regenerating protein thiols, or, in the case of ascorbate, more highly oxidized species. The implications of this reactivity on detection methods for protein sulfenic acids and S‐nitrosothiols are discussed.

While radiation therapy is commonly used for treating cancer, radiation resistance can limit long-term control of the disease. In this study, we investigated the reprogramming of the energy metabolism in radiosensitive and radioresistant head and neck squamous cell carcinomas (HNSCC) using a preclinical matched model of radiation resistance. Our investigation found that radioresistant rSCC-61 cells: 1. They display increased glucose uptake and decreased fatty acid uptake; 2. They deviate from the classical Warburg effect by diverting the glycolytic flux into the pentose phosphate pathway; 3. They are more dependent on glucose than glutamine metabolism to support growth; 4. They have decreased mitochondrial oxidative phosphorylation; 5. They have enhanced fatty acid biosynthesis by increasing the expression of fatty acid synthase; and 6. They utilize endogenous fatty acids to meet the energy demands for proliferation. Inhibition of fatty acid synthase with orlistat or FASN siRNA resulted in increased cytotoxicity and sensitivity to radiation in rSCC-61 cells. These results demonstrate the potential of combination therapy using radiation and orlistat or other inhibitors of lipid and energy metabolism for treating radiation resistance in HNSCC.

AMPK is a key regulator at the molecular level for maintaining energy metabolism homeostasis. Mammalian AMPK is a heterotrimeric complex, and its catalytic α subunit exists in 2 isoforms: AMPKα1 and AMPKα2. Recent studies suggest a role of AMPKα overactivation in Alzheimer’s disease–associated (AD-associated) synaptic failure. However, whether AD-associated dementia can be improved by targeting AMPK remains unclear, and roles of AMPKα isoforms in AD pathophysiology are not understood. Here, we showed distinct disruption of hippocampal AMPKα isoform expression patterns in postmortem human AD patients and AD model mice. We further investigated the effects of brain- and isoform-specific AMPKα repression on AD pathophysiology. We found that repression of AMPKα1 alleviated cognitive deficits and synaptic failure displayed in 2 separate lines of AD model mice. In contrast, AMPKα2 suppression did not alter AD pathophysiology. Using unbiased mass spectrometry–based proteomics analysis, we identified distinct patterns of protein expression associated with specific AMPKα isoform suppression in AD model mice. Further, AD-associated hyperphosphorylation of eukaryotic elongation factor 2 (eEF2) was blunted with selective AMPKα1 inhibition. Our findings reveal isoform-specific roles of AMPKα in AD pathophysiology, thus providing insights into potential therapeutic strategies for AD and related dementia syndromes.

Selective modification of proteins at cysteine residues by reactive oxygen, nitrogen or sulfur species formed under physiological and pathological states is emerging as a critical regulator of protein activity impacting cellular function. This review focuses primarily on protein sulfenylation (–SOH), a metastable reversible modification connecting reduced cysteine thiols to many products of cysteine oxidation. An overview is first provided on the chemistry principles underlining synthesis, stability and reactivity of sulfenic acids in model compounds and proteins, followed by a brief description of analytical methods currently employed to characterize these oxidative species. The following chapters present a selection of redox-regulated proteins for which the –SOH formation was experimentally confirmed and linked to protein function. These chapters are organized based on the participation of these proteins in the regulation of signaling, metabolism and epigenetics. The last chapter discusses the therapeutic implications of altered redox microenvironment and protein oxidation in disease.

Resistance to radiation therapy constitutes a significant challenge in the treatment of head and neck squamous cell cancer (HNSCC). Alteration in DNA methylation is thought to play a role in this resistance. Here, we analyzed DNA methylation changes in a matched model of radiation resistance for HNSCC using the Illumina HumanMethylation450 BeadChip. Our results show that compared to radiation-sensitive cells (SCC-61), radiation-resistant cells (rSCC-61) had a significant increase in DNA methylation. After combining these results with microarray gene expression data, we identified 84 differentially methylated and expressed genes between these 2 cell lines. Ingenuity Pathway Analysis revealed ILK signaling, glucocorticoid receptor signaling, fatty acid α-oxidation, and cell cycle regulation as top canonical pathways associated with radiation resistance. Validation studies focused on CCND2, a protein involved in cell cycle regulation, which was identified as hypermethylated in the promoter region and downregulated in rSCC-61 relative to SCC-61 cells. Treatment of rSCC-61 and SCC-61 with the DNA hypomethylating agent 5-aza-2'deoxycitidine increased CCND2 levels only in rSCC-61 cells, while treatment with the control reagent cytosine arabinoside did not influence the expression of this gene. Further analysis of HNSCC data from The Cancer Genome Atlas found increased methylation in radiation-resistant tumors, consistent with the cell culture data. Our findings point to global DNA methylation status as a biomarker of radiation resistance in HNSCC, and suggest a need for targeted manipulation of DNA methylation to increase radiation response in HNSCC.