Joseph I. Kang

5-Halogenated pyrimidine lesions within a CpG sequence context mimic 5-methylcytosine by enhancing the binding of the methyl-CpG-binding domain of methyl-CpG-binding protein 2 (MeCP2)

Perturbations in cytosine methylation signals are observed in the majority of human tumors; however, it is as yet unknown how methylation patterns become altered. Epigenetic changes can result in the activation of transforming genes as well as in the silencing of tumor suppressor genes. We report that methyl-CpG-binding proteins (MBPs), specific for methyl-CpG dinucleotides, bind with high affinity to halogenated pyrimidine lesions, previously shown to result from peroxidase-mediated inflammatory processes. Emerging data suggest that the initial binding of MBPs to methyl-CpG sequences may be a seeding event that recruits chromatin-modifying enzymes and DNA methyltransferase, initiating a cascade of events that result in gene silencing. MBD4, a protein with both methyl-binding and glycosylase activity demonstrated repair activity against a series of 5-substituted pyrimidines, with the greatest efficiency against 5-chlorouracil, but undetectable activity against 5-chlorocytosine. The data presented here suggest that halogenated pyrimidine damage products can potentially accumulate and mimic endogenous methylation signals.

Hypochlorous acid damages histone proteins forming 3-chlorotyrosine and 3,5-dichlorotyrosine.

While the last 30 years chronicles an extensive effort to understand the damage to DNA caused by reactive oxygen species (ROS), little research has examined the chemical damage to the histone proteins found in chromatin. Hypochlorous acid (HOCl), the primary product of activated neutrophils, is known to damage both DNA and proteins. This article describes the use of mass spectrometry to quantitate the formation of 3-chlorotyrosine and 3,5-dichlorotyrosine, stable and unique markers of protein damage caused by HOCl, in the core histone proteins. Our results indicate that up to 25% of the tyrosine in histone proteins become chlorinated by excess HOCl. We also observe significant formation of 3-chlorotyrosine and 3,5-dichlorotyrosine at low HOCl concentrations and short reaction times. We use mass spectrometry to identify the tyrosine residues on each histone protein that are chlorinated based on the observation of chlorine-containing peptides following protease digestion of histone proteins exposed to HOCl. The tyrosine residues preferentially chlorinated by HOCl are generally within three residues of a lysine or histidine residue, further implicating the initial formation of chloramines in the efficient chlorination of tyrosine residues. The methods and results described here should further our understanding of how HOCl produced at sites of inflammation might damage chromatin.

Examination of Hypochlorous Acid-Induced Damage to Cytosine Residues in a CpG Dinucleotide in DNA

Inflammation-mediated, neutrophil-derived hypochlorous acid can damage DNA and result in the chlorination damage products 5-chlorocytosine and 5-chlorouracil as well as the oxidation damage products 5-hydroxycytosine and 5-hydroxyuracil. While 5-chlorocytosine could potentially perturb epigenetic signals if formed at a CpG dinucleotide, the remaining products are miscoding and could result in transition mutations. In this article, we have investigated the reaction of hypochlorous acid with an oligonucleotide site-specifically enriched with 15N to probe the reactivity of cytosine at CpG. These experiments demonstrate directly the formation of 5-chlorocytosine at a CpG dinucleotide in duplex DNA. We observe that chlorination relative to oxidation damage is greater at CpG by a factor of approximately two, whereas similar amounts of 5-chlorocytosine and 5-hydroxycytosine are formed at two non-CpG sites examined. The relative amounts of deamination of the cytosine to uracil derivatives are similar at CpG and non-CpG sites. Overall, we observe that the reactivity of cytosine at CpG and non-CpG sites toward hypochlorous acid induced damage is similar (5-chlorocytosine > 5-hydroxycytosine > 5-hydroxyuracil > 5-chlorouracil), with a greater proportion of chlorination damage at CpG sites. These results are in accord with the potential of inflammation-mediated DNA damage to both induce transition mutations and to perturb epigenetic signals.

Proton therapy for gastrointestinal cancers

Proton beam therapy provides an opportunity to deliver ionizing radiation with improved dose conformity. It has gained popularity as a means of more localized radiation delivery. However, proton therapy data are still lacking, as there are still relatively few proton treatment centers worldwide. This paucity of data is particularly evident in gastrointestinal (GI) cancers. Most GI cancers are located in close proximity to or abut critical organs. The ability to deliver an appropriate dose to a target in this area is challenging; normal organ toxicities often limit the amount of radiation that can be delivered to achieve a therapeutic dose. The modern trend in treatment of GI cancers is toward multimodality treatment. However, there is an increased risk of toxicity when combining modalities such as radiotherapy, chemotherapy, and surgery, thus placing an even greater emphasis or normal-tissue toxicities. Improvements in radiation treatment techniques over the past few decades have allowed dose escalation with improved normal-tissue sparing. The driving force behind improving treatment conformity is the significant short- and long-term morbidity of normal tissue toxicity during and after radiation treatment. The degree of normal-tissue sparing within individuals undergoing radiation treatment is highly variable and depends on tumor type and region. Tumors of the esophagus, for example, are surrounded by lung and spinal cord, while anal cancers are in close proximity to the bladder and rectum. Each subsite of the GI tract requires different techniques and approaches to maximize normal-organ sparing while delivering adequate amounts of radiation to the tumor. The physical properties of proton radiation may offer a distinct benefit in treating GI malignancies.

Proton therapy for esophageal cancer

Trimodality therapy (chemotherapy, radiotherapy and surgery) for esophageal cancer has improved patient outcomes and is favored by the current standard of practice for surgical patients. Neoadjuvant chemoradiotherapy followed by surgery appears to increase resectability, induce tumor downstaging, and improve local control, disease-free survival and overall survival compared to surgery alone. For non-surgical candidates, the addition of concurrent chemotherapy to radiotherapy provides a chance for long-term survival. However, these improved outcomes have come at the cost of increased side-effects. The standard dose of radiotherapy given with concurrent chemotherapy remains up to 50 Gy in both the preoperative setting and in the definitive setting for non-surgical patients despite previous attempts at dose escalation. Even with this modest dose, the majority of patients receiving concurrent chemoradiotherapy with conventional techniques still experience grade 3 or greater toxicity. The challenge for the radiation oncologist is to achieve an adequate dose to gross tumor volume located in the posterior mediastinum while limiting dose to nearby critical organs at risk. Due to their mass (about 1800 times an electron) and charge, proton beams can be shaped and controlled in three dimensions in order to more accurately deliver the radiation dose within target volumes while simultaneously reducing dose to surrounding non-target tissue. Proton beams are characterized by a narrow penumbra. Additionally, protons display a Bragg peak where the deposited dose from a single proton beam is low upon entrance and slowly increases until the desired depth in tissue at which point the majority of the dose is deposited with minimal dose distal to the target. Given the esophagus’s central location near several critical structures, including the lungs, heart, and spinal cord, the ability of proton therapy to conform high radiation dose to the tumor volume while reducing the dose of radiation to adjacent normal tissue has the potential to decrease radiation-related toxicity and provide a means for dose escalation. Modern radiotherapy techniques have to assess the risk of organ-specific radiation toxicity. Spinal cord toxicity risk would be low with a standard prescribed dose of 50.4 Gy for esophageal cancer,as current data suggests that the probability of myelopathy is 0.03% at 45 Gy and 0.2% at 50 Gy. However, a higher risk can be expected with a dose escalation approach. Late cardiac toxicity commonly manifests as pericardial disease and increases the risk of acute myocardial infarction. Wei et al. reported that the pericardium volume irradiated by a dose of 30 Gy or higher (V30) was a significant predictor of pericardial effusion risk. Inferior left ventricular ischemia is commonly found in patients irradiated for distal esophageal tumors, usually occurring in volumes of myocardium irradiated to a dose of 45 Gy or more. Lung toxicity dosimetric parameters from several studies show an increased risk of radiation pneumonitis correlating with mean lung dose and the percentage of lung volume receiving at least 20 Gy (V20), 10 Gy (V10) and 5 Gy (V5). Graham et al. found a strong correlation between parameter V20 and pneumonitis in lung cancer patients with the incidence of grade 2 or greater pneumonitis of 7%, 13% and 36% for patients with V20 in the range of 22–31%,32–40% and greater than 40% respectively. However, more stringent parameters may be required when evaluating patients in the neoadjuvant setting, as pulmonary toxicity is critical in the setting of esophagectomy. One study reported a 15% rate of significant pulmonary complications accounting for 55% of mortality following esophagectomy. Lee et al. noted higher pulmonary toxicity in trimodality therapy (35% vs. 8%, P = 0.014) when pulmonary V10 was at least 40% versus less than 40%. A follow-up study by Wang et al. found, on multivariate analysis, that the volume of lung spared from doses of at least 5 Gy was the only independent dosimetric parameter predictive of pulmonary toxicity. These data suggest that seemingly insignificant low doses of radiation can impact postoperative pulmonary

Stereotactic body proton therapy for liver metastases

Although surgical resection is the gold standard, stereotactic body radiotherapy (SBRT) is often the preferred treatment for metastatic liver tumors due to size, multi-focality, tumor distribution or patient co-morbidities limiting surgical options. The major dose-limiting concern in the use of SBRT for liver tumors is the risk of radiation-induced liver disease (RILD). This can limit the number of patients who may be candidates for conventional SBRT. The use of protons for SBRT is potentially attractive given the dosimetric advantages inherent to proton radiotherapy potentially offering a way to maximize tumor control via dose escalation while avoiding excessive radiation to the remaining liver and other organs at risk. In this review we discuss the physical attributes and rationale for stereotactic body proton therapy (SBPT) for the treatment of liver metastases.