Circulating tumor DNA (ctDNA) has emerged as a useful diagnostic and prognostic biomarker in many cancers. Here, we conducted a study to investigate the potential use of ctDNA methylation markers for the diagnosis and prognostication of colorectal cancer (CRC) and used a prospective cohort to validate their effectiveness in screening patients at high risk of CRC. We first identified CRC-specific methylation signatures by comparing CRC tissues to normal blood leukocytes. Then, we applied a machine learning algorithm to develop a predictive diagnostic and a prognostic model using cell-free DNA (cfDNA) samples from a cohort of 801 patients with CRC and 1021 normal controls. The obtained diagnostic prediction model discriminated patients with CRC from normal controls with high accuracy (area under curve = 0.96). The prognostic prediction model also effectively predicted the prognosis and survival of patients with CRC (P < 0.001). In addition, we generated a ctDNA-based molecular classification of CRC using an unsupervised clustering method and obtained two subgroups of patients with CRC with significantly different overall survival (P = 0.011 in validation cohort). Last, we found that a single ctDNA methylation marker, cg10673833, could yield high sensitivity (89.7%) and specificity (86.8%) for detection of CRC and precancerous lesions in a high-risk population of 1493 participants in a prospective cohort study. Together, our findings showed the value of ctDNA methylation markers in the diagnosis, surveillance, and prognosis of CRC.

The abnormal de novo methylation of promoter CpG islands in numerous tumor suppressor and other cancer-related genes has been shown to be associated with their silencing during carcinogenesis (3,18,19,30). This frequent alteration in human cancer cells may represent an alterative mechanism to mutations and chromosomal deletions for gene inactivation during tumorigenesis. The prevalence of aberrant methylation in cancer has encouraged the search for therapeutic agents which can inhibit methylation and which may thus be utilized to reverse this effect by reactivating genes which have become abnormally silenced.5-Azacytidine (5-Aza-CR) and its deoxy analog, 5-aza-2Ј-deoxycytidine (5-Aza-CdR), are two of the most well-known DNA methylation inhibitors (15,20). Both drugs are nucleoside analogs which have been widely used for studying the role of DNA methylation in biological processes as well as for clinical treatment of patients with acute myeloid leukemia and myelodysplastic syndrome (28,34,42). 5-Aza-CR and 5-AzaCdR are potent mechanism-based inhibitors of DNA methyltransferases (DNMTs) and function by substituting for cytosine residues during DNA replication and forming covalent bonds with the DNMT, which ultimately leads to the inhibition of the DNMT's activity (8,12,31,39). Unfortunately, these drugs are both unstable in aqueous solutions and toxic (4, 7, 39), and these characteristics have complicated their clinical use; hence there is the need for an effective, stable, and minimally toxic inhibitor of DNA methylation.Previously, we characterized zebularine [1-(␤-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one) as a novel inhibitor of DNA methylation which is stable and minimally toxic both in vitro and in vivo (6). Zebularine is a cytidine analog containing a 2-(1H)-pyrimidinone ring that was originally developed as a cytidine deaminase inhibitor because it lacks an amino group on position 4 of the ring (22,24). Studies with synthetic oligonucleotides containing the 2-(1H)-pyrimidinone ring have demonstrated the formation of a tight complex with bacterial methyltransferases in vitro (17), and this was further corroborated by a recent study demonstrating the crystallization of a bacterial DNA methyltransferase with the 2-(1H)-pyrimidinone ring forming a covalent bond at the active site (43). In a previous study, we have shown that zebularine can be orally administered to cause reactivation and demethylation of a silenced and hypermethylated p16 gene in human bladder tumor cells grown in nude mice (6). Nonetheless, one of the major challenges with the usage and application of nucleoside analogs as inhibitors of DNA methylation is the problem of remethylation of genes that were demethylated after treatment with these agents, which eventually results in their resilencing (5). This phenomenon of remethylation following cessation of drug treatment makes the clinical application of these drugs quite limited.Here we demonstrate that the single treatment of T24 bladder carcinoma cells with zebularine resulted in a rapid i...

The ideal treatment of localized cancer should directly cause an irreversible and complete death of tumor cells without damage to surrounding normal tissue. High intensity focused ultrasound (HIFU) is such a potential treatment, which induces a complete coagulative necrosis of a tumor at depth through the intact skin. The idea that using an extracorporeal source of therapeutic ultrasound was introduced more than 50 years ago [J. Gen. Physiol. 26 (1942) 179]. However, up to now, most of the studies on HIFU have been dealing with animal experiments because this extracorporeal technique is very complicated in clinical applications. The purpose of this study is to introduce Chinese clinical experience of using extracorporeal HIFU for the treatment of patients with various kinds of solid tumor. From December 1997 to October 2001, a total of 1038 patients with solid tumors underwent HIFU ablation in China. Among them, 313 patients were treated at the Chongqing University of Medical Sciences, China. Pathological examination showed that the target region presented clear evidence of cellular destruction. Small blood vessels less than 2 mm in diameter were severely damaged. Follow-up diagnostic imaging revealed that there was no, or reduced, blood supply, and no uptake of radioisotope in the treated tumor after HIFU, both indicating a positive therapeutic response and an absence of viable tumor. Imaging at 6-12 months showed obvious regression of the lesion. Four-year follow-up data were significantly observed in patients with hepatocellular carcinoma, osteosarcoma, and breast cancer. An extremely low major complication rate was noted. It is concluded that HIFU ablation is a safe, effective, and feasible modality for the ablation of carcinomas.

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Propionylation has been identified recently as a new type of protein post-translational modification. Bacterial propionylCoA synthetase and human histone H4 are propionylated at specific lysine residues that have been known previously to be acetylated. However, other proteins subject to this modification remain to be identified, and the modifying enzymes involved need to be characterized. In this work, we report the discovery of histone H3 propionylation in mammalian cells. Propionylation at H3 lysine Lys 23 was detected in the leukemia cell line U937 by mass spectrometry and Western analysis using a specific antibody. In this cell line, the propionylated form of Lys 23 accounted for 7%, a level at least 6-fold higher than in other leukemia cell lines (HL-60 and THP-1) or non-leukemia cell lines (HeLa and IMR-90). The propionylation level in U937 cells decreased remarkably during monocytic differentiation, indicating that this modification is dynamically regulated. Moreover, in vitro assays demonstrated that histone acetyltransferase p300 can catalyze H3 Lys 23 propionylation, whereas histone deacetylase Sir2 can remove this modification in the presence of NAD ؉ .These results suggest that histone propionylation might be generated by the same set of enzymes as for histone acetylation and that selection of donor molecules (propionyl-CoA versus acetylCoA) may determine the difference of modifications. Because like acetyl-CoA, propionyl-CoA is an important intermediate in biosynthesis and energy production, histone H3 Lys 23 propionylation may provide a novel epigenetic regulatory mark for cell metabolism.Eukaryotic histones are rich in multiple post-translational modifications, the combinatory array of which is the basis for epigenetic regulation of gene expression (1). Appropriate histone modifications are required for normal cell growth and differentiation, whereas aberrant histone modifications contribute to tumor formation such as malignant hematopoiesis (2). For example, genome-wide alterations of histone modification patterns are found in prostate cancer and are predictive of clinical outcomes (3); histone H3 Lys 79 (H3K79) hypermethylation and the activation of oncogenic HOX genes are prominent causes for leukemia with chromosome translocation involving the AF10 gene (4, 5), whereas global hypomethylation at H3K79 sites has been proposed to cause genome instability in AF10-related leukemia cancers (6). Alteration in histone modification patterns serves as a hallmark of cancer that provides clues for cancer diagnosis and treatment (7).Lysine propionylation has recently been identified as a new type of post-translational modification. It was first discovered in human histone H4 enriched for acetylation using anti-acetyl H4 antibodies (9). The propionyl-CoA synthetase of Salmonella enterica was also found to be propionylated at lysine 592, which inactivates the enzyme activity in vivo (8). In vitro experiments showed that histone acetyltransferase p300 and CREBbinding protein (CBP) can propionylate histone H4,...

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While methylcytosines serve as the fifth base encoding epigenetic information, they are also a dangerous endogenous mutagen due to their intrinsic instability. Methylcytosine undergoes spontaneous deamination, at a rate much higher than cytosine, to generate thymine. In mammals, two repair enzymes, thymine DNA glycosylase (TDG) and methyl-CpG binding domain 4 (MBD4), have evolved to counteract the mutagenic effect of methylcytosines. Both recognize G/T mismatches arising from methylcytosine deamination and initiate base-excision repair that corrects them to G/C pairs. However, the mechanism by which the methylation status of the repaired cytosines is restored has remained unknown. We show here that the DNA methyltransferase Dnmt3a interacts with TDG. Both the PWWP domain and the catalytic domain of Dnmt3a are able to mediate the interaction with TDG at its N-terminus. The interaction affects the enzymatic activity of both proteins: Dnmt3a positively regulates the glycosylase activity of TDG, while TDG inhibits the methylation activity of Dnmt3a in vitro. These data suggest a mechanistic link between DNA repair and remethylation at sites affected by methylcytosine deamination.

Chronic renal failure (CRF) is a major public health problem worldwide. Hydrogen sulfide (H2S) plays important roles in renal physiological and pathophysiological processes. However, whether H2S could protect against CRF in rats remains unclear. In this study, we found that H2S alleviated gentamicin-induced nephrotoxicity by reducing reactive oxygen species (ROS)-mediated apoptosis in normal rat kidney-52E cells. We demonstrated that H2S significantly improved the kidney structure and function of CRF rats. We found that H2S decreased the protein levels of Bax, Caspase-3, and Cleaved-caspase-3, but increased the expression of Bcl-2. Treatment with H2S reduced the levels of malondialdehyde and ROS and increased the activities of superoxide dismutase and glutathione peroxidase. H2S significantly abolished the phosphorylation of extracellular signal-regulated protein kinase 1/2, c-Jun N-terminal kinase, and p38 in the kidney of CRF rats. Furthermore, H2S decreased the expression levels of tumor necrosis factor-α, interleukin (IL)-6, IL-10, and monocyte chemoattractant protein-1, as well as the protein levels of p50, p65, and p-p65 in the kidney of CRF rats. In conclusion, H2S could ameliorate adenine-induced CRF in rats by inhibiting apoptosis and inflammation through ROS/mitogen-activated protein kinase and nuclear factor-kappa B signaling pathways.