Reactive oxygen species (ROS)-induced DNA damage is repaired by the base excision repair pathway. However, the effect of chromatin structure on BER protein recruitment to DNA damage sites in living cells is poorly understood. To address this problem, we developed a method to specifically produce ROS-induced DNA damage by fusing KillerRed (KR), a light-stimulated ROS-inducer, to a tet-repressor (tetR-KR) or a transcription activator (TA-KR). TetR-KR or TA-KR, bound to a TRE cassette (∼90 kb) integrated at a defined genomic locus in U2OS cells, was used to induce ROS damage in hetero- or euchromatin, respectively. We found that DNA glycosylases were efficiently recruited to DNA damage in heterochromatin, as well as in euchromatin. PARP1 was recruited to DNA damage within condensed chromatin more efficiently than in active chromatin. In contrast, recruitment of FEN1 was highly enriched at sites of DNA damage within active chromatin in a PCNA- and transcription activation-dependent manner. These results indicate that oxidative DNA damage is differentially processed within hetero or euchromatin.

Intravenous leiomyomatosis (IVL) is a rare benign tumor. The study aimed to assess outcomes of patients treated surgically for IVL.Between November 2002 and January 2015, 76 patients were treated for IVL. The stage of IVL was evaluated preoperatively by echocardiography and enhanced computerized tomography (CT) scan, and graded into 4 stages according to intravascular tumor progression. We recorded age, lower limb edema before surgery, surgical parameters, and hospitalization expenses. Patients were followed up every 6 months and tumor recurrence was assessed by CT and ultrasound. Patients were followed up for a mean of 4.5 ± 2.5 years (range 1–13 years) and there was no operative, hospital, or long-term mortality or were lost to follow-up.The rate of lower extremity edema, amount of blood loss, postoperative transfusion, length of intensive care unit (ICU) stay, postoperative hospitalization, and hospitalization expenses differed significantly between patients at different presurgery stages. Tumors recurred in 4 of 7 patients with stage I IVL that opted for surgery that preserved the ovaries and uterus. No recurrence was observed in patients graded stage II or more, in all of which the uterus and ovaries were removed. Recurrence was observed in only 4 of 76 cases of IVL, all of whom opted for surgery that spared the ovaries and uterus.Different surgical strategies should be decided based on the staging to completely remove the tumor and ensure the safety of patients. Removal of both ovaries is necessary for inhibiting tumor growth and avoiding recurrence.

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Cellular DNA is organized into chromosomes and capped by a unique nucleoprotein structure, the telomere. Both oxidative stress and telomere shortening/dysfunction cause aging-related degenerative pathologies and increase cancer risk. However, a direct connection between oxidative damage to telomeric DNA, comprising <1% of the genome, and telomere dysfunction has not been established. By fusing the KillerRed chromophore with the telomere repeat binding factor 1, TRF1, we developed a novel approach to generate localized damage to telomere DNA and to monitor the real time damage response at the single telomere level. We found that DNA damage at long telomeres in U2OS cells is not repaired efficiently compared to DNA damage in non-telomeric regions of the same length in heterochromatin. Telomeric DNA damage shortens the average length of telomeres and leads to cell senescence in HeLa cells and cell death in HeLa, U2OS and IMR90 cells, when DNA damage at non-telomeric regions is undetectable. Telomere-specific damage induces chromosomal aberrations, including chromatid telomere loss and telomere associations, distinct from the damage induced by ionizing irradiation. Taken together, our results demonstrate that oxidative damage induces telomere dysfunction and underline the importance of maintaining telomere integrity upon oxidative damage.

dEfficient DNA double-strand break (DSB) repair is critical for the maintenance of genome stability. Unrepaired or misrepaired DSBs cause chromosomal rearrangements that can result in severe consequences, such as tumorigenesis. RAD6 is an E2 ubiquitin-conjugating enzyme that plays a pivotal role in repairing UV-induced DNA damage. Here, we present evidence that RAD6 is also required for DNA DSB repair via homologous recombination (HR) by specifically regulating the degradation of heterochromatin protein 1␣ (HP1␣). Our study indicates that RAD6 physically interacts with HP1␣ and ubiquitinates HP1␣ at residue K154, thereby promoting HP1␣ degradation through the autophagy pathway and eventually leading to an open chromatin structure that facilitates efficient HR DSB repair. Furthermore, bioinformatics studies have indicated that the expression of RAD6 and HP1␣ exhibits an inverse relationship and correlates with the survival rate of patients. Double-strand breaks (DSBs) in DNA are considered the most deleterious types of DNA damage and pose a great threat to the integrity of the genome. Two pathways, homologous recombination (HR) and nonhomologous end joining (NHEJ), have evolved in mammals to repair the broken ends that characterize DSBs (1). The HR pathway is a precise repair pathway, wherein missing and damaged sequence information is copied from sister chromatids to catalyze the repair (2, 3). In contrast, the repair of DNA DSBs by NHEJ is more error prone and often leads to insertions, deletions, or other types of chromosomal rearrangements. The accumulation of DNA mutations, due to either unrepaired broken ends or improper repair, is thought to increase the incidence rate of cancer and other types of diseases (4, 5).Mounting evidence indicates that the ubiquitination of DSB repair proteins plays an important role in regulating DSB repair in mammals (6-8). Ubiquitination is classified into two types, monoubiquitination and polyubiquitination, depending on the number of ubiquitin molecules that become posttranslationally attached to target proteins. Monoubiquitinated proteins have been shown to participate in nonproteolytic pathways such as receptor trafficking, signal transduction, gene transcription, and DNA repair, while the polyubiquitination of substrates often leads to protein degradation either through the 26S proteasome pathway or through the autophagy pathway (9-12).Ubiquitination is catalyzed by a series of enzymes that includes the ubiquitin activation enzyme (E1), the ubiquitin-conjugating enzyme (E2), and the ubiquitin ligase (E3) (13). RAD6 is an E2 ubiquitin-conjugating enzyme with a well-described role in stimulating the repair of UV-induced DNA damage (7,14). In budding yeast, RAD6 interacts with RAD18 to catalyze the monoubiquitination of proliferating cell nuclear antigen (PCNA) on lysine 164, thereby promoting the error-prone DNA damage repair pathway by recruiting low-fidelity polymerases. Interestingly, the interaction between the RAD6-RAD18 complex and the Ubc13-MMS2-Rad5 complex facilita...

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Neutrophil extracellular traps (NETs) are upregulated and promote thrombosis in Behçet’s disease (BD). However, whether NETs promote autoinflammation in BD remains unclear. This study aimed to investigate the potential role of NETs in promoting macrophage activation in BD. Firstly, we quantified NETs by measuring double-stranded DNA (dsDNA) using PicoGreen and calculating the proportion of NETosis. Then macrophages were stimulated with BD- or healthy controls (HC)-derived NETs, and IL-8 and TNF-α production and IFN-γ+ CD4+ T cells differentiation were measured using ELISA and flow cytometry, respectively. The protein components in NETs were analyzed by western blot. Macrophages were stimulated with Histone H4 neutralized NETs, and IL-8 and TNF-α production were measured using ELISA. The level of 8-hydroxydeoxyguanosine (8-OHdG) DNA in NETs was measured using ELISA. The levels of reactive oxygen species (ROS) in serum and neutrophils were measured using ROS probes by a microplate reader and flow cytometry. We found that circulating NETs and neutrophil-derived NETs were significantly higher in BD than HC. BD NETs stimulated macrophages produced higher levels of IL-8 and TNF-α, and promoted IFN-γ+ CD4+ T cells differentiation. BD NETs were enriched in Histone H4, and neutralizing Histone H4 abrogated the BD NETs-mediated IL-8 production by macrophages, but not TNF-α. Also, BD neutrophils produced more 8-OHdG DNA than HC neutrophils, and the percentage of 8-OHdG DNA in dsDNA from BD neutrophils was also higher than that of HC neutrophils. The ROS levels in serum and neutrophils were both higher in BD than HC. Our findings suggested that excessive BD NETs promoted macrophages activation and facilitated IFN-γ+ CD4+ T cells differentiation. Higher levels of Histone H4 and oxidized DNA in BD NETs might mediate macrophages hyperactivation.

Epidermal growth factor receptor (EGFR) is a rational target for cancer therapy, because its overexpression plays an important oncogenic role in a variety of solid tumors; however, EGFR‐targeted antibody–drug conjugate (ADC) therapy for esophageal squamous cell carcinoma (ESCC) is exceedingly rare. LR004 is a novel anti‐EGFR antibody with the advantages of improved safety and fewer hypersensitivity reactions. It may be of great value as a carrier in ADCs with high binding affinity and internalization ability. Here, we prepared an EGFR‐targeting ADC, LR004‐VC‐MMAE, and evaluated its antitumor activities against ESCC and EGFR‐positive cells. LR004 was covalently conjugated with monomethyl auristatin E (MMAE) via a VC linker by antibody interchain disulfide bond reduction. VC‐MMAE was conjugated with LR004 with approximately 4.0 MMAE molecules per ADC. LR004‐VC‐MMAE showed a potent antitumor effect against ESCC and other EGFR‐positive cells with IC 50 values of nM concentrations in vitro. The in vivo antitumor effects of LR004‐VC‐MMAE were investigated in ESCC KYSE520 and A431 xenograft nude mice models. Significant activity was seen at 5 mg·kg−1, and complete tumor regression was observed at 15 mg·kg−1 in the KYSE520 xenograft nude mice after four injections, while the naked antibody LR004 had little effect on inhibiting tumor growth. Similar promising results were obtained in the A431 models. In addition, the tumors also remained responsive to LR004‐VC‐MMAE for large tumor experiments (tumor volume 400–500 mm3). The study results demonstrated that LR004‐VC‐MMAE could be a potential therapeutic agent for ESCC and other EGFR‐expressing malignancies. We also evaluated PK profile of LR004‐VC‐MMAE ADC in the mice model, which would provide qualitative guiding significance for the further research.