Radiofrequency EMF irradiation effects on pre-B lymphocytes undergoing somatic recombination

Intense electromagnetic fields (EMFs) induce DNA double stranded breaks (DSBs) in exposed lymphocytes.We study developing pre-B lymphocytes following V(D)J recombination at their Immunoglobulin light chain loci (IgL). Recombination physiologically induces DNA DSBs, and we tested if low doses of EMF irradiation affect this developmental stage. Recombining pre-B cells, were exposed for 48 h to low intensity EMFs (maximal radiative power density flux S of 9.5 µW/cm2 and electric field intensity 3 V/m) from waves of frequencies ranging from 720 to 1224 MHz. Irradiated pre-B cells show decreased levels of recombination, reduction which is dependent upon the power dose and most remarkably upon the frequency of the applied EMF. Although 50% recombination reduction cannot be obtained even for an S of 9.5 µW/cm2 in cells irradiated at 720 MHz, such an effect is reached in cells exposed to only 0.45 µW/cm2 power with 950 and 1000 MHz waves. A maximal four-fold recombination reduction was measured in cells exposed to 1000 MHz waves with S from 0.2 to 4.5 µW/cm2 displaying normal levels of γH2AX phosphorylated histone. Our findings show that developing B cells exposure to low intensity EMFs can affect the levels of production and diversity of their antibodies repertoire.

Elevated Expression of the RAGE Variant-V in SCLC Mitigates the Effect of Chemotherapeutic Drugs

Small cell lung carcinoma (SCLC) is a highly aggressive malignancy with a very high mortality rate. A prominent part of this is because these carcinomas are refractory to chemotherapies, such as etoposide or cisplatin, making effective treatment almost impossible. Here, we report that elevated expression of the RAGE variant-V in SCLC promotes homology-directed DNA DSBs repair when challenged with anti-cancer drugs. This variant exclusively localizes to the nucleus, interacts with members of the double-strand break (DSB) repair machinery and thus promotes the recruitment of DSBs repair factors at the site of damage. Increased expression of this variant thus, promotes timely DNA repair. Congruently, the tumor cells expressing high levels of variant-V can tolerate chemotherapeutic drug treatment better than the RAGE depleted cells. Our findings reveal a yet undisclosed role of the RAGE variant-V in the homology-directed DNA repair. This variant thus can be a potential target to be considered for future therapeutic approaches in advanced SSLC.

DNA damage and necroptosis induced by peroxidase from proso millet in human colorectal cancer cells

Purpose: To investigate the effects of cationic peroxidase from proso millet (PmPOD) on DNA damage and necroptosis in human colon cancer HCT116 and HT29 cells. Methods: Cell necroptosis and cell cycle was stained using Annexin V-FITC and cell cycle kits, respectively, and evaluated by flow cytometry. Lipid raft on the membrane was disrupted by cholesterol depletion and the location of PmPOD observed by confocal microscopy. Comet assays were used to detect DNA damage, and different inhibitors were also used. Knockdown of p53 or ectopic p53 expression in HCT116 cells were transfected p53 siRNA and pCMV3-TP53-myc plasmid, and p53 expression analyzed by western blotting. Results: Pre-treatment of HCT116 and HT29 cell lines with the specific necroptosis inhibitor Nec-1 prevented PmPOD-induced necroptosis, whereas the apoptosis inhibitor, z-VAD-fmk, had no effect. The entry of PmPOD is necessary for induction of DNA damage and necroptosis. Furthermore, PmPOD induced cell cycle arrest at S phase, as well as DNA DSBs in vivo, as reflected by numerous γ-H2AX foci in CRC cells. However, the tumor suppressor protein, p53, alleviated PmPOD-induced DNA damage and necroptosis. Conclusions: These results demonstrate that PmPOD-induced DNA DSBs in CRC cells is the main cause of necroptosis, and that the tumor suppressor protein, p53, alleviates PmPOD-induced necroptosis by promoting p53-mediated repair pathways.

Pharmacologic ascorbate enhances the therapeutic index of ATM-inhibitor based chemoradiation for colorectal cancer.

3609 Background: Ataxia telangectasia mutated protein (ATM) is one of the key sensors of DNA damage and specific inhibitors of ATM are potent radiosensitizers. However, their clinical utility with radiation (RT) is limited because they lack tissue specificity and increase normal tissue injury. Pharmacologic (high dose) ascorbate (P-AscH-) selectively increases oxidative stress in tumors while functioning as a donor antioxidant and reducing RT damage in normal tissues. We hypothesized that P-AscH- could enhance the therapeutic index of ATM-inhibitor based chemoradiation (CRT) for colorectal cancer (CRC) by simultaneously enhancing efficacy and reducing RT bowel injury. Methods: Human HCT116, SW480, and HT29 and murine CT26 and MC38 CRC models were used. Clonogenic survival was assessed following single-fraction RT (2-8 Gy) +/- P-AscH- (5 pM/cell) +/- veliparib (PARP), VE821 (ATR), or KU60019 (ATM). Catalase expression was induced using HCT116 cells expressing a doxycycline inducible catalase transgene. DNA double strand breaks (DSBs) were quantified using neutral comet assays 0-24 hours post RT. Cell cycle phases were assessed using flow cytometry. ATM and pATM localization were assessed using IF. Jejunal toxicity was assessed using IHC in fixed tissues following single fraction (10 Gy) whole abdominal RT in c57blj/6 mice. Tumor growth delay was assessed following RT (5 Gy x 3) +/- drug treatment in unilateral flank tumors. Results: Veliparib, VE821, and KU60019 were potent radiosensitizers in HCT116, SW480, HT29, MC38, and CT26 CRC tumor models and P-AscH- further reduced clonogenic survival with DRIs in all lines except for HT29. In contrast, P-AscH- enhanced survival of cultured HUVEC and FHs-74 cells exposed to RT. Enhanced cell kill with P-AscH- is H202 mediated as it is completely attenuated by inducible catalase expression. P-AscH- significantly increased the number of DNA DSBs in tumors after RT in vitro. Despite the increase in DNA DSBs, P-AscH-significantly decreased nuclear localization of activated P-ATM after RT and significantly decreased the fraction of cells in G2/M phases of the cell cycle. In vivo, RT + P-AscH- + KU60019 induced more tumor growth delay/clearance than all other combinations in unilateral MC38 or HCT116 flank tumors. Finally, P-AscH- significantly reduced loss of jejunal crypt cell density, epithelial architecture, and markers of lipid and protein oxidation following whole abdominal RT. Conclusions: P-AscH- selectively enhances the efficacy of ATM-based CRT in CRC tumor models while simultaneously decreasing RT-mediated small bowel toxicity. In tumors, P-AscH- enhances DNA DSBs by stimulating an H202 flux and prevents activation of DNA repair pathways and cell cycle checkpoints by inhibiting RT-induced activation of ATM. Selective radioprotectors like P-AscH- could facilitate the clinical translation ATM inhibitors as radiosensitizers.

FOXO3a protects glioma cells against temozolomide-induced DNA double strand breaks via promotion of BNIP3-mediated mitophagy

FOXO3a (forkhead box transcription factor 3a) is involved in regulating multiple biological processes in cancer cells. BNIP3 (Bcl-2/adenovirus E1B 19-kDa-interacting protein 3) is a receptor accounting for priming damaged mitochondria for autophagic removal. In this study we investigated the role of FOXO3a in regulating the sensitivity of glioma cells to temozolomide (TMZ) and its relationship with BNIP3-mediated mitophagy. We showed that TMZ dosage-dependently inhibited the viability of human U87, U251, T98G, LN18 and rat C6 glioma cells with IC50 values of 135.75, 128.26, 142.65, 155.73 and 111.60 μM, respectively. In U87 and U251 cells, TMZ (200 μM) induced DNA double strand breaks (DSBs) and nuclear translocation of apoptosis inducing factor (AIF), which was accompanied by BNIP3-mediated mitophagy and FOXO3a accumulation in nucleus. TMZ treatment induced intracellular ROS accumulation in U87 and U251 cells via enhancing mitochondrial superoxide, which not only contributed to DNA DSBs and exacerbated mitochondrial dysfunction, but also upregulated FOXO3a expression. Knockdown of FOXO3a aggravated TMZ-induced DNA DSBs and mitochondrial damage, as well as glioma cell death. TMZ treatment not only upregulated BNIP3 and activated autophagy, but also triggered mitophagy by prompting BNIP3 translocation to mitochondria and reinforcing BNIP3 interaction with LC3BII. Inhibition of mitophagy by knocking down BNIP3 with SiRNA or blocking autophagy with 3MA or bafilomycin A1 exacerbated mitochondrial superoxide and intracellular ROS accumulation. Moreover, FOXO3a knockdown inhibited TMZ-induced BNIP3 upregulation and autophagy activation. In addition, we showed that treatment with TMZ (100 mg·kg−1·d−1, ip) for 12 days in C6 cell xenograft mice markedly inhibited tumor growth accompanied by inducing FOXO3a upregulation, oxidative stress and BNIP3-mediated mitophagy in tumor tissues. These results demonstrate that FOXO3a attenuates temozolomide-induced DNA double strand breaks in human glioma cells via promoting BNIP3-mediated mitophagy.

A Role for RAGE in DNA Double Strand Breaks (DSBs) Detected in Pathological Placentas and Trophoblast Cells

Impaired DNA damage responses are associated with several diseases, including pregnancy complications. Recent research identified an ATM-kinase dependent function for the nuclear isoform of the receptor for advanced glycation end-products (RAGE) during double strand break (DSB)-repair. RAGE contributes to end-resectioning of broken DNA sites by binding with the MRE11-Rad50-Nbs1 (MRN) complex. Placental research is limited regarding the impact of genomic instability and the mechanism for potential repair. We tested the hypothesis regarding the involvement of RAGE during the repair of placental DNA-DSBs. We first identified that the pregnancy complications of PE and preterm labor (PTL) experience loss of genomic integrity and an in vitro trophoblast cell model was used to characterize trophoblast DSBs. Colocalized immunofluorescence of γ-H2AX and RAGE support the potential involvement of RAGE in cellular responses to DNA-DSBs. Immunoblotting for both molecules in PE and PTL placenta samples and in trophoblast cells validated a connection. Co-immunoprecipitation studies revealed interactions between RAGE and pATM and MRE11 during DNA-DSBs. Reduced cellular invasion confirmed the role of genomic instability in trophoblastic function. Collectively, these experiments identified genomic instability in pregnancy complications, the impact of defective DNA on trophoblast function, and a possible RAGE-mediated mechanism during DNA-DSB repair.

Double-strand breaks induce short scale DNA replication and damage amplification in the fully-grown mouse oocytes

Break-induced replication (BIR) is essential for the repair of DNA double-strand breaks (DSBs) with single ends. DSBs-induced microhomology-mediated BIR (mmBIR) and template-switching can increase the risk of complex genome rearrangement. In addition, DSBs can also induce the multi-invasion-mediated DSB amplification. The mmBIR-induced genomic rearrangement has been identified in cancer cells and patients with rare diseases. However, when and how mmBIR are initiated haven’t been fully and deeply studied. Furthermore, it is not well understood about the conditions for initiation of multi-invasion-mediated DSB amplification. In the G2 phase oocyte of mouse, we identified a type of short scale BIR (ssBIR) using the DNA replication indicator 5-ethynyl-2´-deoxyuridine (EdU). These ssBIRs could only be induced in the fully-grown oocytes but not the growing oocytes. If the DSB oocytes were treated with Rad51 or Chek1/2 inhibitors, both EdU signals and DSB marker γH2A.X foci would decrease. In addition, the DNA polymerase inhibitor Aphidicolin could inhibit the ssBIR and another inhibitor ddATP could reduce the number of γH2A.X foci in the DSB oocytes. In conclusion, our results showed that DNA DSBs in the fully-grown oocytes can initiate ssBIR and be amplified by Rad51 or DNA replication.

Multiple classes of bactericidal antibiotics cause DNA double strand breaks in Staphylococcus aureus

Antibiotics inhibit essential bacterial processes, resulting in arrest of growth and in some cases cell death. Many antibiotics are also reported to trigger endogenous production of reactive oxygen species (ROS), which damage DNA and other macromolecules. However, the type of DNA damage that arises and the mechanisms used by bacteria to repair it are largely unclear. We found that several different classes of antibiotic triggered dose-dependent DNA damage in Staphylococcus aureus, including some bacteriostatic drugs. Damage was heterogenous and varied in magnitude between strains. However, antibiotic-triggered DNA damage led to double strand breaks, the processing of which by the RexAB helicase/nuclease complex triggered the SOS response and reduced staphylococcal susceptibility to most of the antibacterials tested. In most cases, DNA DSBs occurred under aerobic but not anaerobic conditions, suggesting a role for ROS. We conclude that DNA double strand breaks are a common occurrence during bacterial exposure to several different antibiotic classes and that repair of this damage by the RexAB complex promotes bacterial survival.

Sirt6-mediated Nrf2/HO-1 activation alleviates angiotensin II-induced DNA DSBs and apoptosis in podocytes

Sirt6 upregulation inhibited Ang II-induced ROS generation and DNA DSBs in podocytes, and it shows the cytoprotective effects in podocytes via the Nrf2/HO-1 pathway. Sirt6 may be a new therapeutic target for the treatment of CKD.