MRE11 liberates cGAS from nucleosome sequestration during tumorigenesis.

Oncogene-induced replication stress generates endogenous DNA damage that activates cGAS-STING-mediated signalling and tumour suppression1-3. However, the precise mechanism of cGAS activation by endogenous DNA damage remains enigmatic, particularly given that high-affinity histone acidic patch (AP) binding constitutively inhibits cGAS by sterically hindering its activation by double-stranded DNA (dsDNA)4-10. Here we report that the DNA double-strand break sensor MRE11 suppresses mammary tumorigenesis through a pivotal role in regulating cGAS activation. We demonstrate that binding of the MRE11-RAD50-NBN complex to nucleosome fragments is necessary to displace cGAS from acidic-patch-mediated sequestration, which enables its mobilization and activation by dsDNA. MRE11 is therefore essential for cGAS activation in response to oncogenic stress, cytosolic dsDNA and ionizing radiation. Furthermore, MRE11-dependent cGAS activation promotes ZBP1-RIPK3-MLKL-mediated necroptosis, which is essential to suppress oncogenic proliferation and breast tumorigenesis. Notably, downregulation of ZBP1 in human triple-negative breast cancer is associated with increased genome instability, immune suppression and poor patient prognosis. These findings establish MRE11 as a crucial mediator that links DNA damage and cGAS activation, resulting in tumour suppression through ZBP1-dependent necroptosis.

Mechanisms of Immune Sensing of DNA Damage.

Genomic stability relies on a multifaceted and evolutionarily conserved DNA damage response (DDR). In multicellular organisms, an integral facet of the DDR involves the activation of the immune system to eliminate cells with persistent DNA damage. Recent research has shed light on a complex array of nucleic acid sensors crucial for innate immune activation in response to oncogenic stress-associated DNA damage, a process vital for suppressing tumor formation. Yet, these immune sensing pathways may also be co-opted to foster tolerance of chromosomal instability, thereby driving cancer progression. This review aims to provide an updated overview of how the innate immune system detects and responds to DNA damage. An improved understanding of the regulatory intricacies governing this immune response may uncover new avenues for cancer prevention and therapeutic intervention.

TPX2 Amplification-Driven Aberrant Mitosis in Culture Adapted Human Embryonic Stem Cells with gain of 20q11.21.

BACKGROUND: Despite highly effective machinery for the maintenance of genome integrity in human embryonic stem cells (hESCs), the frequency of genetic aberrations during in-vitro culture has been a serious issue for future clinical applications. METHOD: By passaging hESCs over a broad range of timepoints (up to 6 years), the isogenic hESC lines with different passage numbers with distinct cellular characteristics, were established. RESULT: We found that mitotic aberrations, such as the delay of mitosis, multipolar centrosomes, and chromosome mis-segregation, were increased in parallel with polyploidy compared to early-passaged hESCs (EP-hESCs) with normal copy number. Through high-resolution genome-wide approaches and transcriptome analysis, we found that culture adapted-hESCs with a minimal amplicon in chromosome 20q11.21 highly expressed TPX2, a key protein for governing spindle assembly and cancer malignancy. Consistent with these findings, the inducible expression of TPX2 in EP-hESCs reproduced aberrant mitotic events, such as the delay of mitotic progression, spindle stabilization, misaligned chromosomes, and polyploidy. CONCLUSION: These studies suggest that the increased transcription of TPX2 in culture adapted hESCs could contribute to an increase in aberrant mitosis due to altered spindle dynamics.

The cGAS/STING/TBK1/IRF3 innate immunity pathway maintains chromosomal stability through regulation of p21 levels.

Chromosomal instability (CIN) in cancer cells has been reported to activate the cGAS-STING innate immunity pathway via micronuclei formation, thus affecting tumor immunity and tumor progression. However, adverse effects of the cGAS/STING pathway as they relate to CIN have not yet been investigated. We addressed this issue using knockdown and add-back approaches to analyze each component of the cGAS/STING/TBK1/IRF3 pathway, and we monitored the extent of CIN by measuring micronuclei formation after release from nocodazole-induced mitotic arrest. Interestingly, knockdown of cGAS (cyclic GMP-AMP synthase) along with induction of mitotic arrest in HeLa and U2OS cancer cells clearly resulted in increased micronuclei formation and chromosome missegregation. Knockdown of STING (stimulator of interferon genes), TBK1 (TANK-binding kinase-1), or IRF3 (interferon regulatory factor-3) also resulted in increased micronuclei formation. Moreover, transfection with cGAMP, the product of cGAS enzymatic activity, as well as add-back of cGAS WT (but not catalytic-dead mutant cGAS), or WT or constitutively active STING (but not an inactive STING mutant) rescued the micronuclei phenotype, demonstrating that all components of the cGAS/STING/TBK1/IRF3 pathway play a role in preventing CIN. Moreover, p21 levels were decreased in cGAS-, STING-, TBK1-, and IRF3-knockdown cells, which was accompanied by the precocious G2/M transition of cells and the enhanced micronuclei phenotype. Overexpression of p21 or inhibition of CDK1 in cGAS-depleted cells reduced micronuclei formation and abrogated the precocious G2/M transition, indicating that the decrease in p21 and the subsequent precocious G2/M transition is the main mechanism underlying the induction of CIN through disruption of cGAS/STING signaling.

Presalting Condition Effects on the Development of Pink Color in Cooked Ground Chicken Breasts.

The effects of presalting conditions (storage temperature and duration) with/without sodium tripolyphosphate (STPP) on the color and pigment characteristics of cooked ground chicken breast were investigated. Meat mixtures containing 2% NaCl (control) or 2% NaCl and 0.5% STPP (STPP treatment) were stored for 0, 3, 5, 7, and 10 d at 2°C or 7°C, followed by cooking to 75°C, and cooling and storage at 2°C-3°C until further analysis. The treatment was the most effective on the pink color defect of all independent variables. The effect of storage temperature was only observed on CIE L* values and percentage myoglobin denaturation (PMD). The control was redder than the STPP treated samples and the CIE a* values increased (p<0.05) from 0 to 5 d in the control and STPP treated samples. Compared to the STPP treatment, the control exhibited increased reducing conditions (more negative oxidation reduction potential), lower undenatured myoglobin, and greater PMD. No differences in the cooking yields of the control and STPP-treated samples were observed for various storage durations. Products with STPP showed higher (p<0.05) pH values than those without STPP, but no differences (p>0.05) in PMD were observed over the storage period in the control and STPP treated samples, except for day 0. Thus, STPP is effective at reducing the pink color in cooked chicken breasts. In addition, presalting for longer than 5 d resulted in increased pink color of the cooked chicken breasts.

The Effects of Addition Timing of NaCl and Sodium Tripolyphosphate and Cooking Rate on Pink Color in Cooked Ground Chicken Breasts.

The current study investigated the effects of timing of NaCl (2%) and sodium tripolyphosphate (STPP, 0.5%) addition and cooking rates on color and pigment properties of ground chicken breasts. Four treatments were tested as follows: treatment 1, no NaCl and STPP added and stored for 7 d; treatment 2, NaCl+STPP added on 0 d and stored for 7 d; treatment 3, NaCl added on 0 d and STPP added on 7 d; and treatment 4, stored for 7 d and NaCl+STPP added. All samples were cooked at a fast (5.67°C/min) or slow cooking rate (2.16°C/min). Regardless of the timing of NaCl and STPP addition, reflectance ratios of nitrosyl hemochrome, cooking yield, pH values, oxidation-reduction potential, and percent myoglobin denaturation were similar (p>0.05) across treatments 2, 3, and 4. The highest CIE a* values were observed in treatment 4 (p<0.05), while treatment 2 was effective in reducing the redness in cooked chicken products. The fast cooking rate resulted in lower CIE a* values and higher CIE L* values and cooking yield in cooked chicken breasts compared to the slow cooking rate. Our results indicate that adding NaCl and STPP to meat, followed by storing and cooking at a fast rate, may result in inhibiting the pink color defect sporadically occurred in cooked ground chicken breasts.

Dual inhibition of DNA-PK and DNA polymerase theta overcomes radiation resistance induced by p53 deficiency.

TP53 deficiency in cancer is associated with poor patient outcomes and resistance to DNA damaging therapies. However, the mechanisms underlying treatment resistance in p53-deficient cells remain poorly characterized. Using live cell imaging of DNA double-strand breaks (DSBs) and cell cycle state transitions, we show that p53-deficient cells exhibit accelerated repair of radiomimetic-induced DSBs arising in S phase. Low-dose DNA-dependent protein kinase (DNA-PK) inhibition increases the S-phase DSB burden in p53-deficient cells, resulting in elevated rates of mitotic catastrophe. However, a subset of p53-deficient cells exhibits intrinsic resistance to radiomimetic-induced DSBs despite DNA-PK inhibition. We show that p53-deficient cells under DNA-PK inhibition utilize DNA polymerase theta (Pol θ)-mediated end joining repair to promote their viability in response to therapy-induced DSBs. Pol θ inhibition selectively increases S-phase DSB burden after radiomimetic therapy and promotes prolonged G2 arrest. Dual inhibition of DNA-PK and Pol θ restores radiation sensitivity in p53-deficient cells as well as in p53-mutant breast cancer cell lines. Thus, combination targeting of DNA-PK- and Pol θ-dependent end joining repair represents a promising strategy for overcoming resistance to DNA damaging therapies in p53-deficient cancers.

Inhibition of PP2A activity by H2O2 during mitosis disrupts nuclear envelope reassembly and alters nuclear shape.

Many types of cancer cells exhibit abnormal nuclear shapes induced by various molecular changes. However, whether reactive oxygen species (ROS) induce nuclear deformation has not been fully addressed. Here, we show that hydrogen peroxide (H2O2) treatment induced concentration-dependent alterations in nuclear shape that were abolished by pretreatment with the antioxidant N-acetyl-L-cysteine or by catalase overexpression. Interestingly, treatment with H2O2 induced nuclear shape alterations significantly more frequently in mitotic cells than in asynchronous cells, suggesting that H2O2 mainly affects nuclear envelope disassembly and/or reassembly processes. Because protein phosphatase 2 A (PP2A) activity is reported to be involved in nuclear envelope reassembly during mitosis, we investigated the possible involvement of PP2A. Indeed, H2O2 reduced the activity of PP2A, an effect that was mimicked by the PP1 and PP2A inhibitor okadaic acid. Moreover, overexpression of PP2A but not PP1 or PP4 partially rescued H2O2-induced alterations in nuclear shape, indicating that the decrease in PP2A activity induced by H2O2 is specifically involved in the observed nuclear shape alterations. We further show that treatment of mitotic cells with H2O2 induced the mislocalization of BAF (barrier-to-autointegration factor), a substrate of PP2A, during telophase. This effect was associated with Lamin A/C mislocalization and was rescued by PP2A overexpression. Collectively, our findings suggest that H2O2 preferentially affects mitotic cells through PP2A inhibition, which induces the subsequent mislocalization of BAF and Lamin A/C during nuclear envelope reassembly, leading to the formation of an abnormal nuclear shape.

MED28 Over-Expression Shortens the Cell Cycle and Induces Genomic Instability.

The mammalian mediator complex subunit 28 (MED28) is overexpressed in a variety of cancers and it regulates cell migration/invasion and epithelial-mesenchymal transition. However, transcription factors that increase MED28 expression have not yet been identified. In this study, we performed a luciferase reporter assay to identify and characterize the prospective transcription factors, namely E2F transcription factor 1, nuclear respiratory factor 1, E-26 transforming sequence 1, and CCAAT/enhancer-binding protein ß, which increased MED28 expression. In addition, the release from the arrest at the G1-S or G2-M phase transition after cell cycle synchronization using thymidine or nocodazole, respectively, showed enhanced MED28 expression at the G1-S transition and mitosis. Furthermore, the overexpression of MED28 significantly decreased the duration of interphase and mitosis. Conversely, a knockdown of MED28 using si-RNA increased the duration of interphase and mitosis. Of note, the overexpression of MED28 significantly increased micronucleus and nuclear budding in HeLa cells. In addition, flow cytometry and fluorescence microscopy analyses showed that the overexpression of MED28 significantly increased aneuploid cells. Taken together, these results suggest that MED28 expression is increased by oncogenic transcription factors and its overexpression disturbs the cell cycle, which results in genomic instability and aneuploidy.

Effect of NaCl Concentration and Cooking Temperature on the Color and Pigment Characteristics of Presalted Ground Chicken Breasts.

This study was conducted to determine the effects of NaCl concentration and cooking temperature on the color and pigment characteristics of presalted ground chicken breasts. Four treatments with different salt concentrations (0%, 1%, 2%, and 3%) were prepared and stored for 7 d prior to cooking. Each sample was cooked to four endpoint temperatures (70°C, 75°C, 80°C, and 85°C). The salt concentration affected the color and pigment properties of the cooked ground chicken breasts. As the salt concentration increased, the cooking yield and residual nitrite content also increased. However, the samples with 1%, 2%, and 3% NaCl showed similar nitrosyl hemochrome and total pigment contents. Among the products containing salt, the samples with 3% NaCl showed the lowest percentage myoglobin denaturation (PMD) and the lowest CIE a* values. The cooking temperature had limited effects on the pigment properties of cooked ground chicken breasts. The oxidation-reduction potential and residual nitrite contents increased with cooking temperature, while the PMD, nitrosyl hemochrome, total pigment contents and CIE a* values were similar in the samples cooked at different temperatures. These results indicated that the addition of up to 2% salt to ground chicken breasts and storage for 7 d could cause the pink color defect of cooked products. However, the addition of 3% NaCl could reduce the redness of the cooked products.

ATP depletion during mitotic arrest induces mitotic slippage and APC/CCdh1-dependent cyclin B1 degradation.

ATP depletion inhibits cell cycle progression, especially during the G1 phase and the G2 to M transition. However, the effect of ATP depletion on mitotic progression remains unclear. We observed that the reduction of ATP after prometaphase by simultaneous treatment with 2-deoxyglucose and NaN3 did not arrest mitotic progression. Interestingly, ATP depletion during nocodazole-induced prometaphase arrest resulted in mitotic slippage, as indicated by a reduction in mitotic cells, APC/C-dependent degradation of cyclin B1, increased cell attachment, and increased nuclear membrane reassembly. Additionally, cells successfully progressed through the cell cycle after mitotic slippage, as indicated by EdU incorporation and time-lapse imaging. Although degradation of cyclin B during normal mitotic progression is primarily regulated by APC/CCdc20, we observed an unexpected decrease in Cdc20 prior to degradation of cyclin B during mitotic slippage. This decrease in Cdc20 was followed by a change in the binding partner preference of APC/C from Cdc20 to Cdh1; consequently, APC/CCdh1, but not APC/CCdc20, facilitated cyclin B degradation following ATP depletion. Pulse-chase analysis revealed that ATP depletion significantly abrogated global translation, including the translation of Cdc20 and Cdh1. Additionally, the half-life of Cdh1 was much longer than that of Cdc20. These data suggest that ATP depletion during mitotic arrest induces mitotic slippage facilitated by APC/CCdh1-dependent cyclin B degradation, which follows a decrease in Cdc20 resulting from reduced global translation and the differences in the half-lives of the Cdc20 and Cdh1 proteins.

Effects of Calcium Powder Mixtures and Binding Ingredients as Substitutes for Synthetic Phosphate on the Quality Properties of Ground Pork Products.

This study aimed to investigate the combined effect of using natural calcium mixtures and various binding ingredients as replacers for synthetic phosphate in ground pork products. We performed seven treatments: control (0.3% phosphate blend), treatment 1 (0.5% natural calcium mixtures [NCM, which comprised 0.2% oyster shell calcium and 0.3% egg shell calcium powder] and 0.25% egg white powder), treatment 2 (0.5% NCM and 0.25% whey protein concentrate), treatment 3 (0.5% NCM and 0.25% concentrated soybean protein), treatment 4 (0.5% NCM and 0.25% isolated soybean protein), treatment 5 (0.5% NCM and 0.25% carrageenan), and treatment 6 (0.5% NCM and 0.25% collagen powder). All the treatment mixtures had higher pH and lower cooking loss than the control, which was treated with phosphate. We found that NCM and binding ingredients had no negative effects on the moisture content, lightness, and yellowness of the cooked ground pork products. Treatments 3 and 4 showed significantly lower CIE a* values than the control. Treatments 2 and 6 improved the textural properties of the products. In conclusion, the combination of NCM with whey protein concentrate or collagen powder could be suitable for producing phosphate-free meat products.

Egg Shell and Oyster Shell Powder as Alternatives for Synthetic Phosphate: Effects on the Quality of Cooked Ground Pork Products.

This study aimed to determine the optimal ratio of natural calcium powders (oyster shell and egg shell calcium) as synthetic phosphate replacers in pork products. Ground pork samples were subjected to six treatments, as follows: control (-) (no phosphate added), control (+) (0.3% phosphate blend added), treatment 1 (0.5% oyster shell calcium powder added), treatment 2 (0.3% oyster shell calcium powder and 0.2% egg shell calcium powder added), treatment 3 (0.2% oyster shell calcium powder and 0.3% egg shell calcium powder added), and treatment 4 (0.5% egg shell calcium powder added). The addition of natural calcium powders resulted in an increase in the pH values of meat products, regardless of whether they were used individually or mixed. The highest cooking loss was observed (p<0.05) in the negative control samples, whereas the cooking loss in samples with natural calcium powder added was similar (p>0.05) to that in the positive control samples. CIE L* values decreased as the amount of added egg shell calcium powder increased. CIE a* values were higher (p<0.05) in samples containing natural calcium powder (treatments 1, 2, 3, and 4) than in the positive control. The combination of oyster shell calcium powder and egg shell powder (treatment 2 or 3) was effective for the improvement of textural properties of the pork products. The findings show that the combined use of 0.2% oyster shell calcium and 0.3% egg shell calcium should enable the replacement of synthetic phosphate in the production of cooked pork products with desirable qualities.

Effects of Various Calcium Powders as Replacers for Synthetic Phosphate on the Quality Properties of Ground Pork Meat Products.

The aim of this study was to identify the optimal and superior type of natural calcium for replacing phosphate in cooked ground pork products. To achieve this, 0.5% eggshell calcium (ESC), oyster shell calcium (OSC), marine algae calcium (MAC), or milk calcium (MC) was added to ground pork meat products. The effect of this substitution was studied by comparing the substituted products with products containing 0.3% phosphate blend (control). ESC was considered an ideal phosphate replacer for minimizing the cooking loss, which likely resulted from the increase in the pH of the product. Among the other natural calcium types, OSC treatment did not cause a significant increase in pH, but it lowered the cooking loss. CIE L* values were higher (p<0.05) in products treated with OSC or MC than the control, and lowest (p<0.05) in the products with ESC. However, products with ESC had higher (p<0.05) CIE a* and CIE b* values than the control and products treated with other powders. Compared to the control, products treated with ESC and OSC had similar substitution effects on the textural properties of the products. Therefore, the results of this study suggested that the combined use of ESC and OSC could be a potentially effective method for replacing synthetic phosphate in ground pork products.

DNA double-strand breaks and Aurora B mislocalization induced by exposure of early mitotic cells to H2O2 appear to increase chromatin bridges and resultant cytokinesis failure.

Aneuploidy, an abnormal number of chromosomes that is a hallmark of cancer cells, can arise from tetraploid/binucleated cells through a failure of cytokinesis. Reactive oxygen species (ROS) have been implicated in various diseases, including cancer. However, the nature and role of ROS in cytokinesis progression and related mechanisms has not been clearly elucidated. Here, using time-lapse analysis of asynchronously growing cells and immunocytochemical analyses of synchronized cells, we found that hydrogen peroxide (H2O2) treatment at early mitosis (primarily prometaphase) significantly induced cytokinesis failure. Cytokinesis failure and the resultant formation of binucleated cells containing nucleoplasmic bridges (NPBs) seemed to be caused by increases in DNA double-strand breaks (DSBs) and subsequent unresolved chromatin bridges. We further found that H2O2 induced mislocalization of Aurora B during mitosis. All of these effects were attenuated by pretreatment with N-acetyl-L-cysteine (NAC) or overexpression of Catalase. Surprisingly, the PARP inhibitor PJ34 also reduced H2O2-induced Aurora B mislocalization and binucleated cell formation. Results of parallel experiments with etoposide, a topoisomerase IIα inhibitor that triggers DNA DSBs, suggested that both DNA DSBs and Aurora B mislocalization contribute to chromatin bridge formation. Aurora B mislocalization also appeared to weaken the "abscission checkpoint". Finally, we showed that KRAS-induced binucleated cell formation appeared to be also H2O2-dependent. In conclusion, we propose that a ROS, mainly H2O2 increases binucleation through unresolved chromatin bridges caused by DNA damage and mislocalization of Aurora B, the latter of which appears to augment the effect of DNA damage on chromatin bridge formation.

A putative pH-dependent nuclear localization signal in the juxtamembrane region of c-Met.

The C-terminal fragment of the c-Met receptor tyrosine kinase is present in the nuclei of some cells irrespective of ligand stimulation, but the responsible nuclear localization signal (NLS) has not been previously reported. Here, we report that two histidine residues separated by a 10-amino-acid spacer (H1068-H1079) located in the juxtamembrane region of c-Met function as a putative novel NLS. Deletion of these sequences significantly abolished the nuclear translocation of c-Met, as did substitution of the histidines with alanines. This substitution also decreased the association of c-Met fragment with importin ß. The putative NLS of c-Met is unique in that it relies on histidines, whose positive charge changes depending on pH, rather than the lysines or arginines, commonly found in classical bipartite NLSs, suggesting the possible 'pH-dependency' of this NLS. Indeed, decreasing the cytosolic pH either with nigericin, an Na(+)/H(+) exchanger or pH 6.5 KRB buffer significantly increased the level of nuclear c-Met and the interaction of the c-Met fragment with importin ß, indicating that low pH itself enhanced nuclear translocation. Consistent with this, nigericin treatment also increased the nuclear level of endogenous c-Met in HeLa cells. The putative aberrant bipartite NLS of c-Met seems to be the first example of what we call a 'pH-dependent' NLS.