Randomized Terminal Linker-dependent PCR: A Versatile and Sensitive Method for Detection of DNA Damage

Objective To design and develop a novel, sensitive and versatile method for in vivo foot printing and studies of DNA damage, such as DNA adducts and strand breaks. Methods Starting with mammalian genomic DNA, single-stranded products were made by repeated primer extension, these products were ligated to a double-stranded linker having a randomized 3 overhang, and used for PCR. DNA breaks in p53 gene produced by restriction endonuclease AfaI were detected by using this new method followed by Southern hybridization with DIG-labeled probe. Results This randomized terminal linker-dependent PCR (RDPCR) method could generate band signals many-fold stronger than conventional ligation-mediated PCR (LMPCR), and it was more rapid, convenient and accurate than the terminal transferase-dependent PCR (TDPCR). Conclusion DNA strand breakage can be detected sensitively in the gene level by RDPCR. Any lesion that blocks primer extension should be detectable.

A mathematical model of a P53 oscillation network triggered by DNA damage

Taking the interaction between a DNA damage repair module, an ATM module, and a P53--MDM2 oscillation module into account, this paper presents a mathematical model of a P53 oscillation network triggered by a DNA damage signal in individual cells. The effects of the DNA damage signal and the delay time of P53-induced MDM2 expression on the behaviours of the P53 oscillation network are studied. In the oscillatory state of the P53--MDM2 oscillator, it is found that the pulse number of P53--P oscillation increases with the increase of the initial DNA damage signal, whereas the amplitude and the period of P53--P oscillation are fixed for different initial DNA damage signals, and the period numbers of P53--P oscillations decrease with the increase of time delay of MDM2 expression induced by P53. These theoretical predictions are consistent with previous experimental results. The combined negative feedback of P53--MDM2 with the time delay of P53-induced MDM2 expression causes oscillation behaviour in the P53 network.

Low-Dose Gamma Radiation Fields Decrease Cell Viability, Damage DNA, and Increase the Expression of Hsp70 and p53 Proteins in Human Leukocytes

Ionizing radiations are tools in diagnosis and treatment of diseases. Leukopenia from exposure to ionizing radiation has been reported. Due to their radiosensitivity, leukocytes are a biological model to analyze cell damage. Therefore, cell viability, DNA damage, and Hsp70 and p53 expression in human leukocytes exposed to low-dose gamma radiation fields from a 137Cs source were evaluated. A decrease in cell viability, DNA damage and an increase in the expression of Hsp70 and p53 proportional to the radiation dose received was found, which was 0.2, 0.4, 0.6, 0.8 and 1.0 mGy.

利用GFP示踪细胞内源性P53活性检测DNA损伤

ＤＮＡ损伤的检测对预防癌症和遗传病等非常重要。采用分子克隆技术，将报告基因—绿色荧光蛋白（ＧＦＰ）置于ＳＶ４０基本启动子调控下，构建成对照载体ｐＳＶ－ＧＦＰ。在ＳＶ４０基本启动子上游插入寡核苷酸Ｐ５３ＲＥ，构建成示踪载体ｐ５３ＲＥ－ＧＦＰ。转染ＮＩＨ３Ｔ３细胞，以ＧＦＰ示踪细胞内源性Ｐ５３的转录激活活性。紫外线照射或Ｈ２Ｏ２处理转化细胞使ＤＮＡ损伤，诱导细胞内源性Ｐ５３的表达。用激光扫描共聚焦成像系统（ＬＳＣＩＳ）对细胞进行红、绿、蓝三色光融合成像，并测定ＧＦＰ经４８８ｎｍ激发后发出的绿色荧光光密度，验证ＧＦＰ示踪Ｐ５３的特异性。ｐ５３ＲＥ－ＧＦＰ转化细胞３Ｔ３－ＲＥＧ经紫外线照射或Ｈ２Ｏ２处理后，ＧＦＰ的表达增高，处理后１ｈｒ光密度即达到最高水平，随后逐渐降低。血清“饥饿”—非ＤＮＡ损伤处理的３Ｔ３－ＲＥＧ细胞，以及经紫外和Ｈ２Ｏ２处理的对照载体ｐＳＶ－ＧＦＰ转化细胞３Ｔ３－ＳＶＧ，ＧＦＰ的表达无明显增强。实验表明：ＧＦＰ示踪内源性Ｐ５３转录激活活性用于检测ＤＮＡ损伤有很高的灵敏度和特异性，适宜推广应用。

Functional analysis of the acetylation of human p53 in DNA damage responses

As a critical tumor suppressor, p53 is inactivated in human cancer cells by somatic gene mutation or disruption of pathways required for its activation. Therefore, it is critical to elucidate the mechanism underlying p53 activation after genotoxic and cellular stresses. Accumulating evidence has indicated the importance of posttranslational modifications such as acetylation in regulating p53 stability and activity. However, the physiological roles of the eight identified acetylation events in regulating p53 responses remain to be fully understood. By employing homologous recombination, we introduced various combinations of missense mutations （lysine to arginine） into eight acetylation sites of the endogenous p53 gene in human embryonic stem cells （hESCs）. By determining the p53 responses to DNA damage in the p53 knock-in mutant hESCs and their derivatives, we demonstrate physiological importance of the acetylation events within the core domain （Kt20 and K164） and at the C-terminus （K370/372/373/381/382/ 386） in regulating human p53 responses to DNA damage.

Activation of DNA Damage Signaling Components by Diagnostic Computed Tomography (CT) Scans Detected in Patient Samples Using an Electrochemiluminescence-Based Assay Platform

Technologies that measure activation of components of the DNA damage response (DDR) have applications in exposure assessment and personalized medicine. The DDR and associated DNA repair pathways encompass hundreds of proteins, making detailed measurement of activation technically challenging and laborious. The purpose of our study was to develop protein-specific assays for certain DDR components on a high-throughput electrochemiluminescence (ECL)-based platform. We developed five working assay pairs for ataxia telangiectasia mutated (ATM), checkpoint kinase 2 (CHK2), phosphorylated-ATM S1981, phosphorylated-CHK2 T68 and phosphorylated-tumor protein p53 (p53) S15. We validated the ECL results against traditional immunoblot and γ-H2AX foci measures in cell and cancer models. In an effort to test the ECL-based technology in a clinical setting, we utilized peripheral blood mononuclear cells (PBMCs) from patients undergoing computed tomography (CT) scans. CT scans represent both a valuable medical imaging diagnostic and a controlled environmental exposure to ionizing radiation for research studies, as they deliver ~2 to 31 millisieverts (mSv) and are known to activate DDR components. In this study, we show that ECL-based technology can measure the basal and damage-induced levels of DDR components in patient PBMC samples. Using a blinded study design and patient matched pre- and post CT scan samples, we show that ECL-derived data can consistently (94% of the time, 15/16 patients) identify PBMCs that have been exposed to low dose ionizing radiation associated with CT scans. Ultimately, the results of our pilot clinical study support the idea that ECL-based technology is applicable for use in clinical and population cohorts that study components of the DDR.

The CXXC finger 5 protein is required for DNA damage-induced p53 activation

The tumor suppressor p53 is a critical component of the DNA damage response pathway that induces a set of genes responsible for cell cycle arrest,senescence,apoptosis,and DNA repair.The ataxia te-langiectasia mutated protein kinase(ATM) responds to DNA-damage stimuli and signals p53 stabiliza-tion and activation,thereby facilitating transactivation of p53 inducible genes and maintainence of genome integrity.In this study,we identified a CXXC zinc finger domain containing protein termed CF5 as a critical component in the DNA damage signaling pathway.CF5 induces p53 transcriptional activity and apoptosis in cells expressing wild type p53 but not in p53-deficient cells.Knockdown of CF5 in-hibits DNA damage-induced p53 activation as well as cell cycle arrest.Furthermore,CF5 physically interacts with ATM and is required for DNA damage-induced ATM phosphorylation but not its recruitment to chromatin.These findings suggest that CF5 plays a crucial role in ATM-p53 signaling in response to DNA damage.

Transcriptomic Landscape Analysis Reveals a Persistent DNA Damage Response in Metabolic Dysfunction-associated Steatohepatitis Post-dietary Intervention

Background and Aims:Metabolic dysfunction-associated steatotic liver disease(MASLD)and its more advanced form,metabolic dysfunction-associated steatohepatitis,have emerged as the most prevalent liver diseases worldwide.Currently,lifestyle modification is the foremost guidelinerecommended management strategy for MASLD.However,it remains unclear which detrimental signals persist in MASLD even after disease remission.Thus,we aimed to examine the persistent changes in liver transcriptomic profiles following this reversal.Methods:Male C57BL/6J mice were divided into three groups:Western diet(WD)feeding,chow diet(CD)feeding,or diet reversal from WD to CD.After 16 weeks of feeding,RNA sequencing was performed on the mice’s livers to identify persistent alterations characteristic of MASLD.Additionally,RNA sequencing databases containing high-fat diet-fed P53-knockout mice and human MASLD samples were utilized.Results:WD-induced MASLD triggered persistent activation of the DNA damage response(DDR)and its primary transcription factor,P53,long after the resolution of the hepatic phenotype through dietary reversal.Elevated levels of P53 might promote apoptosis,thereby exacerbating metabolic dysfunction-associated steatohepatitis,as they strongly correlated with hepatocyte ballooning,an indicator of apoptosis activation.Moreover,P53 knockout in mice led to downregulated expression of apoptosis signaling in the liver.Mechanistically,P53 may regulate apoptosis by transcriptionally activating the expression of apoptosis-enhancing nuclease(AEN).Consistently,P53,AEN,and the apoptosis process all exhibited persistently elevated expression and showed a strong inter-correlation in the liver following dietary reversal.Conclusions:The liver demonstrated upregulation of DDR signaling and the P53-AEN-apoptosis axis both during and after exposure to WD.Our findings provide new insights into the mechanisms of MASLD relapse,highlighting DDR signaling as a promising target to prevent MASLD recurrence.

Role of p53 in Anticancer Drug Treatment- and Radiation-Induced Injury in Normal Small Intestine

In the human gastrointestinal tract,the functional mucosa of the small intestine has the highest capacity for absorption of nutrients and rapid proliferation rates,making it vulnerable to chemoradiotherapy.Recent understanding of the protective role of p53- mediated cell cycle arrest in the small intestinal mucosa has led researchers to explore new avenues to mitigate mucosal injury during cancer treatment.A traditional p53 inhibitor and two other molecules that exhibit strong protective effects on normal small intestinal epithelium during anticancer drug treatment and radiation therapy are introduced in this work.The objective of this review was to update current knowledge regarding potential mechanisms and targets that inhibit the side effects induced by chemoradiotherapy.

Drugging in the absence of p53

Inactivation of the p53 gene is a key driver of tumorigenesis in various cancer cohorts and types.The quest for a successful p53-based therapy that holds the promise of treating more than half of the cancer population has culminated in extensive knowledge about the role and function of p53 and led to new proposed innovative strategies against p53-defective cancers.We will discuss some of these latest studies with a focus on metabolic regulation and DNA damage response and also highlight novel functions of p53 in these pathways that may provide a contemporary rationale for targeting p53 loss in tumors.

Inhibition of p53 and/or AKT as a new therapeutic approach specifically targeting ALT cancers

While the majority of all human cancers court teract telomere shortening by expressing telomerase,-15%of all cancers maintain telomere length by a telomerase?independent mechanism known as alternative lengthening of telomeres(ALT).Here,we show that high load of intrinsic DNA damage is present in ALT cancer cells,leading to apoptosis stress by activating p53-independent,but JNK/c-IVIyc-dependent apoptotic pathway.Notably,ALT cells expressing wild-type p53 show much lower apoptosis than p53-deficient ALT cells.Mechanistically,we find that intrinsic DNA damage in ALT cells induces low level of p53 that is insufficient to initiate the transcription of apoptosis-related genes,but is sufficient to stimulate the expression of key components of mTORC2(mTOR and Rictor),which in turn leads to phosphorylation of AKT.Activated AKT(p-AKT)thereby stimulates downstream anti-apoptotic events.Therefore,p53 and AKT are the key factors that suppress sponta?neous apoptosis in ALT cells.Indeed,inhibition of p53 or AKT selectively induces rapid death of ALT cells in vitro,and p53 inhibitor severely suppresses the growth of ALT-cell xenograft tumors in mice.These findings reveal a previously unrecognized function of p53 in antiapoptosis and identify that the inhibition of p53 or AKT has a potential as therapeutics for specifically targeting ALT cancers.

细胞对DNA损伤的反应

细胞在其ＤＮＡ受损时，利用“关卡控制”阻断细胞周期的进行，以便于对受损的ＤＮＡ进行修复，避免了突变的产生．此外，在多细胞生物中，ＤＮＡ的严重损伤还会诱发细胞凋亡，以个别ＤＮＡ受损细胞的死亡为代价保护机体遗传信息的稳定性．在上述过程中，两种人类抑癌基因———Ｐ５３和ＡＴＭ基因的产物发挥着重要作用．

Human umbilical cord mesenchymal stem cells attenuate diabetic nephropathy through the IGF1R-CHK2-p53 signalling axis in male rats with type 2 diabetes mellitus

diabetes mellitus(DM)is a disease syndrome characterized by chronic hyperglycaemia.A long-term high-glucose environment leads to reactive oxygen species(ROS)production and nuclear DNA damage.human umbilical cord mesenchymal stem cell(HUcMSC)infusion induces significant antidiabetic effects in type 2 diabetes mellitus(T2DM)rats.Insulin-like growth factor 1(IGF1)receptor(IGF1R)is important in promoting glucose metabolism in diabetes;however,the mechanism by which HUcMSC can treat diabetes through IGF1R and DNA damage repair remains unclear.In this study,a DM rat model was induced with high-fat diet feeding and streptozotocin(STZ)administration and rats were infused four times with HUcMSC.Blood glucose,interleukin-6(IL-6),IL-10,glomerular basement membrane,and renal function were examined.Proteins that interacted with IGF1R were determined through coimmunoprecipitation assays.The expression of IGF1R,phosphorylated checkpoint kinase 2(p-CHK2),and phosphorylated protein 53(p-p53)was examined using immunohistochemistry(IHC)and western blot analysis.Enzyme-linked immunosorbent assay(ELISA)was used to determine the serum levels of 8-hydroxydeoxyguanosine(8-OHdG).Flow cytometry experiments were used to detect the surface markers of HUcMSC.The identification of the morphology and phenotype of HUcMSC was performed by way of oil red“O”staining and Alizarin red staining.DM rats exhibited abnormal blood glucose and IL-6/10 levels and renal function changes in the glomerular basement membrane,increased the expression of IGF1 and IGF1R.IGF1R interacted with CHK2,and the expression of p-CHK2 was significantly decreased in IGF1R-knockdown cells.When cisplatin was used to induce DNA damage,the expression of p-CHK2 was higher than that in the IGF1R-knockdown group without cisplatin treatment.HUcMSC infusion ameliorated abnormalities and preserved kidney structure and function in DM rats.The expression of IGF1,IGF1R,p-CHK2,and p-p53,and the level of 8-OHdG in the DM group increased significantly compared with those in the control group,and decreased after HUcMSC treatment.Our results suggested that IGF1R could interact with CHK2 and mediate DNA damage.HUcMSC infusion protected against kidney injury in DM rats.The underlying mechanisms may include HUcMSC-mediated enhancement of diabetes treatment via the IGF1R-CHK2-p53 signalling pathway.

DNA Damage Response in Hematopoietic Stem Cell Ageing

Maintenance of tissue-specific stem cells is vital for organ homeostasis and organismal longevity.Hematopoietic stem cells（HSCs） are the most primitive cell type in the hematopoietic system.They divide asymmetrically and give rise to daughter cells with HSC identity（selfrenewal） and progenitor progenies（differentiation）,which further proliferate and differentiate into full hematopoietic lineages.Mammalian ageing process is accompanied with abnormalities in the HSC self-renewal and differentiation.Transcriptional changes and epigenetic modulations have been implicated as the key regulators in HSC ageing process.The DNA damage response（DDR）in the cells involves an orchestrated signaling pathway,consisting of cell cycle regulation,cell death and senescence,transcriptional regulation,as well as chromatin remodeling.Recent studies employing DNA repair-deficient mouse models indicate that DDR could intrinsically and extrinsically regulate HSC maintenance and play important roles in tissue homeostasis of the hematopoietic system.In this review,we summarize the current understanding of how the DDR determines the HSC fates and finally contributes to organismal ageing.

Senescence in adipose-derived stem cells and its implications in nerve regeneration

Adult mesenchymal stem cells, specifically adipose-derived stem cells have self-renewal and multiple differentiation potentials and have shown to be the ideal candidate for therapeutic applications in regenerative medicine, particularly in peripheral nerve regeneration. Adipose-de- rived stem cells are easily harvested, although they may show the effects of aging, hence their potential in nerve repair may be limited by cellular senescence or donor age. Cellular senescence is a complex process whereby stem cells grow old as consequence of intrinsic events （e.g., DNA damage） or environmental cues （e.g., stressful stimuli or diseases）, which determine a permanent growth arrest. Several mechanisms are implicated in stem cell senescence, although no one is exclusive of the others. In this review we report some of the most important factors modulating the senescence process, which can influence adipose-derived stem cell morphology and function, and compromise their clinical application for peripheral nerve regenerative cell therapy.

Genotoxic Effects of PAH Containing Sludge Extracts in Chinese Hamster Ovary Cell Cultures

Objective Many studies have been conducted in order to evaluate the genotoxicity of chemicals and waste materials, which utilized in vivo test protocols. The use of animals for routine toxicity testing is now questioned by a growing segment of society. Methods Keeping the above fact in mind, we have conducted in the present study the genotoxicity evaluation of oily sludge samples generated from a petroleum refinery and petrochemical industry and ETP sludge from petroleum refinery using DNA damage, chromosomal aberration, p53 protein induction and apoptosis in short term in vitro mammalian Chinese Hamster Ovary cell cultures. Results It is evident from the results that the oily sludge compounds derived from petroleum refinery and petrochemical industry could cause DNA damage, chromosomal aberration, p53 protein accumulation and apoptotic cell death on exposure to oily sludge extracts in the presence of metabolic activation system (S-9 mix), however, ETP sludge extract could not cause significant genotoxicity in comparison to oily sludge extract and negative control. Conclusion The effect may be attributed to polycyclic aromatic hydrocarbons present in the samples as evidenced from GC-MS.

Cellular responding kinetics based on a model of gene regulatory networks under radiotherapy

Radiotherapy can cause DNA damage into cells, triggering the cell cycle arrest and cell apop-tosis through complicated interactions among vital genes and their signal pathways. In order to in-depth study the complicated cellular res- ponses under such a circumstance, a novel mo- del for P53 stress response networks is pro- posed. It can be successfully used to simulate the dynamic processes of DNA damage trans-ferring, ATM and ARF activation, regulations of P53-MDM2 feedback loop, as well as the toxins degradation. Particularly, it has become feasible to predict the outcomes of cellular response in fighting against genome stresses. Consequently, the new model has provided a reasonable framework for analyzing the complicated regu-lations of P53 stress response networks, as well as investigating the mechanisms of the cellular self-defense under radiotherapy.

Nucleo-cytoplasmic communication in apoptotic response to genotoxic and inflammatorystress

Genotoxic agents or inflammatory cytokines activate cellular stress responses and trigger programmed cell death. We have identified a signal transduction module, including three nuclear proteins that participate in the regulation of cell death induced by chemotherapeutic agents and tumor necrosis factor (TNF). In this nuclear signaling module, retino- blastoma protein (Rb) functions as an inhibitor of apoptotic signal transduction. Inactivation of Rb by phosphorylation or caspase-dependent cleavage/degradation is required for cell death to occur. Rb inhibits the Abl tyrosine kinase. Thus, Rb inactivation is a pre-requisite for Abl activation by DNA damage or TNF. Activation of nuclear Abl and its down- stream effector p73 induces mitochondriadependent cell death. The involvement of these nuclear signal transducers in TNF induced apoptosis, which does not require new gene expression, indicates that nuclear events other than transcrip- tion can contribute to extrinsic apoptotic signal transduction.

Modeling of DSBs Generation and Repair Process under Ion Radiation

Under acute perturbations from outside, cell can trigger the self-defense mechanisms in fighting against these genome stresses. To simulate the investigation of the complicated mechanisms of cellular responding DNA damage at single cell level, a model of the double strand breaks (DSBs) generation and repair process is proposed under continuous effect of acute IR. Under different IR dose domains, this model can be used to simulate the complicated interactions among vital components within the cell, and the plausible outcomes of cellular response in fighting against DNA damage.

Measurement of Protein 53 Diffusion Coefficient in Live HeLa Cells Using Raster Image Correlation Spectroscopy (RICS)

We have applied Raster Image Correlation Spectroscopy (RICS) technique to characterize the dynamics of protein 53 (p53) in living cells before and after the treatment with DNA damaging agents. HeLa cells expressing Green Fluores-cent Protein (GFP) tagged p53 were incubated with and without DNA damaging agents, cisplatin or eptoposide, which are widely used as chemotherapeutic drugs. Then, the diffusion coefficient of GFP-p53 was determined by RICS and it was significantly reduced after the drug treatment while that of the one without drug treatment was not. It is suggested that the drugs induced the interaction of p53 with either other proteins or DNA. Together, our results demonstrated that RICS is able to detect the protein dynamics which may be associated with protein-protein or protein-DNA interactions in living cells and it may be useful for the drug screening.