Alpha‐phellandrene‐induced DNA damage and affect DNA repair protein expression in WEHI‐3 murine leukemia cells in vitro

Although there are few reports regarding α‐phellandrene (α‐PA), a natural compound from Schinus molle L. essential oil, there is no report to show that α‐PA induced DNA damage and affected DNA repair associated protein expression. Herein, we investigated the effects of α‐PA on DNA damage and repair associated protein expression in murine leukemia cells. Flow cytometric assay was used to measure the effects of α‐PA on total cell viability and the results indicated that α‐PA induced cell death. Comet assay and 4,6‐diamidino‐2‐phenylindole dihydrochloride staining were used for measuring DNA damage and condensation, respectively, and the results indicated that α‐PA induced DNA damage and condensation in a concentration‐dependent manner. DNA gel electrophoresis was used to examine the DNA damage and the results showed that α‐PA induced DNA damage in WEHI‐3 cells. Western blotting assay was used to measure the changes of DNA damage and repair associated protein expression and the results indicated that α‐PA increased p‐p53, p‐H2A.X, 14‐3‐3‐σ, and MDC1 protein expression but inhibited the protein of p53, MGMT, DNA‐PK, and BRCA‐1. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1322–1330, 2015.     

Cyclin-dependent kinases (cdks) and the DNA damage response: rationale for cdk inhibitor–chemotherapy combinations as an anticancer strategy for solid tumors

Importance of the field: The eukaryotic cell division cycle is a tightly regulated series of events coordinated by the periodic activation of multiple cyclin-dependent kinases (cdks). Small-molecule cdk-inhibitory compounds have demonstrated preclinical synergism with DNA-damaging agents in solid tumor models. An improved understanding of how cdks regulate the DNA damage response now provides an opportunity for optimization of combinations of cdk inhibitors and DNA damaging chemotherapy agents that can be translated to clinical settings.

Areas covered in this review: Here, we discuss novel work uncovering multiple roles for cdks in the DNA-damage-response network. First, they activate DNA damage checkpoint and repair pathways. Later their activity is turned off, resulting in cell cycle arrest, allowing time for DNA repair to occur. Recent clinical data on cdk inhibitor–DNA-damaging agent combinations are also discussed.

What the reader will gain: Readers will learn about novel areas of cdk biology, the complexity of DNA damage signaling networks and clinical implications.

Take home message: New data demonstrate that cdks are ‘master’ regulators of DNA damage checkpoint and repair pathways. Cdk inhibition may therefore provide a means of potentiating the clinical activity of DNA-damaging chemotherapeutic agents for the treatment of cancer.     

Determination of UV-induced DNA damages to suppress protein expression using reporter gene assay-based single cell cotransfection imaging cytometry

Effects of UVB or UVC-induced DNA damage were simultaneously monitored at single cellular level by analyzing the change of yellow fluorescent protein (YFP) and red fluorescent protein (RFP) expression in human embryonic kidney (HEK) 293 cells using multicolor single-cell imaging cytometry. The method is based on the idea that there exists a quantitative correlation between the degree of UV-induced DNA damage and protein expression. A cotransfection assay was performed using UVB irradiated YFP and UVC irradiated RFP genes to eliminate cell-to-cell variation in protein expression yield. Up to an UVB irradiation dose of 50 kJ/m², YFP expression yield did not change compared to control. On the other hand, RFP expression yield decreased remarkably as the UVC dose increased from 79.5 to 159 J/m². The results showed that a certain level of DNA damage is efficiently repaired by intracellular repair mechanism and does not influence protein mutation. In addition, it was found that the amount of DNA damage induced by UVB in sunlight would not interfere with normal protein expression in the human body. Single-cell imaging cytometry is a cell lysis-free approach to directly monitor the intracellular correlation between the degree of UV-induced DNA damage and protein expression.     

Response to environmental carcinogens in DNA-repair-deficient disorders

An increased mutation rate in human cells is a critical contributing factor for the development of malignancy. Many autosomal recessive genetic disorders are known, in which an increased mutation rate and predisposition for cancer can be attributed to a deficiency in DNA repair or associated processes. Some of these DNA repair deficiencies are manifested at the level of the mitotic chromosome as increased breakage, particularly after treatment with specific mutagens. The examination of the response of cells from patients with these disorders to carcinogens offers the opportunity to elucidate the mechanisms operating in human cells to combat DNA damage and mutation. Over the last few years, the underlying genes in many of these syndromes have been identified, enabling a much more detailed definition of the processes disturbed. This review concentrates on two of the chromosomal breakage syndromes, Fanconi anaemia and Nijmegen breakage syndrome, which have both distinct and common cellular features.     

螺旋藻多糖对紫外线诱发的人胚肺二倍体细胞DNA损伤修复的影响

目的：观察螺旋藻多糖（polysaaccharide from Spirulina platensis,PSP)对紫外线诱发的人胚肺二倍体细胞DNA损伤修复的影响。方法：使用单细胞凝胶电泳技术（single cell gel clectrophoresis assay SCGE),检测紫外线（UV）照射30min及照射后30,60,90min各组细胞DNA的损伤情况,以DNA迁移距离（migrate length of DNA,MLD)作为损伤指标。结果PSP能预防UV诱发的DNA损伤,并能促进UV诱发损伤后的DNA修复合成。结论：PSP有增强核酸内切酶和连接酶活性的作用,并且这种作用呈剂量依赖性。     

Effects of methylmercury exposure on neuronal differentiation of mouse and human embryonic stem cells

Sulphur mustard (SM) is a blistering agent that causes debilitating damage to the skin, eyes and respiratory system. In cases of severe exposure, immunodepletion can occur as well as death, due to secondary infections. The toxicity of SM is thought to be mediated in part by the alkylation of nucleic acids and proteins, although the exact mechanisms are not clear. In addition, although the first known use of SM was in military conflict nearly 100 years ago, there are still no effective treatments or preventative measures. In order to develop treatments it is necessary to have a detailed understanding of the cellular biochemical changes induced by SM as well as information on the mechanisms that cells employ to protect against SM toxicity. We have previously demonstrated that the homologous recombination (HR) DNA repair pathway promotes cell survival after SM. This study investigated the role of other DNA repair pathways in the cellular response to SM, specifically base excision repair (BER), nucleotide excision repair (NER) and non-homologous end joining (NHEJ) as well as studying the activation and regulation of DNA damage signalling pathways. Our data confirmed that HR is the major repair pathway protecting against acute SM toxicity, with NER and NHEJ also contributing to cell survival. In addition, this study demonstrated the dose- and time-dependent activation of DNA damage signalling pathways after SM in human TK6 lymphoblastoid cells, in particular the phosphorylation of CHK1, CHK2 and p53. These phosphorylation events were orchestrated by a combination of the ATM and ATR protein kinases.     

Low-LET-induced radioprotective mechanisms within a stochastic two-stage cancer model

A stochastic two-stage cancer model with clonal expansion was used to investigate the potential impact on human lung cancer incidence of some aspects of the hormesis mechanisms suggested by Feinendegen (Health Phys. 52 663-669, 1987). The model was applied to low doses of low-LET radiation delivered at low dose rates. Non-linear responses arise in the model because radiologically induced adaptations in radical scavenging and DNA repair may reduce the biological consequences of DNA damage formed by endogenous processes and ionizing radiation. Sensitivity studies were conducted to identify critical model inputs and to help define the changes in cellular defense mechanisms necessary to produce a lifetime probability for lung cancer that deviates from a linear no-threshold (LNT) type of response. Our studies suggest that lung cancer risk predictions may be very sensitive to the induction of DNA damage by endogenous processes. For doses comparable to background radiation levels, endogenous DNA damage may account for as much as 50 to 80% of the predicted lung cancers. For an additional lifetime dose of 1 Gy from low-LET radiation, endogenous processes may still account for as much as 20% of the predicted cancers (Fig. 2). When both repair and scavengers are considered as inducible, radiation must enhance DNA repair and radical scavenging in excess of 30 to 40% of the baseline values to produce lifetime probabilities for lung cancer outside the range expected for endogenous processes and background radiation.     

Oxidative stress and oxidative damage in chemical carcinogenesis

Reactive oxygen species (ROS) are induced through a variety of endogenous and exogenous sources. Overwhelming of antioxidant and DNA repair mechanisms in the cell by ROS may result in oxidative stress and oxidative damage to the cell. This resulting oxidative stress can damage critical cellular macromolecules and/or modulate gene expression pathways. Cancer induction by chemical and physical agents involves a multi-step process. This process includes multiple molecular and cellular events to transform a normal cell to a malignant neoplastic cell. Oxidative damage resulting from ROS generation can participate in all stages of the cancer process. An association of ROS generation and human cancer induction has been shown. It appears that oxidative stress may both cause as well as modify the cancer process. Recently association between polymorphisms in oxidative DNA repair genes and antioxidant genes (single nucleotide polymorphisms) and human cancer susceptibility has been shown.     

DNA damage response to the Mdm2 inhibitor Nutlin-3

Mdm2 inhibitors represent a promising class of p53 activating compounds that may be useful in cancer treatment and prevention. However, the consequences of pharmacological p53 activation are not entirely clear. We observed that Nutlin-3 triggered a DNA damage response in azoxymethane-induced mouse AJ02-NM₀ colon cancer cells, characterized by the phosphorylation of H2AX (at Ser-139) and p53 (at Ser-15). The DNA damage response was highest in cells showing robust p53 stabilization, it could be triggered by the active but not the inactive Nutlin-3 enantiomer, and it was also activated by another pharmacological Mdm2 inhibitor (Caylin-1). Quantification of γH2AX-positive cells following Nutlin-3 exposure showed that approximately 17% of cells in late S and G2/M were mounting a DNA damage response (compared to a ∼50% response to 5-fluorouracil). Nutlin-3 treatment caused the formation of double-strand DNA strand breaks, promoted the formation of micronuclei, accentuated strand breakage induced by doxorubicin and sensitized the mouse colon cancer cells to DNA break-inducing topoisomerase II inhibitors. Although the HCT116 colon cancer cells did not mount a significant DNA damage response following Nutlin-3 treatment, Nutlin-3 enhanced the DNA damage response to the nucleotide synthesis inhibitor hydroxyurea in a p53-dependent manner. Finally, p21 deletion also sensitized HCT116 cells to the Nutlin-3-induced DNA damage response, suggesting that cell cycle checkpoint abnormalities may promote this response. We propose that p53 activation by Mdm2 inhibitors can result in the slowing of double-stranded DNA repair. Although this effect may suppress illegitimate homologous recombination repair, it may also increase the risk of clastogenic events.     

The protective effects of hydroxytyrosol against ortho-phenylphenol-induced DNA damage in HepG2 cells

Ortho-phenylphenol (OPP) has been found to cause carcinomas in the urinary tract of rats. Since OPP is a potent genotoxic compound, and used as fungicides and antibacterial agents in fruits and fruit products, search for newer, better agents for protection against toxicity of OPP is required. In this study, the chemoprotective effect of hydroxytyrosol (HT) against OPP-induced DNA damage in HepG2 cells was investigated. Comet assay was used to detect the DNA damage induced by OPP. To elucidate the possible mechanisms, we tested lysosomal membrane stability, mitochondrial membrane potential, intracellular generation of reactive oxygen species (ROS), and reduced glutathione (GSH). Results showed that HT significantly reduced the DNA strand breaks caused by OPP. Moreover, HT effectively suppressed OPP-induced ROS formation, and increased the GSH level. Lysosomal membrane and mitochondrial membrane were also protected when cells were pretreated with HT. These results suggested that the disruption of lysosomal membrane integrity and the oxidative stress, leading to DNA fragmentation, may be the mechanism of DNA damage induced by OPP. The antioxidant activity of HT may play an important part in attenuating the DNA damage of OPP.     

Compromised DNA repair enhances sensitivity of the yeast RNR3-lacZ genotoxicity testing system.

The RNR3-lacZ genotoxicity testing system was developed based on the induction of a Saccharomyces cerevisiae RNR3-lacZ reporter gene in response to a broad range of DNA-damaging agents. In order to enhance the sensitivity of the RNR3-lacZ system, several deletion mutant strains representing different repair pathways were created and examined for their effects on RNR3-lacZ expression. It was found that inactivation of different DNA repair pathways has profound effects on the DNA damage induction of RNR3 expression. Although deletion of MAG1 in the base excision repair pathway enhances the detection sensitivity to DNA-alkylating agents, and deletion of RAD2 in the nucleotide excision repair pathway enhances the detection sensitivity to ultraviolet and agents that produce bulky lesions, inactivation of genes involved in the recombination repair and postreplication repair variably reduces RNR3-lacZ induction. This study not only helps to establish a more sensitive genotoxicity testing system but also suggests that certain eukaryotic DNA repair pathways are required for gene regulation in response to DNA damage and probably serve as sensors in the signal transduction cascade.     

Suppression of a DNA base excision repair gene, hOGG1, increases bleomycin sensitivity of human lung cancer cell line

Bleomycin (BLM) has been found to induce 8-oxoguanine and DNA strand breaks through producing oxidative free radicals, thereby leading to cell cycle arrest, apoptosis and cell death. Cellular DNA damage repair mechanisms such as single strand DNA break repair/base excision repair (BER) are responsible for removing bleomycin-induced DNA damage, therefore confer chemotherapeutic resistance to bleomycin. In this study, we have investigated if down-regulation of human 8-oxoguanine DNA glycosylase (hOGG1), an important BER enzyme, could alter cellular sensitivity to bleomycin, thereby reducing chemotherapeutic resistance in human tumor cell. A human lung cancer cell line with hOGG1 deficiency (A549-R) was created by ribozyme gene knockdown technique. Bleomycin cellular sensitivity and DNA/chromosomal damages were examined using MTT, colony forming assay, comet assay as well as micronucleus assay. We demonstrated that hOGG1 gene knockdown enhanced bleomycin cytotoxicity and reduced the ability of colony formation of the lung cancer cell lines. We further demonstrated that bleomycin-induced DNA strand breaks resulted in an increase of micronucleus rate. hOGG1 deficiency significantly reduced DNA damage repair capacity of the lung cancer cell lines. Our results indicated that hOGG1 deficiency allowed the accumulation of bleomycin-induced DNA damage and chromosomal breaks by compromising DNA damage repair capacity, thereby increasing cellular sensitivity to bleomycin.     

Inhibition of nucleotide excision repair (NER) by microcystin-LR in CHO-K1 cells

Microcystin-LR (MC-LR), a potent inhibitor of PP1 and PP2A protein phosphatases, is related to tumor promotion and initiation. Although the genotoxic properties of this toxin have been extensively investigated with a variety of non-mammalian and mammalian test systems, the existing results are contradictory. Based on our previous results regarding the impact of MC-LR on the processes of DNA repair we decided to examine in greater detail its effect on the capacity of nucleotide excision repair (NER). CHO-K1 cells were pre-treated with increasing doses of MC-LR (1, 10 and 20 μg/ml) and then exposed to UV radiation (25 J/m²). Apoptosis was analyzed to exclude the possibility of false positive results in the comet assay. The results suggest that MC-LR targets the nucleotide excision repair mechanisms by interference with the incision/excision phase as well as the rejoining phase of NER and leads to an increased level of UV-induced cytogenetic DNA damage in CHO-K1 cells.     

DNA damage-triggered apoptosis: critical role of DNA repair, double-strand breaks, cell proliferation and signaling

Genotoxic DNA damaging agents may activate both membrane death receptors and the endogenous mitochondrial damage pathway leading to cell death via apoptosis. Here, apoptotic responses in cells exhibiting a defect in various DNA repair pathways such as alkyltransferase, base excision repair, nucleotide excision repair and mismatch repair are reviewed. The HSVTk/ganciclovir and VZV/BVDU suicide system will also be discussed. Data are available to show that critical DNA damage triggers apoptosis in a DNA replication dependent way by activating the mitochondrial damage pathway in fibroblasts. It is proposed that DNA double-strand breaks (DSBs) are common ultimate apoptosis-triggering lesions arising from primary DNA lesions during DNA replication. Thus, DNA replication is a necessary component in DNA damage-triggered apoptosis, at least in fibroblasts treated with genotoxins not inducing DSBs themselves. For methylating agents inducing O(6)-methylguanine, an additional requirement is mismatch repair provoking DSB formation that triggers Bcl-2 decline and caspase-9/-3 activation. This occurs independent of p53 since most of the repair deficient cell lines under study were mutated for p53. Moreover, p53 knockout fibroblasts are more sensitive to methylating agents and UV light than p53 wt cells, suggesting p53 to play a protective rather than a pro-apoptotic role in this cell system, probably by its involvement in DNA repair. However, for lymphoblastoid cells p53 wt variants are more sensitive to DNA damage indicating that p53 participates in apoptotic signaling in a cell type-specific fashion. The role of topoisomerase II inhibitors and c-Fos/AP-1 in apoptosis will also be discussed.     

Non-problematic risks from low-dose radiation-induced DNA damage clusters

Radiation-induced DNA damage clusters have been proposed and are usually considered to pose the threat of serious biological damage. This has been attributed to DNA repair debilitation or cessation arising from the complexity of cluster damage. It will be shown here, contrary to both previous suggestions and perceived wisdom, that radiation induced damage clusters contribute to non-problematic risks in the low-dose, low-LET regime. The very complexity of cluster damage which inhibits and/or compromises DNA repair will ultimately be responsible for the elimination and/or diminution of precancerous and cancerous cells.     

Characterization of DDRI-18 (3,3'-(1H,3'H-5,5'-bibenzo[d]imidazole-2,2'-diyl)dianiline), a novel small molecule inhibitor modulating the DNA damage response

BACKGROUND AND PURPOSE

Recently, the DNA damage response (DDR) has emerged as a promising target for anticancer drug development. In our previous study, we identified several DDR-inhibiting compounds via high-content screening of a small molecule library using γH2AX foci as a biomarker. Here, we studied the effects of the DNA damage response inhibitor DDRI-18 (3,3′-(1H,3′H-5,5′-bibenzo[d]imidazole-2,2′-diyl)dianiline) on DDR.

EXPERIMENTAL APPROACH

Osteosarcoma U2OS cells were treated with etoposide to induce DDR. The nuclear foci of γH2AX and other signalling molecules in DDR were visualized by immunofluorescence and quantified using an IN Cell Analyzer. The DNA repair capacity of cells was analysed using the comet assay and in vivo DNA end-joining assay. Cell survival after drug treatment was quantified using the MTT assay, and apoptotic cell death was analysed by Annexin V staining and flow cytometry.

KEY RESULTS

DDRI-18 inhibited the non-homologous end-joining (NHEJ) DNA repair process and delayed the resolution of DNA damage-related proteins (γH2AX, ATM and BRCA1) from DNA lesions at a later phase of DDR. Furthermore, DDRI-18 enhanced the cytotoxic effects of anticancer DNA-damaging drugs, including etoposide, camptothecin, doxorubicin and bleomycin. This synergistic effect on cell death was shown to be due to caspase-dependent apoptosis.

CONCLUSIONS AND IMPLICATIONS

We identified a chemical compound, DDRI-18, that has chemosensitization activity. Although the target molecule and mechanism of action of DDRI-18 remain unknown, DDRI-18 is an effective chemosensitizing agent and may improve the therapy with classical anticancer drugs.     

Mitochondrial and nuclear DNA damage induced by curcumin in human hepatoma G2 cells.

Curcumin is extensively used as a spice and pigment and has anticarcinogenic effects that could be linked to its antioxidant properties. However, some studies suggest that this natural compound possesses both pro- and antioxidative effects. In this study, we found that curcumin induced DNA damage to both the mitochondrial and nuclear genomes in human hepatoma G2 cells. Using quantitative polymerase chain reaction and immunocytochemistry staining of 8-hydroxydeoxyguanosine, we demonstrated that curcumin induced dose-dependent damage in both the mitochondrial and nuclear genomes and that the mitochondrial damage was more extensive. Nuclear DNA fragments were also evident in comet assays. The mechanism underlies the elevated level of reactive oxygen species and lipid peroxidation generated by curcumin. The lack of DNA damage at low doses suggested that low levels of curcumin does not induce DNA damage and may play an antioxidant role in carcinogenesis. But at high doses, we found that curcumin imposed oxidative stress and damaged DNA. These data reinforce the hypothesis that curcumin plays a conflicting dual role in carcinogenesis. Also, the extensive mitochondrial DNA damage might be an initial event triggering curcumin-induced cell death.     

异佛尔酮二异氰酸酯诱发DNA损伤的研究

对异佛尔酮二异氰酸醋(Isophorone Diisocyanate,IPDI)诱导大肠杆菌SOS反应、人淋巴细胞程序外DNA合成(UDS)和小鼠骨髓细胞DNA合成的影响进行了初步研究。IPDI对大肠杆菌PQ_(35)和PQ_(37)两个菌株均无明显的诱导SOS反应的能力,但IPDI可明显诱发人淋巴细胞UDS,对培养小鼠骨髓细胞DNA的合成具有明显的抑制作用,而且呈剂量-效应关系。IPDI对哺乳动物细胞DNA具有损伤作用。     

Excess ribonucleotide reductase R2 subunits coordinate the S phase checkpoint to facilitate DNA damage repair and recovery from replication stress

Ribonucleotide reductase (RNR), which consists of R1 and R2 subunits, catalyzes a key step of deoxyribonucleoside triphosphate (dNTP) synthesis for DNA replication and repair. The R2 subunit is controlled in a cell cycle-specific manner for timely DNA synthesis and is negatively regulated by p53 in response to DNA damage. Herein we demonstrate that the presence of excess R2 subunits in p53(-/-) HCT-116 human colon cancer cells protects against DNA damage and replication stress. siRNA-mediated stable knockdown (>80%) of excess R2 subunits has no effect on proliferative growth but results in enhanced accumulation of gamma-H2Ax and delayed recovery from DNA lesions inflicted by exposure to cisplatin and Triapine. This accentuated induction of gamma-H2Ax in R2-knockdown cells is attributed to reduced ability to repair damaged DNA and overcome replication blockage. The lack of excess R2 subunits consequently augments chk1 activation and cdc25A degradation, causing impeded cell progression through the S phase and enhanced apoptosis in response to DNA damage and replication stress. In contrast, the level of R1 subunits appears to be limiting, since depletion of the R1 subunit directly activates the S phase checkpoint due to replication stress associated with impaired RNR activity. These findings suggest that excess R2 subunits facilitate DNA damage repair and recovery from replication stress through coordination with the S phase checkpoint in the absence of functional p53. Thus, the level of the R2 subunit constitutes an important determinant of the chemosensitivity of cancer cells and serves as a potential target for enhancement of DNA-damage based therapy.