DNA replication stress and cancer chemotherapy

DNA replication is one of the fundamental biological processes in which dysregulation can cause genome instability. This instability is one of the hallmarks of cancer and confers genetic diversity during tumorigenesis. Numerous experimental and clinical studies have indicated that most tumors have experienced and overcome the stresses caused by the perturbation of DNA replication, which is also referred to as DNA replication stress (DRS). When we consider therapeutic approaches for tumors, it is important to exploit the differences in DRS between tumor and normal cells. In this review, we introduce the current understanding of DRS in tumors and discuss the underlying mechanism of cancer therapy from the aspect of DRS.     

RRM1 maintains centrosomal integrity via CHK1 and CDK1 signaling during replication stress

DNA lesion-induced centrosomal abnormalities during the replication phase are relatively unknown. Here, we report that RNAi-mediated depletion of RRM1 induces cell-cycle arrest at the replication phase, along with severe DNA damage and centrosomal amplification. Interestingly, CHK1 depletion synergistically increased RRM1-depletion-induced centrosomal amplification. In response to hydroxyurea, CHK1 was delocalized from the centrosome by RRM1 depletion. Moreover, CDK1, which functions in centrosome separation and is inhibited by CHK1, was found to be essential for RRMI1-depletion-induced centrosomal amplification. Thus, we herein demonstrate that RRM1 preserves chromosomal stability via the CHK1- and CDK1-dependent stabilization of the centrosomal integrity at the replication stage.     

Whole genome RNAi screens reveal a critical role of REV3 in coping with replication stress

REV3, the catalytic subunit of translesion polymerase zeta (polζ), is commonly associated with DNA damage bypass and repair. Despite sharing accessory subunits with replicative polymerase δ, very little is known about the role of polζ in DNA replication. We previously demonstrated that inhibition of REV3 expression induces persistent DNA damage and growth arrest in cancer cells. To reveal determinants of this sensitivity and obtain insights into the cellular function of REV3, we performed whole human genome RNAi library screens aimed at identification of synthetic lethal interactions with REV3 in A549 lung cancer cells. The top confirmed hit was RRM1, the large subunit of ribonucleotide reductase (RNR), a critical enzyme of de novo nucleotide synthesis. Treatment with the RNR‐inhibitor hydroxyurea (HU) synergistically increased the fraction of REV3‐deficient cells containing single stranded DNA (ssDNA) as indicated by an increase in replication protein A (RPA). However, this increase was not accompanied by accumulation of the DNA damage marker γH2AX suggesting a role of REV3 in counteracting HU‐induced replication stress (RS). Consistent with a role of REV3 in DNA replication, increased RPA staining was confined to HU‐treated S‐phase cells. Additionally, we found genes related to RS to be significantly enriched among the top hits of the synthetic sickness/lethality (SSL) screen further corroborating the importance of REV3 for DNA replication under conditions of RS.     

A study of pyrimidine base damage in relation to oxidative stress and cancer

Background:

A long-standing hypothesis is that oxidative stress is a risk factor for cancer. Support for this hypothesis comes from observations of higher levels of oxidative damage in the DNA of WBC of cancer patients compared with healthy controls.

Methods:

Two generally overlooked types of DNA damage, the formamide modification and the thymine glycol modification, both derived from pyrimidine bases, were assayed as markers of oxidative stress. Damage levels were measured in the DNA of WBC of ovarian cancer patients and of healthy controls.

Results:

The levels of both modifications were higher in ovarian cancer patients than in healthy controls although in the case of the formamide modification age could not be ruled out as a factor.

Conclusion:

Our results in combination with other published measurements of oxidative DNA damage support the hypothesis that oxidative damage, on average, is higher in WBC of cancer patients than in healthy controls.     

Arsenic trioxide induces oxidative stress,DNA damage,and mitochondrial pathway of apoptosis in human leukemia (HL-60) cells

Background

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML), which accounts for approximately 10% of all acute myloid leukemia cases. It is a blood cancer that is formed by chromosomal mutation. Each year in the United States, APL affects about 1,500 patients of all age groups and causes approximately 1.2% of cancer deaths. Arsenic trioxide (ATO) has been used successfully for treatment of APL patients, and both induction and consolidated therapy have resulted in complete remission. Recently published studies from our laboratory have demonstrated that ATO pharmacology as an anti-leukemic drug is associated with cytotoxic and genotoxic effects in leukemia cells.

Methods

In the present study, we further investigated the detailed molecular mechanism of ATO-mediated intrinsic pathway of apoptosis; using HL-60 cells as a test model. Oxidative stress was assessed by spectrophotometric measurements of MDA and GSH levels while genotoxicity was determined by single cell gel electrophoresis (Comet assay). Apoptosis pathway was analyzed by Western blot analysis of Bax, Bcl2 and caspase 3 expression, as well as immunocytochemistry and confocal imaging of Bax and Cyt c translocation and mitochondrial membrane potential depolarization.

Results

ATO significantly (p < 0.05) induces oxidative stress, DNA damage, and caspase 3 activityin HL-60 cells in a dose-dependent manner. It also activated the intrinsic pathway of apoptosis by significantly modulating (p < 0.05) the expression and translocation of apoptotic molecules and decreasing the mitochondrial membrane potential in leukemia cells.

Conclusion

Taken together, our research demonstrated that ATO induces mitochondrial pathway of apoptosis in HL-60 cells. This apoptotic signaling is modulated via oxidative stress, DNA damage, and change in mitochondrial membrane potential, translocation and upregulation of apoptotic proteins leading programmed cell death.     

Early ROS-mediated DNA damage and oxidative stress biomarkers in Monoclonal B Lymphocytosis

Monoclonal B Lymphocytosis (MBL) is defined as asymptomatic monoclonal B-cell expansion characterised by a CLL-phenotype, but with less than 5×10(9)/l circulating cells. Reactive oxygen species (ROS)-mediated cell damage plays a critical role in the initiation of carcinogenesis as well as in malignant transformation. The goal of this study was to perform an analysis of the oxidative stress statuses of patients affected by MBL and chronic lymphocytic leukaemia (CLL). We examined peripheral blood and urine specimens from 29 patients with MBL, 55 with CLL and 31 healthy subjects. There was a significant increase in the occurrence of the mutagenic base 8-oxo-2'-deoxiguanosine (8-oxo-dG) in the lymphocytes and urine of MBL and CLL patients compared with controls. Significant differences were also observed in the levels of the lipid peroxidation product malondialdehyde (MDA) and in the oxidised/reduced glutathione (GSSG/GSH) ratio, although an increase in 8-isoprostane was not detected. Interestingly, the antioxidant catalase activity of circulating lymphocytes decreased in the patient groups. In conclusion, early oxidative stress exists in patients with MBL and CLL, causing damage to DNA and lipid structures. The higher levels of 8-oxo-dG in lymphocytes than in urine may be related to a decrease in the capacity of DNA repair systems. There were no differences in the oxidative statuses of the MBL and CLL patients, suggesting that oxidative injuries appear during a pre-leukaemic state of the disease.     

Therapeutic targeting of ATR yields durable regressions in small cell lung cancers with high replication stress

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PM2.5对斑马鱼胚胎发育早期氧化应激和DNA甲基化

目的：探讨细颗粒物（PM2.5）暴露后,斑马鱼胚胎发育过程中氧化应激和DNA甲基化基因表达的改变。方法：使用大功率大气PM2.5采样器收集PM2.5样品,超声后冷冻提取PM2.5,分别以0、5、20 μg/mL的浓度作用于斑马鱼胚胎。在暴露后2、6、24、48 h提取胚胎RNA,用荧光定量PCR法检测氧化应激相关基因超氧化物歧化酶1（sod1）和8-羟基鸟嘌呤DNA糖苷酶（ogg1）以及DNA甲基化相关基因DNA羟化酶1（tet1）、DNA甲基转移酶（dnmt1） mRNA的表达。结果：斑马鱼胚胎暴露于0、5、20 μg/mL的PM2.5后2~6 h,sod1、tet1和dnmt1的mRNA相对表达量均随着剂量升高而明显升高,呈剂量-效应关系（r依次=0.98、0.98、0.99,P均 < 0.05）;PM2.5暴露后2~6 h ogg1的mRNA相对表达量在5 μg/mL剂量组显著升高（P < 0.05）。而PM2.5暴露后24~48 h,sod1、ogg1、tet1和dnmt1的mRNA相对表达量相对2~6 h均明显下降（P < 0.05）。结论：PM2.5对斑马鱼胚胎的氧化应激相关基因sod1和ogg1以及DNA甲基化相关基因tet1和dnmt1的mRNA表达均有影响。     

Effects of topoisomerase inhibitors that induce DNA damage response on glucose metabolism and PI3K/Akt/mTOR signaling in multiple myeloma cells

Hallmarks of cancer cells comprise altered glucose metabolism (aerobic glycolysis) and differences in DNA damage response (DDR). Glucose transporters (GLUT), glycolytic enzymes such as hexokinase (HK) and metabolic pathways (e.g. PI3K/Akt/mTor) have been shown to be upregulated in multiple myeloma and other cancer cell lines. Here we have investigated the effects of clinically used inhibitors of topoisomerases, of DDR and of the PI3K/Akt/mTor pathway on glucose metabolism and on cell survival in multiple myeloma cells. The effects of DNA damaging topoisomerase inhibitors (doxorubicin, etoposide, topotecan), non-DNA damaging agents (bortezomib, vincristine) as well as of molecular inhibitors of DNA damage related kinases PIKKs (KU55933 [ATM], NU7026 [DNA-PKCs]) and PI3K/Akt/mTor signaling (BEZ235 [PI3K/mTor], MK-2206 [Akt]) were analyzed 24 hours after treatment of OPM-2 multiple myeloma cells. For this purpose we monitored [18F]-FDG uptake, cell viability using an ATP assay and expression of GLUT-1, hexokinase II (HKII), cleaved caspase-3 and cleaved PARP via Western-blotting. All topoisomerase inhibitors used could upregulate expression of GLUT-1 and HKII in OPM-2 cells, resulting in elevated [18F]-FDG uptake and promotion of cell survival. In contrast, bortezomib and vincristine induced a decline in [18F]-FDG uptake combined with early induction of apoptosis. Combination treatment with topoisomerase inhibitors and molecular inhibitors of PIKK and PI3K could reverse elevated [18F]-FDG uptake, as observed after application of topoisomerase inhibitors only, and aggravate induction of apoptosis. Thus, elevated glucose consumption in OPM-2 cells can be reversed by targeting both DDR and PI3K/Akt/mTOR signaling, thus providing a promising strategy in the treatment of cancer.