TOPK Inhibition Enhances the Sensitivity of Colorectal Cancer Cells to Radiotherapy by Reducing the DNA Damage Response

Objective Abnormal expression of T-lymphokine-activated killer cell-originated protein kinase(TOPK)was reported to be closely related to the resistance of prostate cancer to radiotherapy and to targeted drug resistance in lung cancer.However,the role of TOPK inhibition in enhancing radiosensitivity of colorectal cancer(CRC)cells is unclear.This study aimed to evaluate the radiosensitization of TOPK knockdown in CRC cells.Methods The expression of TOPK was detected in CRC tissues by immunohistochemistry,and the effect of TOPK knockdown was detected in CRC cells by Western blotting.CCK-8 and clonogenic assays were used to detect the growth and clonogenic ability of CRC cells after TOPK knockdown combined with radiotherapy in CRC cells.Furthermore,proteomic analysis showed that the phosphorylation of TOPK downstream proteins changed after radiotherapy.DNA damage was detected by the comet assay.Changes in the DNA damage response signaling pathway were analyzed by Western blotting,and apoptosis was detected by flow cytometry.Results The expression of TOPK was significantly greater in CRC tissues at grades 2–4 than in those at grade 1.After irradiation,CRC cells with genetically silenced TOPK had shorter comet tails and reduced expression levels of DNA damage response-associated proteins,including phospho-cyclin-dependent kinase 1(p-CDK1),phospho-ataxia telangiectasia-mutated(p-ATM),poly ADP-ribose polymerase(PARP),and meiotic recombination 11 homolog 1(MRE11).Conclusions TOPK was overexpressed in patients with moderately to poorly differentiated CRC.Moreover,TOPK knockdown significantly enhanced the radiosensitivity of CRC cells by reducing the DNA damage response.

Tissue specificity of DNA damage response and tumorigenesis

The genome of cells is constantly challenged by DNA damages from endogenous metabolism and environmental agents.These damages could potentially lead to genomic instability and thus to tumorigenesis.To cope with the threats, cells have evolved an intricate network, namely DNA damage response(DDR) system that senses and deals with the lesions of DNA.Although the DDR operates by relatively uniform principles, different tissues give rise to distinct types of DNA damages combined with high diversity of microenvironments across tissues.In this review, we discuss recent findings on specific DNA damage among different tissues as well as the main DNA repair way in corresponding microenvironments, highlighting tissue specificity of DDR and tumorigenesis.We hope the current review will provide further insights into molecular process of tumorigenesis and generate new strategies for cancer treatment.

Decoupling of DNA damage response signaling from DNA damages underlies temozolomide resistance in glioblastoma cells

Glioblastoma multiforme （GBM） is the most aggressive primary brain tumor in adults.Current therapy includes surgery,radiation and chemotherapy with temozolomide （TMZ）.Major determinants of clinical response to TMZ include methylation status of the O6-methylguanine-DNA methyltransferase （MGMT） promoter and mismatch repair （MMR） status.Though the MGMT promoter is methylated in 45% of cases,for the first nine months of follow-up,TMZ does not change survival outcome.Furthermore,MMR deficiency makes little contribution to clinical resistance,suggesting that there exist unrecognized mechanisms of resistance.We generated paired GBM cell lines whose resistance was attributed to neither MGMT nor MMR.We show that,responding to TMZ,these cells exhibit a decoupling of DNA damage response （DDR） from ongoing DNA damages.They display methylation-resistant synthesis in which ongoing DNA synthesis is not inhibited.They are also defective in the activation of the S and G2 phase checkpoint.DDR proteins ATM,Chk2,MDC1,NBS1 and gammaH2AX also fail to form discrete foci.These results demonstrate that failure of DDR may play an active role in chemoresistance to TMZ.DNA damages by TMZ are repaired by MMR proteins in a futile,reiterative process,which activates DDR signaling network that ultimately leads to the onset of cell death.GBM cells may survive genetic insults in the absence of DDR.We anticipate that our findings will lead to more studies that seek to further define the role of DDR in ultimately determining the fate of a tumor cell in response to TMZ and other DNA methylators.

Poly(ADP-ribosyl)ation of Apoptosis Antagonizing Transcription Factor Involved in Hydroquinone-Induced DNA Damage Response

The molecular mechanism of DNA damage induced by hydroquinone （HQ） remains unclear. Poly（ADP-ribose） polymerase-1 （PARP-1） usually works as a DNA damage sensor, and hence, it is possible that PARP-1 is involved in the DNA damage response induced by HQ. In TK6 cells treated with HQ, PARP activity as well as the expression of apoptosis antagonizing transcription factor （AATF）, PARP-1, and phosphorylated H2AX （v-H2AX） were maximum at 0.5 h, 6 h, 3 h, and 3 h, respectively. To explore the detailed mechanisms underlying the prompt DNA repair reaction, the above indicators were investigated in PARP-l-silenced cells. PARP activity and expression of AATF and PARP-1 decreased to 36%, 32%, and 33%, respectively, in the cells; however, y-H2AX expression increased to 265%. Co-immunoprecipitation （co-IP） assays were employed to determine whether PARP-1 and AATF formed protein complexes. The interaction between these proteins together with the results from IP assays and confocal microscopy indicated that poly（ADP-ribosyl）ation {PARylation） regulated AATF expression, in conclusion, PARP-1 was involved in the DNA damage repair induced by HQ via increasing the accumulation of AATF through PARylation.

DNA damage responses in cancer stem cells: Implications for cancer therapeutic strategies

The identification of cancer stem cells(CSCs) that are responsible for tumor initiation, growth, metastasis, and therapeutic resistance might lead to a new thinking on cancer treatments. Similar to stem cells,CSCs also display high resistance to radiotherapy and chemotherapy with genotoxic agents. Thus, conventional therapy may shrink the tumor volume but cannot eliminate cancer. Eradiation of CSCs represents a novel therapeutic strategy. CSCs possess a highly efficient DNA damage response(DDR) system, which is considered as a contributor to the resistance of these cells from exposures to DNA damaging agents. Targeting of enhanced DDR in CSCs is thus proposed to facilitate the eradication of CSCs by conventional therapeutics. To achieve this aim, a better understanding of the cellular responses to DNA damage in CSCs is needed. In addition to the protein kinases and enzymes that are involved in DDR, other processes that affect the DDR including chromatin remodeling should also be explored.

RECQL4 regulates DNA damage response and redox homeostasis in esophageal cancer

Objective:RECQL4(a member of the RECQ helicase family)upregulation has been reported to be associated with tumor progression in several malignancies.However,whether RECQL4 sustains esophageal squamous cell carcinoma(ESCC)has not been elucidated.In this study,we determined the functional role for RECQL4 in ESCC progression.Methods:RECQL4 expression in clinical samples of ESCC was examined by immunohistochemistry.Cell proliferation,cellular senescence,the epithelial-mesenchymal transition(EMT),DNA damage,and reactive oxygen species in ESCC cell lines with RECQL4 depletion or overexpression were analyzed.The levels of proteins involved in the DNA damage response(DDR),cell cycle progression,survival,and the EMT were determined by Western blot analyses.Results:RECQL4 was highly expressed in tumor tissues when compared to adjacent non-tumor tissues in ESCC(P<0.001)and positively correlated with poor differentiation(P=0.011),enhanced invasion(P=0.033),and metastasis(P=0.048).RECQL4 was positively associated with proliferation and migration in ESCC cells.Depletion of RECQL4 also inhibited growth of tumor xenografts in vivo.RECQL4 depletion induced G0/G1 phase arrest and cellular senescence.Importantly,the levels of DNA damage and reactive oxygen species were increased when RECQL4 was depleted.DDR,as measured by the activation of ATM,ATR,CHK1,and CHK2,was impaired.RECQL4 was also shown to promote the activation of AKT,ERK,and NF-k B in ESCC cells.Conclusions:The results indicated that RECQL4 was highly expressed in ESCC and played critical roles in the regulation of DDR,redox homeostasis,and cell survival.

DNA Damage Response in Resting and Proliferating Peripheral Blood Lymphocytes Treated by Camptothecin or X-ray

DNA damage response （DDR） in different cell cycle status of human peripheral blood lymphocytes （PBLs） and the role of H2AX in DDR were investigated. The PBLs were stimulated into cell cycle with phytohemagglutinin （PHA）. The apoptotic ratio and the phosphorylation H2AX （S139） were flow cytometrically measured in resting and proliferating PBLs after treatment with camptothecin （CPT） or X-ray. The expressions of γH2AX, Bcl-2, caspase-3 and caspase-9 were detected by Western blotting. DDR in 293T cells was detected after H2AX was silenced by RNAi method. Our results showed that DNA double strand breaks （DSBs） were both induced in quiescent and proliferating PBLs after CPT or X-ray treatment. The phosphorylation of H2AX and apoptosis were more sensitive in proliferating PBLs compared with quiescent lymphocytes （P0.05）. The expression levels of anti-apoptotic proteins Bcl-2 were reduced and cleaved caspase-3 and caspase-9 were increased. No significant changes were observed in CPT-induced apoptosis in 293T cells between H2AX knocking down group and controls. It was concluded that proliferating PBLs were more vulnerable to DNA damage compared to non-stimulated lymphocytes and had higher apoptosis rates. γH2AX may only serve as a marker of DNA damage but exert no effect on apoptosis regulation.

Evaluation of 30 DNA damage response and 6 mismatch repair gene mutations as biomarkers for immunotherapy outcomes across multiple solid tumor types

Objective:DNA damage response(DDR)genes have low mutation rates,which may restrict their clinical applications in predicting the outcomes of immune checkpoint inhibitor(ICI)treatment.Thus,a systemic analysis of multiple DDR genes is needed to identify potential biomarkers of ICI efficacy.Methods:A total of 39,631 patients with mutation data were selected from the cBioPortal database.A total of 155 patients with mutation data were obtained from the Fudan University Shanghai Cancer Center(FUSCC).A total of 1,660 patients from the MSK-IMPACT cohort who underwent ICI treatment were selected for survival analysis.A total of 249 patients who underwent ICI treatment from the Dana-Farber Cancer Institute(DFCI)cohort were obtained from a published dataset.The Cancer Genome Atlas(TCGA)level 3 RNA-Seq version 2 RSEM data for gastric cancer were downloaded from cBioPortal.Results:Six MMR and 30 DDR genes were included in this study.Six MMR and 20 DDR gene mutations were found to predict the therapeutic efficacy of ICI,and most of them predicted the therapeutic efficacy of ICI,in a manner dependent on TMB,except for 4 combined DDR gene mutations,which were associated with the therapeutic efficacy of ICI independently of the TMB.Single MMR/DDR genes showed low mutation rates;however,the mutation rate of all the MMR/DDR genes associated with the therapeutic efficacy of ICI was relatively high,reaching 10%–30%in several cancer types.Conclusions:Coanalysis of multiple MMR/DDR mutations aids in selecting patients who are potential candidates for immunotherapy.

Cellular processing determinants for the activation of damage signals in response to topoisomerase I-linked DNA breakage

Recent studies have suggested an involvement of processing pathways for the initiation of cellular responses induced by topoisomerase-targeting drugs. Here, we showed that cellular exposure to camptothecin （CPT） induced formation of topoisomerase I cleavable complex （TOPlcc）, degradation of TOP1 and activation of DNA damage responses （DDR）. Transcription and proteasome-dependent proteolysis, but not replication, were involved in CPTo indneed TOPl degradation, while none of above three processing activities affected TOPlcc formation. Replication- and transcription-initiated proeessing （RIP and TIP） of TOPlee were identified as two independent pathways, which contribute distinctly to various CPT-activated DDR. Specifically, in cycling cells, RIP-processed TOPlec triggered the CPT-induced RPA pbosphorylation. At higher CPT dosages, the TIP pathway is required for other DDR activation, including ATM, p53 and Chkl/2 phosphorylation. The TIP pathway was further demonstrated to be S-phase independent by using three nonreplicating cell models. Furthermore, the effect of proteasome inhibitors mimicked that of transcription inhibition on the CPT-induced activation of DDR, suggesting the involvement of proteasome in the TIP pathway. Interestingly, the TIP pathway was important for TOPlcc-activated, but not ionization radiationactivated ATM, p53 and Chk2 phosphorylation. We have also found that pharmacological interferences of TIP and RIP pathways distinctively modulated the CPT-induced cell killing with treatments at low and high dosages, respec- tively. Together, our results support that both RIP and TIP pathways of TOPlcc are required for the activation of CPT-induced DDR and cytotoxicity.

cGAS regulates the DNA damage response to maintain proliferative signaling in gastric cancer cells

The activation of some oncogenes promote cancer cell proliferation and growth,facilitate cancer progression and metastasis by induce DNA replication stress,even genome instability.Activation of the cyclic GMP-AMP synthase(cGAS)mediates classical DNA sensing,is involved in genome instability,and is linked to various tumor development or therapy.However,the function of cGAS in gastric cancer remains elusive.In this study,the TCGA database and retrospective immunohistochemical analyses revealed substantially high cGAS expression in gastric cancer tissues and cell lines.By employing cGAS high-expression gastric cancer cell lines,including AGS and MKN45,ectopic silencing of cGAS caused a significant reduction in the proliferation of the cells,tumor growth,and mass in xenograft mice.Mechanistically,database analysis predicted a possible involvement of cGAS in the DNA damage response(DDR),further data through cells revealed protein interactions of the cGAS and MRE11-RAD50-NBN(MRN)complex,which activated cell cycle checkpoints,even increased genome instability in gastric cancer cells,thereby contributing to gastric cancer progression and sensitivity to treatment with DNA damaging agents.Furthermore,the upregulation of cGAS significantly exacerbated the prognosis of gastric cancer patients while improving radiotherapeutic outcomes.Therefore,we concluded that cGAS is involved in gastric cancer progression by fueling genome instability,implying that intervening in the cGAS pathway could be a practicable therapeutic approach for gastric cancer.

Kinetochore protein MAD1 participates in the DNA damage response through ataxia-telangiectasia mutated kinase-mediated phosphorylation and enhanced interaction with KU80

Objective:Mitotic arrest-deficient protein 1(MAD1)is a kinetochore protein essential for the mitotic spindle checkpoint.Proteomic studies have indicated that MAD1 is a component of the DNA damage response(DDR)pathway.However,whether and how MAD1 might be directly involved in the DDR is largely unknown.Methods:We ectopically expressed the wild type,or a phosphorylation-site--mutated form of MAD1 in MAD1 knockdown cells to look for complementation effects.We used the comet assay,colony formation assay,immunofluorescence staining,and flow cytometry to assess the DDR,radiosensitivity,and the G2/M checkpoint.We employed co-immunoprecipitation followed by mass spectrometry to identify MAD1 interacting proteins.Data were analyzed using the unpaired Student'st-test.Results:We showed that MAD1 was required for an optimal DDR,as knocking down MAD1 resulted in impaired DNA repair and hypersensitivity to ionizing radiation(IR).We found that IR-induced serine 214 phosphorylation was ataxia-telangiectasia mutated(ATM)kinase-dependent.Mutation of serine 214 to alanine failed to rescue the phenotypes of MAD1 knockdown cells in response to IR.Using mass spectrometry,we identified a protein complex mediated by MAD1 serine 214 phosphorylation in response to IR.Among them,we showed that KU80 was a key protein that displayed enhanced interaction with MAD1 after DNA damage.Finally,we showed that MAD1 interaction with KU80 required serine 214 phosphorylation,and it was essential for activation of DNA protein kinases catalytic subunit(DNA-PKcs).Conclusions:MAD1 serine 214 phosphorylation mediated by ATM kinase in response to IR was required for the interaction with KU80 and activation of DNA-PKCs.

Strategies for targeting the DNA damage response for cancer therapeutics

The DNA damage response is critical for cells to maintain genome stability and survival. In this review, we discuss approaches to targeting critical elements of the DNA damage response for radiosensitization and chemosensitization. In addition, we also discuss strategies for targeting DNA damage response and DNA repair defects in cancer cells for synthetic lethality.

DNA损伤修复相关通路的合成致死靶点研究及其在卵巢癌中的应用和前景

DNA损伤引发细胞启动一系列DNA损伤应答(DNA damage response,DDR),包括DNA损伤修复、细胞周期检查点激活、细胞周期阻滞、各种细胞内信号转导途径的活化和细胞凋亡等。DNA损伤修复(DNA damage repair)是细胞维持基因组稳定性的重要机制,于2015年获得诺贝尔化学奖。DNA损伤修复途径主要包括:碱基切除修复(base-excision repair,BER)、核苷酸切除修复(nucleotide excision repair,NER)、错配修复(mismatch repair,MMR)、同源重组(homologous recombination,HR)和非同源末端连接(non-homologous end joining,NHEJ)等,分别在DNA单链断裂(single-strand break,SSB)或双链断裂(double-strand break,DSB)等损伤修复中发挥重要作用。DNA损伤修复缺陷与肿瘤发生发展密切相关,同时也是肿瘤治疗的重要靶点。DNA损伤修复通路的多聚ADP核糖聚合酶(poly-ADP-ribose polymerase,PARP)与乳腺癌易感基因BRCA 1/2等存在合成致死(synthetic lethality)作用,使PARP抑制剂(PARP inhibitor,PARPi)成为第一个也是目前唯一上市的肿瘤治疗合成致死靶药。PARPi在卵巢癌及多种实体瘤治疗中疗效良好,使DNA损伤修复及相关DDR通路的合成致死靶药研发成为热点,其他在研靶点主要包括:共济失调毛细血管扩张突变蛋白(ataxia telangiectasia-mutated protein,ATM)、共济失调毛细血管扩张与RAD3相关蛋白(ataxia telangiectasia and Rad3 related protein,ATR)、DNA依赖性蛋白质激酶催化亚单位(DNA-dependent protein kinase catalytic subunit,DNA-PKcs)、细胞周期检测点激酶1(checkpoint kinase1,CHK1)、细胞周期检测点激酶2(checkpoint kinase 2,CHK2)、阻止有丝分裂的蛋白质激酶WEE1等。PARPi与其他DDR靶药、抗血管生成药物及免疫检查点抑制剂的联用,有可能成为克服PARPi耐药、提高疗效的有效手段和发展前景。本文针对DNA损伤修复及相关DDR通路的关键分子和潜在肿瘤治疗靶点进行综述,阐述了DNA损伤修复相关通路的合成致死靶点研究及在卵巢癌的应用和前景,为基础研究及临床应用提供指导。

DNA损伤与肿瘤免疫治疗的联合策略——现状与展望

免疫治疗,尤其是免疫检查点抑制,已成为癌症治疗的重要手段。然而,大多数癌症中仅有少数患者对免疫检查点阻断有反应。随着对肿瘤免疫治疗认识的深入,DNA损伤与修复机制在肿瘤免疫逃逸中的作用愈发显著。基因组不稳定性和DNA损伤应答的缺陷,尤其是在放射治疗和化学治疗的背景下,能够增强免疫检查点阻断的疗效。尽管研究已取得积极进展,但如何精准地调控DNA损伤应答以及优化联合治疗策略,提高疗效和减少毒副作用,仍是未来研究的重点。本文详细讨论放射治疗和化学治疗等传统基因毒性治疗与免疫检查点阻断联合治疗策略的免疫调节机制,探讨干扰素基因刺激因子激动剂和DNA损伤应答抑制剂在联合治疗中的潜力,还提出基于DNA损伤应答靶向抑制与免疫治疗联合应用的新策略,强调了结合传统治疗和新型免疫调节策略以及开发生物标志物的重要性。

Ataxia telangiectasia-mutated-Rad3-related DNA damage checkpoint signaling pathway triggered by hepatitis B virus infection

AIM： To explore whether acute cellular DNA damage response is induced upon hepatitis B virus （HBV） infection and the effects of the HBV infection. METHODS： We incubated HL7702 hepatocytes with HBV-positive serum, mimicking a natural HBV infection process. We used immunoblotting to evaluate protein expression levels in HBV-infected cells or in non-infected cells; immunofluorescence to show ATR foci ands Chkl phosphorylation foci formation; flow cytometry to analyze the cell cycle and apoptosis; ultraviolet （UV） radiation and ionizing radiation （IR）-treated cells to mimic DNA damage; and Trypan blue staining to count the viable cells. RESULTS： We found that HBV infection induced an increased steady state of ATR protein and increased phosphorylation of multiple downstream targets including Chkl, p53 and H2AX. In contrast to ATR and its target, the phosphorylated form of ATM at Ser-1981 and its downstream substrate Chk2 phosphorylation at Thr-68 did not visibly increase upon infection. However, the level of Mre11 and p21 were reduced beginning at 0.5 h aEer HBV-positive serum addition. Also, HBV infection led to transient cell cycle arrest in the S and the G2 phases without accompanying increasedapoptosis. Research on cell survival changes upon radiation following HBV infection showed that survival of UV-treated host cells was greatly increased by HBV infection, owing to the reduced apoptosis. Meanwhile, survival of IR-treated host cells was reduced by HBV infection. CONCLUSION： HBV infection activates ATR DNA damage response to replication stress and abrogates the checkpoint signaling controlled by DNA damage response.

西达本胺联合(+)-JQ-1通过破坏DNA损伤反应途径杀伤MLL重排急性髓系白血病细胞

目的:探讨西达本胺与BRD4抑制剂(+)-JQ-1联合对混合谱系白血病基因重排急性髓系白血病(MLL-r AML)细胞的DNA损伤及修复机制的影响。方法:分别将MLL-r AML细胞系Molm-13、MV4-11细胞和非MLL-r AML细胞系Kasumi细胞分成对照组(contr)、西达本胺组(chida)、(+)-JQ-1组、联合组(combi) 4组。采用CCK-8检测Molm-13细胞活力,以确定西达本胺和(+)-JQ-1的给药浓度。采用流式细胞术检测细胞周期变化,Western blot检测凋亡相关因子Bcl-2、Bax、caspase-3的表达。利用免疫荧光检测DNA损伤标志物γH2AX;Western blot检测DNA损伤因子γH2AX,DNA损伤检查点激酶p-ATR、p-CHK1、p-ATM、p-CHK2和DNA损伤修复因子Rad51、53BP1蛋白的表达情况;qRT-PCR检测DNA损伤修复因子Rad51、53BP1 mRNA的表达情况。结果:在药物西达本胺(300 nmol/L)和(+)-JQ-1(400 nmol/L)的联合作用下,MLL-r AML细胞系Molm-13、MV4-11中,与contr组相比,combi组G1期细胞比例升高;非MLL-r AML细胞系Kasumi中,与contr组相比,combi组G1期细胞比例升高(P <0.05)。在Molm-13、MV4-11细胞系中,与contr组相比,combi组DNA损伤标志物γH2AX表达水平升高(P <0.05),DNA损伤检查点和损伤修复因子p-ATR、p-CHK1、p-ATM、p-CHK2、Rad51、53BP1表达水平降低(P <0.05);而在Kasumi细胞系中,与contr组相比,combi组上述因子部分表达无明显变化(P>0.05),部分因子的表达趋势与MLL-r AML细胞系相反。在MLL-r AML细胞系Molm-13、MV4-11中,与contr组相比,combi组Bax和caspase-3蛋白表达水平升高,而Bcl-2蛋白表达水平下降(P <0.05);非MLL-r AML细胞系Kasumi细胞中,与contr组相比,combi组凋亡因子的表达无明显变化(P>0.05)。结论:西达本胺联合(+)-JQ-1可抑制MLL-r AML细胞的增殖,并通过抑制DNA损伤反应途径抑制此类白血病细胞保护性自我修复的启动,最终增加细胞的凋亡,而非MLL-r AML细胞却无类似的结果。

Rad7 E3 Ubiquitin Ligase Attenuates Polyubiquitylation of Rpn10 and Dsk2 Following DNA Damage in <i>Saccharomyces cerevisiae</i>

During Nucleotide Excision Repair (NER) in the yeast S. cerevisiae, ubiquitylation of Rad4 is carried out by the E3 ubiquitin ligase that includes Rad7-Elc1-Cul3 and is critical to optimal NER. Rad7 E3 activity targets Rad4 for degradation by the proteaseome but, in principle, could also trigger other DNA damage responses. We observed increased nuclear ubiquitin foci (Ub-RFP) formation in S. cerevisiae containing a Rad7 E3 ligase mutant (rad7SOCS) in response to DNA damage by benzo[a]pyrenediolepoxide (BPDE) in dividing cells. Immunoblots reveal that ubiquitin conjugates of Rpn10 and Dsk2 accumulate in greater abundance in rad7SOCS compared to RAD7 in dividing cells in response to BPDE which makes Rpn10 and Dsk2 candidates for being the ubiquitylated species observed in our microscopy experiments. Microscopy analysis with strains containing Dsk2-GFP shows that Dsk2-GFP and Dsk2-GFP/Ub-RFP colocalized in nuclear foci form to an increased extent in a rad7SOCS mutant background in dividing cells than in a RAD7 wild-type strain. Further, Dsk2-GFP in the rad7SOCS strain formed more foci at the plasma membrane following BPDE treatment in dividing cells relative to strains containing RAD7 or a rad7△?deletion mutant. In response to a different agent, UV irradiation, levels of ubiquitylated proteins were increased in rad7SOCS relative to RAD7, and the proteasomal deubiquitylase subunit, Rpn11 was even monoubiquitylated in the absence of damaging agents. Together these data show that Rad7 E3 activity attenuates ubiq-uitylation of proteins regulating the shuttling of polyubiquitylated proteins to the proteasome (Dsk2 and Rpn10) and removal of ubiquitin chains just prior to degradation (Rpn11). Since Rad7 E3 ligase activity has been shown to increase ubiquitylation of its target proteins, yet our results show increased ubiquitylation in the absence of Rad7 E3, we suggest that Rad7 E3 action regulates ubiquitin ligase and deubiquitylase (DUB) activities that act on Rpn10, Dsk2 and Rpn11.

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.

DNA损伤反应缺陷与高风险神经母细胞瘤

神经母细胞瘤(neuroblastoma,NB)是儿童最常见的颅外实体肿瘤。其中高风险患者进展快、预后差。分子遗传学上,高风险NB特征性表现为染色体不稳定性,以DNA损伤反应(DNA damage response,DDR)缺陷为主。目前已有靶向DDR的抑制剂(如ATR、ATM、PARP、CHK1和WEE1抑制剂)用于高风险NB的治疗。应用DDR缺陷及靶向抑制剂等方式可为高风险NB的精准诊断及治疗提供参考,有望成为治疗高风险NB的新策略。

基于DNA损伤应答的肝细胞癌预后基因筛选和预后模型构建

目的筛选差异表达的DDR相关基因(differential expressed-DNA damage response associated genes,DEDDR)构建肝细胞癌(HCC)预后模型。方法使用limma R软件包从TCGA-LIHC数据集中筛选HCC样本和正常样本之间的差异表达基因(DEGs),将其与276个DDR基因取交集获得差异表达DEDDR。通过单因素Cox分析和Lasso回归分析以确定DEDDR预后模型,ROC曲线评估模型的准确性。单因素、多因素Cox回归分析DEDDR预后模型风险评分是否为HCC的预后独立危险因素,并采用国际癌症基因组联盟(ICGC)中LIHC数据作为外部数据进行验证。最后对low-DEDDR组和high-DEDDR组进行GSEA和免疫浸润分析。结果1361个DEGs与276个DDR基因取交集获得25个DEDDR,Lasso回归分析得到4个DEDDR(TTK、NSMCE2、NUDT1、NEIL3)用于构建预后模型。low-DEDDR组比high-DEDDR组患者具有更高的生存率。ROC曲线显示该模型预测HCC患者1年、2年和3年生存率的AUC值分别为0.77、0.70和0.68;单因素和多因素Cox回归分析结果显示DEDDR预后模型风险评分是HCC患者的独立预后危险因素,且其AUC值高于其他临床病理特征,ICGC中LIHC数据进一步验证了该模型的准确性和临床适用性较好。GSEA分析显示high-DEDDR组主要富集细胞周期、细胞衰老、DNA复制等信号通路,此外high-DEDDR组中2型辅助性T细胞(Th2)丰度更高,而嗜酸性粒细胞丰度更低。结论DEDDR预后模型在预测HCC患者的预后方面具有较好的表现,可为评估HCC患者预后、识别高危患者提供参考。