BCR/ABL-transformed chronic myeloid leukemia (CML) cells accumulate numerous DNA double-strand breaks (DSB) induced by reactive oxygen species (ROS) and genotoxic agents. To repair these lesions BCR/ABL stimulate unfaithful DSB repair pathways， homologous recombination repair (HRR)， nonhomologous end-joining (NHEJ)， and single-strand annealing (SSA). Here， we show that BCR/ABL enhances the expression and increase nuclear localization of WRN (mutated in Werner syndrome)， which is required for processing DSB ends during the repair. Other fusion tyrosine kinases (FTK)， such as TEL/ABL， TEL/JAK2， TEL/PDGFβR， and NPM/ALK also elevate WRN. BCR/ABL induces WRN mRNA and protein expression in part by c-MYC-mediated activation of transcription and Bcl-xL-dependent inhibition of caspase-dependent cleavage， respectively. WRN is in complex with BCR/ABL resulting in WRN tyrosine phosphorylation and stimulation of its helicase and exonuclease activities. Activated WRN protects BCR/ABL-positive cells from the lethal effect of oxidative and genotoxic stresses， which causes DSBs. In addition， WRN promotes unfaithful recombination-dependent repair mechanisms HRR and SSA， and enhances the loss of DNA bases during NHEJ in leukemia cells. In summary， we postulate that BCR/ABL-mediated stimulation of WRN modulates the efficiency and fidelity of major DSB repair mechanisms to protect leukemia cells from apoptosis and to facilitate genomic instability.
目的 探讨胃癌组织中硫酸酯酶2(SULF2)和WRN基因启动子甲基化对伊立替康药物敏感性以及胃癌患者临床病理特征的关系.方法 采用药物敏感性实验检测伊立替康对102例胃癌新鲜组织的抑制率.采用甲基化特异性聚合酶链反应检测SULF2和WRN基因启动子的甲基化状况，并分析 SULF2和WRN基因的甲基化与胃癌患者临床病理特征以及伊立替康敏感性的关系.建立裸鼠移植瘤模型，检测胃癌组织中SULF2基因的甲基化对伊立替康 敏感性的影响.结果 102例胃癌组织中，SULF2和WRN基因的甲基化率分别为28.4％ (29/102)和23.5％ (24/102).SULF2和WRN基因的甲基化与胃癌患者的年龄、性别、病理类型、分化程度、淋巴结转移以及TNM分期均无关(均P ＞0.05).102例胃癌组织中，伊立替康敏感组30例，耐药组72例.敏感组和耐药组胃癌组织中SULF2基因的甲基化率分别为46.7％ (14/30)和20.8％ (15/72)，差异有统计学意义(P=0.008)；WRN基因的甲基化率分别为33.3％ (10/30)和19.4％(14/72)，差异无统计学意义(P =0.214).SULF2和WRN基因均甲基化的胃癌组织对伊立替康的敏感性更高.SULF2基因甲基化胃癌组织的裸鼠移植瘤对于伊立替康的敏感性更 高，平均抑瘤率为75.3％.结论 SULF2和WRN基因启动子区甲基化检测可能可以为筛选出适宜伊立替康化疗的胃癌患者提供依据.
Abstract Werner syndrome is a hereditary disorder characterized by the early onset of age-related symptoms， including cancer. The absence of a p53-WRN helicase interaction may disrupt the signal to direct S-phase cells into apoptosis for programmed cell death and contribute to the pronounced genomic instability and cancer predisposition in Werner syndrome cells. Results from coimmunoprecipitation studies indicate that WRN is associated with replication protein A (RPA) and p53 in vivo before and after treatment with the replication inhibitor hydroxyurea or gamma-irradiation that introduces DNA strand breaks. Analysis of the protein interactions among purified recombinant WRN， RPA， and p53 proteins indicate that all three protein pairs bind with similar affinity in the low nanomolar range. In vitro studies show that p53 inhibits RPA-stimulated WRN helicase activity on an 849-bp M13 partial duplex substrate. p53 also inhibited WRN unwinding of a short (19-bp) forked duplex substrate in the absence of RPA. WRN unwinding of the forked duplex substrate was specific， because helicase inhibition mediated by p53 was retained in the presence of excess competitor DNA and was significantly reduced or absent in helicase reactions catalyzed by a WRN helicase domain fragment lacking the p53 binding site or the human RECQ1 DNA helicase， respectively. p53 effectively inhibited WRN helicase activity on model DNA substrate intermediates of replication/repair， a 5' ssDNA flap structure and a synthetic replication fork. Regulation of WRN helicase activity by p53 is likely to play an important role in genomic integrity surveillance， a vital function in the prevention of tumor progression.
Perturbed replication induced genome wide or at common fragile sites is differently managed in the absence of WRN.
The Werner syndrome protein (WRN) is a member of the RecQ helicase family. Loss of WRN results in a human disease， the Werner syndrome (WS)， characterized by high genomic instability， elevated cancer risk and premature aging. WRN is crucial for the recovery of stalled replication forks and possesses both helicase and exonuclease enzymatic activities of uncertain biological significance. Previous work revealed that WRN promotes formation of MUS81-dependent double strand breaks (DSBs) at HU-induced stalled forks， allowing replication restart at the expense of chromosome stability. Here， using cells expressing the helicase- or exonuclease-dead WRN mutant， we show that both activities of WRN are required to prevent MUS81-dependent breakage after HU-induced replication arrest. Moreover， we provide evidence that， in WS cells， DSBs generated by MUS81 do not require RAD51 activity for their formation. Surprisingly， when replication is specifically perturbed at common fragile sites (CFS) by aphidicolin， WRN limits accumulation of ssDNA gaps and no MUS81-dependent DSBs are detected. However， in both cases， RAD51 is essential to ensure viability of WS cells， although by different mechanisms. Thus， the role of WRN in response to perturbation of replication along CFS is functionally distinct from that carried out at stalled forks genome wide. Our results contribute to unveil two different mechanisms used by the cell to overcome the absence of WRN.
The human WRN and BLM RecQ helicases differentially regulate cell proliferation and survival after chemotherapeutic DNA damage.
Loss-of-function mutations in the human RecQ helicase genes WRN and BLM respectively cause the genetic instability/cancer predisposition syndromes Werner syndrome and Bloom syndrome. To identify common and unique functions of WRN and BLM， we systematically analyzed cell proliferation， cell survival， and genomic damage in isogenic cell lines depleted of WRN， BLM， or both proteins. Cell proliferation and survival were assessed before and after treatment with camptothecin， cis-diamminedichloroplatinum(II)， hydroxyurea， or 5-fluorouracil. Genomic damage was assessed， before and after replication arrest， by gamma-H2AX staining， which was quantified at the single-cell level by flow cytometry. Cell proliferation was affected strongly by the extent of WRN and/or BLM depletion， and more strongly by BLM than by WRN depletion (P = 0.005). The proliferation of WRN/BLM-codepleted cells， in contrast， did not differ from BLM-depleted cells (P = 0.34). BLM-depleted and WRN/BLM-codepleted cells had comparably impaired survival after DNA damage， whereas WRN-depleted cells displayed a distinct pattern of sensitivity to DNA damage. BLM-depleted and WRN/BLM-codepleted cells had similar， significantly higher gamma-H2AX induction levels than did WRN-depleted cells. Our results provide new information on the role of WRN and BLM in determining cell proliferation， cell survival， and genomic damage after chemotherapeutic DNA damage or replication arrest. We also provide new information on functional redundancy between WRN and BLM. These results provide a strong rationale for further developing WRN and BLM as biomarkers of tumor chemotherapeutic responsiveness.
WRN protein as a novel erythroblast immunohistochemical marker with applications for the diagnosis of Werner syndrome
Genetic testing for mutations in the WRN gene is critical for the diagnosis of Werner syndrome (WS); however， these tests cannot be performed in a clinical setting. Nearly all of the WRN mutations result in expression of truncated WRN proteins that are missing the C-terminal nuclear localization signal. We evaluated the use of WRN protein immunohistochemistry for diagnosing WS using paraffin-embedded bone marrow sections. Using a well-defined commercially available polyclonal antibody against the C terminus of WRN， we found that of all the cell types tested， bone marrow erythroid precursors showed the strongest nuclear expression of WRN. Immunohistochemical analysis of bone marrow samples from 120 patients with non-WS hematological disorders (age range， 7days-90years) revealed WRN staining of the nuclei of CD71-positive early and late erythroid precursors. Erythroblasts negative for WRN immunostaining were only observed in two patients， both of whom were diagnosed with WS： one with concomitant myelodysplastic syndrome and the other with erythroleukemia with overexpression of TP53. Western blot analysis and immunocytochemistry indicated WRN was localized in the nuclei of the four positive control cell lines from non-WS patients but not in the five cell lines from WS patients， who had three different types of WRN mutations. Thus， immunohistochemical detection of WRN in erythroblasts from bone marrow paraffin sections could be useful in screening of WS cases and worthy of further molecular confirmation.
RECQL1 and WRN proteins are RecQ DNA helicases that participate in suppression of DNA hyper-recombination and repair. In this study， we report evidence supporting their candidacy as cancer therapeutic targets. In hypopharyngeal carcinomas， which have the worst prognosis among head and neck squamous cell carcinomas (HNSCC) that are rapidly rising in incidence， we found that RECQL1 and WRN proteins are highly expressed and that siRNA-mediated silencing of either gene suppressed carcinoma cell growth in vitro. Similarly， siRNA administration in a murine xenograft model of hypopharyngeal carcinoma markedly inhibited tumor growth. Moreover， combining either siRNA with cis-platinum (II) diammine dichloride significantly augmented the in vivo anticancer effects of this drug that is used commonly in HNSCC treatment. Notably， we observed no recurrence of some tumors following siRNA treatment in this model. Our findings offer a preclinical proof of concept for RECQL1 and WRN proteins as novel therapeutic targets to treat aggressive HNSCC and perhaps other cancers.
目的探讨苯代谢物氢醌(HQ) 对人白血病细胞K562中解旋酶WRN基因mRNA转录及蛋白表达的影响。方法常规培养白血病细胞K562至生长对数期，低剂量HQ组、中剂量HQ组、高 剂量HQ组分别以15、30、60μmol/L浓度HQ重复间隔染毒48及72 h，以等体积的PBS培养的细胞组为空白对照组。采用单细胞凝胶电泳法(SCGE)检测细胞DNA损伤;采用Taqman探针实时荧光定量PCR法检测 WRN基因mRNA水平，采用免疫印迹分析WRN基因蛋白表达水平，计算mRNA及蛋白的相对表达量。结果 (1)HQ可导致细胞DNA损伤，且损伤效应随染毒浓度增加而增大，72 h比48 h的DNA损伤程度增加，呈时间-剂量依赖性;(2)HQ染毒48 h，WRN基因mRNA在各组差异均无统计学意义(P0.05);HQ染毒72 h，各剂量组与空白对照组比较差异有统计学意义(P0.05)，低、中剂量组与高剂量组比较，差异有统计学意义(P0.05)，低剂量组与中剂量组组间差 异无统计学意义(P0.05);(3)HQ染毒48 h，WRN蛋白相对表达量在各组差异无统计学意义(P0.05);HQ染毒72 h，各剂量组与空白对照组比较，蛋白表达随着染毒剂量增大而降低，差异有统计学意义(P0.05)，HQ各剂量组之间两两比较差异无统计学意义 (P0.05)。结论 HQ可导致K562细胞DNA损伤，且呈时间-剂量依赖性，其机制可能与HQ下调WRN基因mRNA转录及蛋白的表达有关。
目的分析永兴县2008— 2011年手足口病的发病动态和流行特征，为今后的防治工作提供依据。方法采用描述流行病学方法，对疫情资料进行三间分布等分析。结果共报告手足口病 2010例，年均发病率为85.468/10万，其中重症20例，死亡3例。发病率以2011年最高，不同年份发病率差异有统计学意义 (χ2=583.192，P=0.000)。各乡镇均有病例报告，其中马田、城关、樟树、柏林和湘阴渡为高发。发病高峰有2个：①4—7月，这一高峰明显 而稳定;②10月前后，尚不清晰。发病以5岁以下儿童居多，占总发病数的94.826%。男女发病率差异有统计学意义 (RR=2.051，95%CI1.860～2.253)，男性高于女性。结论永兴县2008—2011年手足口病呈高度散发和上升趋势，有明显的时间、 地区和人群差异，手足口病已成为当地主要传染病，应针对性地加大防治力度。
RecQ helicase family members are involved in multiple DNA repair pathways， protecting the genome from incorrect recombination during mitosis and maintaining its stability. Deficiencies in genes encoding the RecQ helicases WRN and BLM lead to rare autosomal recessive diseases， Werner and Bloom syndromes， which have been implicated in early onset of aging， and predisposition to various types of cancer. We investigated associations of ， and -associated protein (/) gene polymorphisms and risk of colorectal cancer (CRC)， genotyping V114I (rs2230009)， L1074F (rs2725362)， C1367R (rs1346044)， S455N (rs1982151) and P868L (rs11852361). A large population-based casecontrol study， including 1795 CRC cases and 1805 controls， found no evidence for an association between the selected allelic variants in DNA repair-related genes and CRC risk. However， we detected a significant association of P868L with an increased rectal cancer risk (odds ratio1.29， 95% confidence interval 1.021.64 and 0.04)， suggesting a potential cancer-site specificity. This is the first study to analyze the associations between polymorphisms in ， and and CRC risk. Although none of them showed a significant association with CRC， the association of P868L with rectal cancer risk requires further investigation.