Interplays between ATM/Tel1 and ATR/Mec1 in sensing and signaling DNA double-strand breaks

DNA double-strand breaks (DSBs) are highly hazardous for genome integrity because they have the potential to cause mutations, chromosomal rearrangements and genomic instability. The cellular response to DSBs is orchestrated by signal transduction pathways, known as DNA damage checkpoints, which are conserved from yeasts to humans. These pathways can sense DNA damage and transduce this information to specific cellular targets, which in turn regulate cell cycle transitions and DNA repair. The mammalian protein kinases ATM and ATR, as well as their budding yeast corresponding orthologs Tel1 and Mec1, act as master regulators of the checkpoint response to DSBs. Here, we review the early steps of DSB processing and the role of DNA-end structures in activating ATM/Tel1 and ATR/Mec1 in an orderly and reciprocal manner.     

Direct measurement of single-stranded DNA intermediates in mammalian cells by quantitative polymerase chain reaction

Single-stranded DNA (ssDNA) intermediates are formed in multiple cellular processes, including DNA replication and recombination. Here, we describe a quantitative polymerase chain reaction (qPCR)-based assay to quantitate ssDNA intermediates, specifically the 3′ ssDNA product of resection at specific DNA double-strand breaks induced by the AsiSI restriction enzyme in human cells. We protect the large mammalian genome from shearing by embedding the cells in low-gelling-point agar during genomic DNA extraction and measure the levels of ssDNA intermediates by qPCR following restriction enzyme digestion. This assay is more quantitative and precise compared with existing immunofluorescence-based methods.     

腹腔镜肝癌切除术与开放式肝癌切除术的疗效比较研究

目的：探讨腹腔镜肝癌切除手术治疗原发性肝癌的可行性及安全性。方法：选取2008年6月至2011年1月在我院行腹腔镜肝癌切除术的30例患者作为研究对象,另外选择同期在我院行开放式肝癌切除术的30例患者作为对照。结果：30例均在腹腔镜下成功地完成手术,其中22例行腹腔镜局部切除术,8例行肝左外叶切除术。手术时间103—142min,出血量60-480mL,术后均未发生严重并发症,术后平均住院8．6d。术后随访18～36个月,局部复发或种植性转移率与对照组无显著差异。结论：腹腔镜肝癌切除术是安全可行治疗原发性肝癌的手术方式.     

Methylation-based molecular margin analysis in hepatocellular carcinoma

The positive surgical margins are associated with postsurgical recurrence in hepatocellular carcinoma patients, and molecular margin analysis is considered more sensitive in detecting preneoplastic lesions than conventional histological margin examination. To evaluate the feasibility of methylation-based molecular margin analysis in HCC and explore its clinical application, we investigated CDKN2A methylation status in the surgical margins of 20 HCC patients using a nested BS-MSP protocol and compared the methylation patterns in resection margins with those in the corresponding tumor and adjacent nonmalignant tissues. The results showed that a considerable frequency (35%, 7 of 20) of CDKN2A methylation was present in histologically negative margins, and methylation pattern analysis might be valuable for studying the cellular origin of recurrent carcinoma. Therefore, methylation-based molecular surgical margin analysis offers a promising tool in prognosis for HCC patients who underwent hepatectomy.     

Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other malignancies of the same tissue type, often in a nearby location. Such field defects have been indicated in colon cancer. The molecular abnormalities that are responsible for a field defect in the colon should be detectable at high frequency in the histologically normal tissue surrounding a colonic adenocarcinoma or surrounding an adenoma with advanced neoplasia (well on the way to a colon cancer), but at low frequency in the colonic mucosa from patients without colonic neoplasia.Using immunohistochemistry, entire crypts within 10 cm on each side of colonic adenocarcinomas or advanced colonic neoplasias were found to be frequently reduced or absent in expression for two DNA repair proteins, Pms2 and/or ERCC1. Pms2 is a dual role protein, active in DNA mismatch repair as well as needed in apoptosis of cells with excess DNA damage. ERCC1 is active in DNA nucleotide excision repair. The reduced or absent expression of both ERCC1 and Pms2 would create cells with both increased ability to survive (apoptosis resistance) and increased level of mutability. The reduced or absent expression of both ERCC1 and Pms2 is likely an early step in progression to colon cancer.DNA repair gene Ku86 (active in DNA non-homologous end joining) and Cytochrome c Oxidase Subunit I (involved in apoptosis) had each been reported to be decreased in expression in mucosal areas close to colon cancers. However, immunohistochemical evaluation of their levels of expression showed only low to modest frequencies of crypts to be deficient in their expression in a field defect surrounding colon cancer or surrounding advanced colonic neoplasia.We show, here, our method of evaluation of crypts for expression of ERCC1, Pms2, Ku86 and CcOI. We show that frequency of entire crypts deficient for Pms2 and ERCC1 is often as great as 70% to 95% in 20 cm long areas surrounding a colonic neoplasia, while frequency of crypts deficient in Ku86 has a median value of 2% and frequency of crypts deficient in CcOI has a median value of 16% in these areas. The entire colon is 150 cm long (about 5 feet) and has about 10 million crypts in its mucosal layer. The defect in Pms2 and ERCC1 surrounding a colon cancer thus may include 1 million crypts. It is from a defective crypt that colon cancer arises.     

肝细胞癌根治性切除术后预后因素分析

目的:探讨影响肝细胞癌患者根治性术后预后相关因素。方法:回顾性分析2004年1月1日至2009年12月31日245例我院行根治性切除术的肝细胞癌患者,采用Kaplan-Meier法和Cox比例风险模型分析临床资料、手术过程、病理特征与预后的关系。结果:多因素分析结果显示术前AFP水平、术中出血量、TNM分期是影响无进展生存时间和总生存时间的独立风险因素。术前AFP水平越高、术中出血量越大、TNM分期越晚则患者无进展生存时间及总生存时间明显缩短。此外,患者出现肿瘤组织局部坏死、门静脉癌栓,则总生存时间明显缩短。结论:术前AFP水平、术中出血量、TNM分期是外科根治性切除术后肝细胞癌患者复发及死亡的相关因素,对于临床医师判断预后及延长术后生存时间具有重要的临床意义。     

三种术式重建食管治疗食管癌和贲门癌的临床研究

目的:提高食管癌和贲门癌的根治切除率和临床治愈率,预防吻合口瘘。方法:设计了Ⅰ、Ⅱ、Ⅲ三种术式:上、中段食管癌采用右胸前外侧切口,经第3或4肋间开胸,左腹直肌旁或左肋弓下斜切口开腹和右颈部切口,保留2～3cm颈段食管,食管次全切除,贲门部或部分胃切除,在颈部食管胃端侧分层吻合(Ⅰ式)。中段食管癌采用左胸前外侧切口,经第4或第5肋间开胸,腹部切口同前,左颈部切口,保留3～4cm颈段食管,食管次全切除,部分胃切除,在左颈部食管胃端侧分层吻合(Ⅱ式)。贲门癌采用左胸前外侧切口,经第5或4肋间开胸,腹部切口同前,中段食管和近半胃或纵半胃切除,在主动脉弓下食管胃端侧分层吻合(Ⅲ式)。尽可能清除区域淋巴结,吻合口均用大网膜包盖加固。结果:食管癌和贲门癌总切除率92．1%(174/189),其中根治性切除率为75．1%(142/189),探查率(未切除)为7．9%(15/189),三种术式的总吻合口瘘发生率为4．0%(7/174),无围手术期死亡。结论:三种术式可提高根治性切除率,大网膜包盖加固吻合口可减少瘘的发生率,食管胃分层吻合法可降低吻合口狭窄的发生率,临床治愈率高,围手术期死亡率低。     

Human Ku70/80 protein blocks exonuclease 1-mediated DNA resection in the presence of human Mre11 or Mre11/Rad50 protein complex

DNA double strand breaks (DSB) are repaired by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Recent genetic data in yeast shows that the choice between these two pathways for the repair of DSBs is via competition between the NHEJ protein, Ku, and the HR protein, Mre11/Rad50/Xrs2 (MRX) complex. To study the interrelationship between human Ku and Mre11 or Mre11/Rad50 (MR), we established an in vitro DNA end resection system using a forked model dsDNA substrate and purified human Ku70/80, Mre11, Mre11/Rad50, and exonuclease 1 (Exo1). Our study shows that the addition of Ku70/80 blocks Exo1-mediated DNA end resection of the forked dsDNA substrate. Although human Mre11 and MR bind to the forked double strand DNA, they could not compete with Ku for DNA ends or actively mediate the displacement of Ku from the DNA end either physically or via its exonuclease or endonuclease activity. Our in vitro studies show that Ku can block DNA resection and suggest that Ku must be actively displaced for DNA end processing to occur and is more complicated than the competition model established in yeast.     

甲状腺大部切除术方法探讨

目的：评价改良甲状腺大部切除术的效果与安全性。方法：回顾性分析1997年3月。2005年10月102例甲状腺良性疾病患者,甲状腺大部切除术后医疗效果。结果：94例痊愈,6例出现术后并发症,2例术后复发。结论：改良的甲状腺大部切除术能明显减轻手术创伤,缩短手术时间,减少术后并发症的发生,提高术后主观满意度,是治疗甲状腺良性疾病的理想术式。     

The N-terminal 45-kDa Domain of Dna2 Endonuclease/Helicase Targets the Enzyme to Secondary Structure DNA

The removal of initiating primers from the 5′-ends of each Okazaki fragment, required for the generation of contiguous daughter strands, can be catalyzed by the combined action of DNA polymerase δ and Fen1. When the flaps generated by displacement of DNA synthesis activity of polymerase δ become long enough to bind replication protein A or form hairpin structures, the helicase/endonuclease enzyme, Dna2, becomes critical because of its ability to remove replication protein A-coated or secondary structure flaps. In this study, we show that the N-terminal 45-kDa domain of Dna2 binds hairpin structures, allowing the enzyme to target secondary structure flap DNA. We found that this activity was essential for the efficient removal of hairpin flaps by the endonuclease activity of Dna2 with the aid of its helicase activity. Thus, the efficient removal of hairpin structure flaps requires the coordinated action of all three functional domains of Dna2. We also found that deletion of the N-terminal 45-kDa domain of Dna2 led to a partial loss of the intra-S-phase checkpoint function and an increased rate of homologous recombination in yeast. We discuss the potential roles of the N-terminal domain of Dna2 in the maintenance of genomic stability.