应用染色质免疫沉淀技术分析DNMT3b和HDAC1蛋白与SLC22A18基因启动子的结合

目的 建立优化的染色质免疫沉淀技术(ChIP),分析乳腺癌细胞MDA-MB-231中DNA甲基转移酶3b(Dnmt3b)和组蛋白去乙酰化酶1(HDAC1)与SLC22A18基因启动子区的结合情况. 方法 建立并优化ChIP实验技术,运用ChIP技术检测DNA甲基转移酶抑制剂5-氮杂-2′-脱氧胞苷(5-aza-dc)和组蛋白去乙酰化酶抑制剂曲古霉素A(TSA)分别单独和联合作用于人乳腺癌细胞MDA-MB-231后,用Dnmt3b和HDAC1特异性抗体沉淀DNA,聚合酶链式反应(PCR)检测SLC22A18基因5＇端特异性序列,免疫印迹法(Western blotting)检测Dnmt3b和HDAC1蛋白的表达情况. 结果 获得了优化的ChIP实验条件,Dnmt3b和HDAC1抗体沉淀的染色质片段中扩增出SLC22A18基因5＇端特异性序列.5-aza-dc、TSA单独用药可抑制DNMT3b、HDAC1与SLC22A18启动子区特异序列的结合,5-aza-dc和TSA联合作用可以明显抑制DNMT3b、HDAC1与SLC22A18启动子的结合;Western blotting结果表明,5-aza-dc、TSA单独及联合用药不影响DNMT3b和HDAC1蛋白的表达. 结论 DNMT3b和HDAC1在MDA-MB-231细胞内结合于SLC22A18基因启动子的特异区域,参与该基因的表达调控.     

胸苷激酶1在肿瘤疾病中的应用研究

胸苷激酶（TK,ATP：thymidine5′-phosphotransferase,EC.2.7.1.21,简称TK）是DNA合成过程中的关键酶之一,在ATP和Mg2＋参与下,催化脱氧胸苷（TdR）为脱氧1-磷酸胸苷酸（dTMP）。TK在人类细胞中以两种同功酶形式存在：细胞质胸苷激酶（TK1）和线粒体胸苷激酶（TK2）。TK1与DNA复制密切相关,在细胞周期G1期含量较低,S期逐渐升高,到G2期最高,临床研究证明,TK1在95%的恶性肿瘤细胞中都有升高,故TK1被认为是一种极具生命潜力的细胞增殖标志物,它在肿瘤早期发现、肿瘤疗效评估、肿瘤预后判断等方面都有广阔的临床应用前景。有鉴于此,本文就TK1在肿瘤疾病中的应用研究作一简要综述。     

抗HIV药物的新发展

随着高效联合抗逆转录病毒治疗（HAART）的进展，副作用、耐药性等问题日益突出，影响了持续治疗。目前正在研究许多新的药物，它们具有较小的毒副作用、更好的抗病毒及耐药毒株活性、服用方便等优点。本文阐述了正在开发中的核苷类逆转录酶抑制（NRTIs）、非核苷类逆转录酶抑制剂（NNRTIs）、蛋白酶抑制剂（Pis）及HIV病毒进入细胞抑制剂，阐述了它们的作用特点、使用剂量、剂型、抗病毒作用的活性及毒副作用，与其它抗病毒药物相互作用的研究。它们大多将会进入大规模临床研究，并可能获得批准而生产上市。     

抗HIV-1活性化合物作用靶点的快速判断方法

目前,艾滋病的防治工作主要依靠抗HIV-1药物,临床使用的抗HIV药物主要是逆转录酶抑制剂、蛋白酶抑制剂、融合抑制剂和整合酶抑制剂。逆转录酶抑制剂包括核苷类逆转录酶抑制剂（NRTI）,如Zidovudine（AZT）、Lamivudine（3TC）、Emtricitabine（FrC）等,非核苷类逆转录酶抑制剂（NNRTI）,如Efavirenz（EFV）、Nevirapine（NVP）、Delavirdine（DLV）等。蛋白酶抑制剂主要有Ritanovir（RTV）、Saquinavir（SQV）、Indinavir（IDV）等。     

急性白血病细胞系中SFRP基因启动子甲基化及去甲基化诱导表达的研究

 目的：探讨分泌型卷曲相关蛋白（SFRP）基因家族启动子CpG岛异常甲基化状态与急性白血病（AL）发生发展的关系,以及DNA甲基转移酶（DNMT）抑制剂5-氮杂-2-脱氧胞苷酸（5-Aza-CdR）去甲基化诱导SFRP基因表达作用的可能机制。方法：采用甲基化特异性PCR检测不同AL细胞系（Molt-4、Jurkat、HL-60和NB4）和不同浓度5-Aza-CdR作用下Jurkat细胞系中SFRP1、SFRP2、SFRP4和SFRP5基因启动子区的甲基化状态,采用实时荧光定量RT-PCR检测SFRP1、SFRP2、SFRP4和SFRP5 mRNA表达,采用半定量RT-PCR检测DNMT1、DNMT3A和DNMT3B mRNA表达。结果：在正常人细胞中不存在SFRP基因的甲基化。SFRP1、SFRP2和SFRP5基因在HL-60、NB4、Molt-4和Jurkat细胞系中均呈完全甲基化状态;SFRP4基因在NB4、Molt-4和Jurkat细胞系中完全甲基化,在HL-60细胞系中则部分甲基化。5-Aza-CdR可逆转SFRP1、SFRP2、SFRP4和SFRP5基因的高甲基化状态,并能够下调DNMT1、DNMT3A和DNMT3B mRNA水平,诱导SFRP基因恢复表达。结论：在AL细胞系中,SFRP1、SFRP2、SFRP4和SFRP5基因出现高甲基化,与AL的发生密切相关,可能成为AL新的基因标志物。5-Aza-CdR通过抑制DNMT表达,逆转SFRP基因的甲基化状态,恢复其表达。     

5-氮杂-2’-脱氧胞苷通过反转原钙黏蛋白10表达抑制人乳腺癌细胞系MDA-MB-231的侵袭和迁移

目的 探讨5-氮杂-2’-脱氧胞苷（5-Aza-CdR）诱导抑癌基因原钙黏蛋白10（PCDH10）重新表达对人乳腺癌细胞MDA-MB-231体外侵袭迁移能力的影响并初步探讨其机制。 方法 体外培养人乳腺癌细胞MDA-MB-231,设置对照组和5-Aza-CdR药物处理组,分别采用反转录聚合酶链反应（RT-PCR）检测PCDH10 mRNA 的表达水平;Transwell法和划痕实验检测细胞的侵袭迁移能力;Western blotting检测PCDH10、DNA甲基转移酶（DNMT）3A、DNMT3B、核因子（NF）-κB p65和基质金属蛋白酶（MMP）-2、MMP-9蛋白表达的变化。 结果 5-Aza-CdR能够反转PCDH10的mRNA和蛋白表达;PCDH10表达恢复后MDA-MB-231细胞的侵袭迁移能力受到抑制;Western blotting检测发现,MDA-MB-231细胞经5-Aza-CdR处理后DNMT3A、DNMT3B、NF-κB p65、MMP-2和MMP-9的表达下调。 结论 5-Aza-CdR可抑制MDA-MB-231细胞DNMT3A和DNMT3B的表达,使抑癌基因PCDH10表达恢复,从而通过阻滞NF-κB p65的活化,下调MMP-2和MMP-9表达而抑制乳腺癌细胞的侵袭转移。     

一氧化氮是GM3激活巨噬细胞介导细胞毒的效应分子

已报道了神经节苷脂ＧＭ３在体外激活小鼠腹腔巨噬细胞介导的细胞毒效应（ＭＴＣ），在此基础上发现ＧＭ３又同时可诱导巨噬细胞合成一氧化氮。后者的合成与巨噬细胞的细胞毒效应呈正相关关系（ｒ＝０．９６４，Ｐ〈０．００１），ＮＯ合酶的抑制剂氨基胍，能显著地减弱ＭＴＣ。上述结果提示，因ＧＭ３激活的ＭＴＣ中，ＮＯ也是效应分子。     

塞来昔布联合阿霉素促胃癌细胞凋亡作用的探讨

目的：探讨非甾体类抗炎药（NSAIDs）中选择性的环氧化酶-2（COX-2）抑制剂塞来昔布与常规化疗药物阿霉素（ADM）小剂量联合用药对MGC-803细胞的促凋亡或抑制增殖作用。方法：传代培养MGc-803细胞，用MTT法检测细胞的增殖程度；用荧光显微镜术，流式细胞术和DNA梯度检测细胞凋亡情况。结果：（1）荧光显微镜下，塞来昔布联合ADM用药组的MGc-803细胞出现典型的细胞凋亡形态学改变；（2）塞来昔布和ADM联合使用可以诱导细胞凋亡发生，DNA梯形条带较单独使用时更加明显：（3）流式细胞术显示单独使用塞来昔布的细胞凋亡率为18．1，阿霉素28．7％，小剂量塞来昔布和阿霉素联合使用为64．9％。结论：小剂量塞来昔布与ADM联合使用可以促进胃癌细胞凋亡。     

加用完形心理治疗及奥氮平对躯体形式障碍的作用

目的：研究单一药物、联合用药、联合用药加完形心理治疗三种治疗方式对躯体形式障碍的疗效。方法：将96例躯体形式障碍患者分为三组，分别采用单一药物5一羟色胺再摄取抑制剂（SSRI）、联合用药（SSRI加小剂量非典型抗精神病药物），以及联合用药加完形心理治疗三种治疗方式。治疗前和治疗3个月后采用汉密尔顿抑郁量表（HAMD）进行测查，评定三种治疗方式的减分率和有效率。结果：（1）三种治疗方式的有效率分别为53．1％、61．8％和96．7％，以联合用药加完形心理治疗组最高，联合用药组次之（82．4％）（X^2=15．5，P〈0．05）；（2）治疗后三组患者的HAMD总分减分率均值，以联合用药加完形心理治疗组最高（82．4％），联合用药组次之（57．9％），单一药物组最低（45．2％），方差分析组问差异显著，Welch统计量为20．92，P〈0．001；Tamhane方法多重比较，联合用药加完形心理治疗组与氟西汀组、联合用药组均有显著性差异；氟西汀组与联合用药组仅躯体化、睡眠、绝望感三因子分有显著性差异。结论：三种治疗方式对躯体形式障碍均有明显疗效；联合用药加完形心理治疗对躯体形式障碍的疗效优于单纯药物治疗。     

阿霉素联合塞来昔布诱导胃癌细胞凋亡的作用研究

目的:研究环氧化酶-2 (COX-2) 抑制剂塞来昔布联合阿霉素对胃癌MGC-803细胞株的凋亡诱导作用，并探讨其相互作用的可能的分子机制。方法:用MTT法检测MGC-803的增殖情况。用荧光显微镜、流式细胞术和DNA梯度电泳检测肿瘤细胞凋亡的情况。结果:随着阿霉素剂量的增加，MGC-803细胞的数量明显减少。细胞大部分静止于G₀/G₁期，S期细胞明显减少。 阿霉素（5 mg/L）联合塞来昔布（25 μmol/L）明显抑制MGC-803细胞的生长。肿瘤细胞经阿霉素或塞来昔布处理后荧光显微镜下可观察到典型的细胞凋亡形态变化。与两者单独用药相比，联合用药后的DNA梯度变化更为明显。联合用药 48 h 后MGC-803细胞堆积在G₀/G₁，而S期减少的细胞数量比两者分别用药时更为显著。结论：塞来昔布和阿霉素具有协调的诱导凋亡的作用，这对于将COX-2抑制剂用于肿瘤的临床辅助化疗具有重要意义。     

Biochemical modulation of 5-fluorouracil with a streptococcal preparation, OK-432, against murine colon-26 carcinoma

Conventional therapy for colorectal carcinoma using 5-fluorouracil (5-FU) has shown limited antitumor action. The purpose of our study was to investigate synergistic antitumor effects of the streptococcal preparation of OK-432 and 5-FU, and to elucidate the mechanisms of interaction between the 2 agents in mice. Biochemical modulation of OK-432 and 5-FU were determined in vivo against colon-26 carcinoma. The concentration of 5-FU and its metabolites, and the activity of thymidylate synthase and thymidine kinase, respectively, were measured using cytosolic extracts of the tumors. Combination treatment with OK-432 produced a significant increase in intratumor 5-FU and 5-FU in RNA (F-RNA) concentrations, increased the thymidylate synthetase inhibition rate, and decreased thymidine kinase activity, as compared with the results observed in the control mice. These additive antitumor effects are obtained by use of the 2 agents; the mechanism of action is considered to be the suppression of both the de novo and the salvage pathway for DNA synthesis, along with the suppression of RNA synthesis.     

The use of thymidylate synthase inhibitors in the treatment of advanced colorectal cancer: current status

The combination of 5-fluorouracil (5-FU) and leucovorin has been the unofficial "standard" therapy for patients with colorectal cancer for over a decade. Recently, however, a number of new agents targeted against the enzyme thymidylate synthase have been synthesized and are in various stages of development. The currently available thymidylate synthase inhibitors are discussed. Enormous efforts have been made over the years to improve the efficacy of 5-FU, the most popular of these agents. Biochemical modulation by leucovorin has been the most successful so far. Continuous infusion schedules also appear to be advantageous over bolus administration. However, marked intra- and interpatient variability, combined with nonlinear elimination kinetics and erratic oral bioavailability are relative limitations to further development of 5-FU. New oral 5-FU prodrugs such as UFT, S-1, and Capecitabine may help to overcome some of these difficulties. Eniluracil, a potent inhibitor of the enzyme dihydropyrimidine dehydrogenase, may also help by overcoming potential 5-FU resistance mechanisms, in addition to increasing its bioavailability. Of the antifolate-based inhibitors, Tomudex is in the most advanced stage of development. Similar efficacy with 5-FU and a convenient schedule may suggest a role in future combination regimens. It is quite likely that even the most optimal thymidylate synthase inhibition will have limitations in terms of clinical efficacy. Novel combinations of 5-FU or its analogs with agents that have different mechanisms of action (e.g., oxaliplatin, irinotecan) could provide important new opportunities for improving the outlook of patients with colorectal cancer.     

5-Chloro-2′-deoxyuridine cytotoxicity results from base excision repair of uracil subsequent to thymidylate synthase inhibition

The lack of a phenotypic alteration of 5-hydroxymethyluracil (hmUra) DNA glycosylase (hmUDG) deficient Chinese hamster V79mut1 cells exposed to DNA-damaging agents known to produce hmUra has raised the question whether there might be DNA substrates other than hmUra for hmUDG. Based on the structural similarity between 5-chlorouracil (ClUra) and hmUra and the observations that 5-chloro-2′-deoxyuridine (CldUrd) induces base excision repair (BER) events, we asked whether hmUDG or some other DNA BER enzyme is responsible for the removal of ClUra from DNA. An in vivo flow cytometry assay with FITC-anti-BrdUrd (which cross-reacts with CldUrd) showed that exogenous CldUrd is incorporated into DNA. However, both in vivo and in vitro experiments indicated that ClUra is not excised from DNA by hmUDG or other DNA glycosylase activities. The absence of removal of ClUra by hmUDG raised the question whether DNA strand breaks occurred subsequent to thymidylate synthase inhibition, leading to deoxyuridine incorporation, followed by cleavage of uracil from DNA by uracil DNA glycosylase (UDG). An in vivo thymidylate synthase activity assay in V79 cells demonstrated that CldUrd treatment inhibits thymidylate synthase as effectively as 5-fluoro-2′-deoxyuridine (FdUrd) treatment. Uracil, a known UDG inhibitor, partially reverses the cytotoxic effects of CldUrd on V79 cells, thus confirming that CldUrd induced cytotoxicity is a result of UDG activity. Our results demonstrated that while CldUrd is not directly repaired from DNA, its cytotoxicity is directly due to the UDG removing uracil subsequent to inhibition of thymidylate synthase by CldUMP.     

胃癌干细胞对氟尿嘧啶敏感性及化疗药物耐受细胞的生物学机制

背景：氟尿嘧啶是胃癌化疗的基础药物,临床上耐药现象较为常见。肿瘤干细胞对化疗药敏感性较低,可能是导致化疗后肿瘤复发进展的重要原因。 目的：探讨胃癌干细胞对氟尿嘧啶敏感性,从细胞生物学角度分析胃癌的化疗耐药机制。 方法：利用免疫组织化学染色法检测69例胃癌组织内干细胞标志物CD44和耐药蛋白胸苷酸合成酶表达情况;基于克隆形态的分选策略,从AGS胃癌细胞系内分离胃癌干细胞克隆,检测CD44和胸苷酸合成酶的表达和自我更新能力;利用CCK-8法检测不同AGS细胞克隆的5-氟尿嘧啶半数抑制浓度(IC50)。 结果与结论：69例胃癌组织标本中,胸苷酸合成酶和CD44的表达阳性率分别为57%(39/69)和61%(42/69),CD44与胸苷酸合成酶的表达之间呈正相关关系(Kappa=0.41,χ²=11.59,P < 0.05)。AGS细胞系的克隆形成率为39%(29/69),其中,副克隆、次克隆、全克隆所占比例分别为 17%(5/29)、69%(20/29)、14%(4/29)。二代克隆形成后,采用胰酶消化克隆并再次低密度接种传代,副克隆均无法传代,次克隆仅少数可连续传代,全克隆均可连续传代。不同浓度5-氟尿嘧啶作用之后,全克隆的生长抑制率均显著低于次克隆和AGS细胞,经比较差异均有显著性意义(P < 0.05)。以上结果表明胃癌干细胞对5-氟尿嘧啶敏感性较低,其对化疗药物耐受,可能是临床胃癌化疗耐药的产生机制之一。     

Induction of invasive and metastatic potential in mouse T-lymphoma cells (BW5147) by treatment with 5-azacytidine

Non-invasive, non-metastatic mouse BW5147 T-lymphoma cells were treated with non-mutagenic concentrations of the hypomethylating agent 5-azacytidine (5-aza-C). Subsequently, invasive variants were selected on monolayers of rat embryo fibroblasts. The estimated frequency of induction of invasive variants was smaller than 1 in 10⁶ cells. We obtained several independent clones that were stable in the expression of the invasive phenotype. In contrast to the parental cell line, the highly invasive clones produced widespread metastases upon tail vein injection in all the syngeneic AKR mice tested, whereas' clones with an intermediate level of invasiveness formed metastases only in part of the mice tested. DNA analysis using the methylation-sensitive and insensitive restriction enzymes, Hpa-II and Msp-I, respectively, showed that the DNA of the invasive variants remained hypomethylated, up to 6 months after 5-aza-C treatment. 5-aza-C is thus able to induce invasive and metastatic potential in the BW5147 T-lymphoma cells, similar to the activated human c-Ha-ras oncogene or human chromosome 7, as studied previously. The acquisition of invasive and metastatic potential is presumably caused by DNA hypomethylation and thus activation of one or more silent invasion controlling genes.     

重组人肿瘤坏死因子与阿霉素抗人结肠癌细胞株（SW480）的序贯作用研究

目的 评价rHuTNF和阿霉素抗人结肠癌细胞株的序贯作用。方法 采用克隆生成试验（Hamburger-Salmon法）,研究rHuTNF和DOX序贯作用于SW480细胞后的抗增殖效果。结果 对结肠癌细胞系SW480,存在DOX至rHuTNF序贯抗增殖作用;除反序贯给药情况外,出现显著的协同作用。结论 在DOX存在情况下,TNF的抗瘤作用增强,从而支持DOX可干扰细胞保护机制以使瘤细胞对TNF细胞毒作用更为敏感的理论。     

Polymorphisms in one-carbon metabolism pathway genes and risk for bladder cancer in a Tunisian population

Cigarette smoking is the most important risk factor for bladder cancer. Moreover, epidemiologic studies have implicated several genetic variations interfering with methyl group metabolisms in susceptibility for a variety of cancers. Examples of these variations can be found in genes of the folate metabolic pathway, which is crucial in the provision of methyl groups for DNA, RNA, and protein methylation, as well as in purine and pyrimidine synthesis. We conducted a case–control study to examine the relationship between the methylenetetrahydrofolate reductase (MTHFR C677 T and MTHFR A1298C), methionine synthase (5-methyltetrahydrofolate-homocysteine methyltransferase, MTR A2756 G), methionine synthase reductase (5-methyltetrahydrofolate-homocysteine methyltransferase reductase, MTRR A66 G and MTRR C524 T), and thymidylate synthase (TYMS 2R→3R and G/C) genotypes and the risk for bladder cancer in a Tunisian population. The isolated MTHFR 677*T, MTRR 66*G and MTRR 524*T variants did not appear to influence bladder cancer susceptibility. The 3R*C/3R*C genotype for the TYMS gene appears to be a protective factor against bladder cancer development (P=0.0001; OR=0.12; 95% CI=0.03–0.40). However, patients heterozygous for MTHFR A1298C or MTR A2756 G genotypes have 1.62- and 2.13-fold higher risk, respectively, of developing bladder cancer. Moreover, the combined study of MTHFR 1298*C and MTR 2756*G variants with either or both MTRR 66GG and TYMS 3R*G/3R*G genotypes suggests a cumulative effect. Finally, this study evidenced that interaction between gene variations involved in folate metabolism and risk of bladder cancer increased dramatically among smokers.     

Base analogue mutagenesis in yeast and its modulation by pyrimidine deoxynucleotide pool imbalances: incorporation of bromodeoxyuridylate and iododeoxyuridylate

Summary Cells of the yeast, Saccharomyces cerevisiae, which are auxotrophic for thymidylate (tmp1) can also incorporate analogues of thymidylate. When the base analogue, 5-bromodeoxyuridylate, is incorporated into tmp1 yeast cells it is lethal and mutagenic. Both lethality and mutation induction can be drastically altered by perturbation of the pyrimidine nucleotide pools. Analysis of mutation induction, bromodeoxyuridylate incorporation into DNA, and cell viability under various conditions revealed: (1) lethality and mutagenesis can be uncoupled, (2) thymidylate enhances mutagenesis and deoxycytidylate suppresses it, (3) mutation induction is not correlated with the magnitude of bromodeoxyuridylate incorporation into DNA. Therefore, in yeast, the pyrimidine nucleotide pools have a powerful effect on bromodeoxyuridylate mutagenesis.Both bromodeoxyuridylate and iododeoxyuridylate are extensively incorporated into the DNA of tmp1 yeast cells; however, iododeoxyuridylate is non-mutagenic. Replication proceeds at the same rate in the presence of the natural substrate or either analogue. When cells are supplied with thymidylate and bromodeoxyuridylate together, there is no discrimination against bromodeoxyuridylate as a DNA precursor. However, in the presence of thymidylate and iododeoxyuridylate, there is a 3 to 1 discrimination against iododeoxyuridylate as compared to thymidylate.Abbreviations BU 5-bromouracil - BrdUrd bromodeoxyuridine - BrdUMP bromodeoxyuridylate - BrdUTP bromodeoxyuridine triphosphate - CDP cytidine diphosphate - dCyt deoxycytidine - dCMP deoxycytidylate - dCDP deoxycytidine diphosphate - dCTP deoxycytidine triphosphate - IdUrd iododeoxyuridine - IdUMP iododeoxyuridylate - dThd deoxythymidine - dTMP deoxythmidylate - dTTP deoxythymidine triphosphate     

Selection of mammalian thymidine auxotrophic cell mutants defective in thymidylate synthase by their reduced sensitivity to methotrexate

Thymidine auxotrophic mutants were selectively isolated from mutagenized mouse FM3A cells by resistance to methotrexate in the presence of thymidine and 5-methyl-tetrahydrofolate with a frequency of 10^–5–10^–6. In most of the thymidine auxotrophs the activity of thymidylate synthase was very low or undetectable, but dihydrofolate reductase activity was normal. Upon starvation of thymidine, the mutant cells immediately stopped growing and started to lyse within one day. In the presence of thymidine, the mutant cells grew quite normally. This phenotype behaved recessively in cell—cell hybrids, and the segregation profile of its marker indicated that the lesions in the mutants are not linked to the X chromosome. Prototrophic revertants could be isolated from these mutants, and they showed almost the normal level of thymidylate synthase activity. The selection method described here should be useful for isolating large numbers of thymidylate synthase-negative mutants from various mammalian cell lines.