O^6—甲基鸟嘌呤—DNA—甲基转移酶与肿瘤预见性化疗

以中国人肿瘤株和活检组织为材料,观察了Ｏ^6－甲基鸟嘌呤－ＤＮＡ－甲基转移酶（ＭＧ ＭＴ）酶活性与亚硝脲药物耐药笥之间的关系,证明ＭＧＭＴｍＲＮＡ高表达是产生耐药笥的原因;体外和临床实验证明链脲菌素和苄基鸟嘌呤可以抑制ＭＧＭＴ酶活笥,克服耐药性;体外实验证明反义寡聚核苷酸可以调节ＭＧＭＴ基因表达,逆转录病毒介导的反义ＲＮＡ可以调节ＭＧＭＴ     

反义RNA调节肿瘤细胞O~6-甲基鸟嘌呤-DNA甲基转移酶活性

报道了逆转录病毒载体介导的反义ＲＮＡ对肿瘤细胞Ｏ６－甲基鸟嘌呤－ＤＮＡ甲基转移酶（ＭＧＭＴ）活性的调节作用．构建了三个表达ＭＧＭＴ反义ＲＮＡ的逆转录病毒载体并用它们转染ＨｅＬａＳ３肿瘤细胞,观察细胞在转染前后ＭＧＭＴｍＲＮＡ水平、ＭＧＭＴ活性及其对ＡＣＮＵ抗药性的变化．发现针对ＭＧＭＴｍＲＮＡ５’端的反义ＲＮＡ能够有效地降低ＭＧＭＴｍＲＮＡ水平和ＭＧＭＴ活性并使细胞对ＡＣＮＵ的敏感性提高４．６倍;针对ＭＧＭＴｍＲＮＡ全长的反义ＲＮＡ也能在一定程度上调节细胞的ＭＧＭＴｍＲＮＡ水平和ＭＧＭＴ活性并增加细胞对ＡＣＮＵ的敏感性,而针对３’端序列的反义ＲＮＡ对ＭＧＭＴ活性没有调节作用．     

用逆转录病毒载体表达人粒细胞－巨噬细胞集落刺激因子

应用基因工程的方法,将含有巨细胞病毒（ＣＭＶ）启动子的基因片段和人粒细胞－巨噬细胞集落刺激因子（ｈＧＭ－ＣＳＦ）的ｃＤＮＡ,克隆进逆转录病毒载体Ｎ２Ａ,得到重组质粒Ｎ２Ａ／ＣＭＶ／ｈＧＭ－ＣＳＦ．经脂质体包装并转染包装细胞,通过Ｇ４１８药物筛选,得到抗性克隆。经ＰＣＲ和Ｓｏｕｔｈｅｍｂｌｏｔ检测证实,ＧＭ－ＣＳＦ基因已整合到该克隆细胞的染色体上,获得的逆转录病毒滴度达１０￣４ＣＦＵ／ｍｌ,克隆细胞培养上清用ＴＦ－１细胞可检测到ＧＭ－ＣＳＦ活性。     

抑制O~6-甲基鸟嘌呤-DNA甲基转移酶活性与肿瘤细胞对亚硝脲药物敏感性关系的研究

80％以上的肿瘤细胞为O~6-甲基鸟嘌吟-DNA甲基转移酶(O~6-MT)活性较高的Mer~+型,能够修复亚硝脲药物(NU)造成的DNA烷化损伤,对NU不敏感。本实验证明,用0.75,0.50和0.25mmol/L甲基亚硝脲(MNU)分别处理Mer~+型的HeLaS3,SMMC-7721和表现Mer~-型特征的Cc801,均能明显降低细胞中O~6-MT活性,从而显著提高了三种细胞对嘧啶亚硝脲和双氯乙亚硝脲的敏感性,提示降低O~6-MT活性是使用NU对Mer~+型肿瘤进行有效治疗的前提。     

用逆转录病毒载体表达人粒细胞—巨噬细胞集落刺激因子

应用基因工程的方法,将含有巨细胞病毒（ＣＭＶ）启动子的基因片段和人粒细胞－茂噬细胞集落刺激因子（ｈＧＭ－ＧＳＦ）的ｃＤＮＡ,克隆进逆转录病毒载体Ｎ２Ａ,得到重组质粒Ｎ２ＱＡ／ＣＭＶ／ｈＧＭ－ＣＳＦ。经脂质体包装并转染包装细胞,通过Ｇ４１８药物筛选,得到抗生克隆。经ＰＣＲ和Ｓｏｕｔｈｅｒｎ ｂｌｏｔ检测证实,ＧＭ－ＣＳＦ基因已整合到该克隆细胞的染色体上,获得的逆转录病毒滴度达１０＾４ＣＦＵ／ｍｌ,克     

人GM—CSF cDNA的克隆和在大肠杆菌中的表达

从诱导的人胚肺细胞ＨＦＬ株中提取总ＲＮＡ．经ＲＴ－ＰＣＲ反应获取了人ＧＭ－ＣＳＦｃＤＮＡ,ＤＮＡ序列测定表明其顺序与文献报道完全一致。为了获得高效表达,应用ＰＣＲ改造了人ＧＭ－ＣＳＦ的ｃＤＮＡ５’端核苷酸序列,并将改造的人ＧＭ－ＣＳＦ基因插入含Ｔ７启动子的质粒ｐＥＴ－１１ｄ构建成表达质粒ｐＥＴＣ－５,将此质粒转化大肠杆菌株ＢＬ２１（ＤＥ３）得到表达菌株ＢＬＥＣ４。表达菌株用０．５ｍｏｌ／ＬＩＰＴＧ诱导２小时后,产生大量重组蛋白并形成包涵体。ＳＤＳ—ＰＡＧＥ电泳图谱扫描结果表明,ｒｈＧＭ－ＣＳＦ产量占菌体总蛋白量的１６％。ＥＬＩＳＡ和ＴＦ－１细胞培养测定表明,初步纯化和复性的ｒｈＧＭ－ＣＳＦ具有天然的ｈＧＭ－ＣＳＦ生物活性。     

重组人MCAF／GM－CSF融合蛋白表达及其生物学活性研究

利用ＰＣＲ技术和ＤＮＡ体外重组方法,把作为导向效应细胞到靶部位的单核细胞趋化激活因子（ＭＣＡＦ）和粒细胞巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）进行基因融合,置于ｐＢＶ２２０载体的λＰＲＰＬ串联启动子下游,构建了ＳＤ序列与ＡＴＧ之间含有不同核苷酸组成的重组质粒ｐＭＧ０１、ｐＭＧ０２和ｐＭＧ０３。ｐＭＧ０１、ｐＭＧ０２和ｐＭＧ０３的翻译起始区都不存在稳定的二级结构,但ＤＨ５α（ｐＭＧ０２、ＤＨ５α（ｐＭＧ０３）的表达水平远远高于ＤＨ５α（ｐＭＧ０１）,ＤＨ５α（ＰＭＧ０１）几乎没有表达。表达产物经Ｗｅｓｔｅｒｎｂｌｏｔ检测表明,它能分别与ＭＣＡＦ和ＧＭ－ＣＳＦ抗体发生特异反应。生物学活性测定表明,表达产物具有明显的单核细胞趋化活性和维持ｈＧＭ－ＣＳＦ依赖的ＴＦ１细胞生长的特性,说明ＭＣＡＦ和ＧＭ－ＣＳＦ的生物学功能是相容的．     

细胞松驰素B促微丝解聚对DNA合成的作用

利用微丝（ｍｉｃｒｏｆｉｌａｍｅｎｔ,ＭＦ）解聚药物细胞松驰素Ｂ（ｃｙｔｏｃｈａｌａｓｉｎＢ,ＣＢ）处理Ｇ_０期小鼠Ｃ_３Ｈ_（１０）Ｔ_（１／２）成纤维细胞,对Ｇ_０至Ｓ期ＤＮＡ合成,胸腺嘧啶核苷激酶（ｔｈｙｍｉｄｉｎｅｋｉｎａｓｅ,ＴＫ）活性、ＴＫ基因表达、钙调素（ｃａｌｍｏｄｕｌｉｎ,ＣａＭ）水平和一些细胞周期早期基因的表达进行了观察,Ｇ_０期细胞经３ｍｇ／ＬＣＢ处理２ｈ,促ＭＦ解聚增强了血清对Ｓ期细胞ＴＫ活性、ＴＫ基因表达和ＤＮＡ合成的刺激作用,并促进细胞提前进入Ｓ期．血清刺激Ｇ_０期细胞进入晚Ｇ_１期和Ｓ期时,ＣａＭ水平明显升高,而ＣＢ预处理则使ＣａＭ含量进一步增加,特别是ＣＢ处理促使Ｓ期ＣａＭ增加向核内转移．ＣＢ处理明显增强血清对ｃ－ｊｕｎ、ｃ－ｆｏｓ和ｃ－ｍｙｃ基因表达的刺激作用,而ＰＫＣ抑制剂Ｈ_７则抑制ＣＢ处理对这些基因转录的刺激作用,说明ＣＢ使Ｇ_０期细胞ＭＦ解聚刺激ｃ－ｊｕｎ、ｃ－ｆｏｓ和ｃ－ｍｙｃ的转录活性与ＰＫＣ的作用有关．结果表明Ｇ_０至Ｓ期早期ＭＦ的重组可促进细胞进入Ｓ期,增强ＤＮＡ合成．     

小鼠金属硫蛋白 Ⅰ cDNA编码序列的克隆及其在昆虫细胞中的表达

将质粒ｐＢＸ－ＭＴ上的小鼠ＭＴ－ⅠｃＤＮＡ片段切下作为模板,通过ＰＣＲ方法删除该片段的非编码序列,将编码序列克隆到质粒ｐＢＳ－ＳＫ中,经ＤＮＡ序列测定后证明其克隆序列正确．再将ＭＴ－ⅠｃＤＮＡ编码序列插入到转移载体ｐＢａｃＰＡＫ８的ＢａｍＨⅠ和ＥｃｏＲⅠ位点之间,通过磷酸钙／ＤＮＡ共转染方法将其导入昆虫细胞Ｓｆ９中,以Ｗｅｓｔｅｒｎｂｌｏｔ和ＤｏｔＥＩＡ方法对表达产物进行了检测,表达量为１ｍｇ／Ｌ     

表达人GM—CSF重组痘苗病毒抗肿瘤作用的实验研究

将ｈＧＭ－ＣＳＦｃＤＮＡ插入含有Ｐ１１ｋ及２５ｋ双向启动子的痘苗病毒载体ＰＪ１２０的Ｐ１１Ｋ启动子下游,而Ｐ２５Ｋ下游则为ＬａｃＺ基因,成表达质粒ｐＪ１２０／ＧＭ－ＣＳＦ。,以此质粒与痘苗病毒天坛株共转染ＴＫ－１４３细胞。在ＢＵdisplay status     

DNA repair protein O6-methylguanine-DNA methyltransferase in testis and testicular tumors as determined by a novel nonradioactive assay

The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT, alkyltransferase) is an important suicide enzyme involved in defense against O6-alkylating endogenous metabolites and environmental carcinogens. It also plays a pivotal role in primary and acquired resistance of tumors to alkylating anticancer drugs targeting the O6-position of guanine (i.e., methylating and chloroethylating agents). MGMT can thus be considered a crucial biomarker for individual susceptibility to alkylating carcinogens and tumor drug resistance. This implies a need for a fast and convenient method for determination of MGMT. Routinely, MGMT is being quantified by radioactive assays which are relatively laborious. Here we report a nonradioactive MGMT enzyme-linked immunosorbent assay (ELISA) for quantification of MGMT in cell and tissue homogenates. We compared the MGMT-ELISA with the standard radioactive assay and found it to be as sensitive but less time consuming. Therefore, it represents an alternative for the quantification of MGMT in cell and tissue homogenates. We applied the assay for determining MGMT in normal and tumor tissue of testes. In both normal and tumor tissue MGMT was quite variable, ranging from zero to 1300 fmol/mg protein. In various tumor samples MGMT was lower than MGMT in the normal tissue from the same patient or was even not detectable. The MGMT-ELISA might become a useful tool for MGMT determination in clinical routine and health control.     

反义RNA在基因治疗中的应用

由于反义RNA作为封闭基因表达的有效手段具有特异性强、安全性高、操作简单、靶基因范围广等特点,已被广泛应用于基因治疗肿瘤和病毒相关疾病的研究,反义RNA治疗肿瘤可以通过抑制癌基因的表达、封闭融合癌基因、抑制肿瘤细胞的耐药性、调节细胞因子的表达量等途径;反义RNA治疗病毒相关疾病多集中在艾滋病上,其手段主要是反义封闭TAR。反义RNA作为基因治疗的新途径具有良好的前景,但在设计上和应用上还存在一些急待解决的问题。     

MDR1反义RNA降低细胞KB_(v200)的耐药性

由ＭＤＲ１基因过度表达所引起的肿瘤细胞对化疗药物的耐药性,是导致化疗失败的主要原因之一．针对ＭＤＲ１中一段包含转录启始位点、翻译启始位点和转录正调控区的序列,设计了反义ＲＮＡ并将其克隆到逆转录病毒载体ｐＬＸＳＮ上．用脂质体包裹载体导入ＭＤＲ１高表达的耐药细胞ＫＢｖ２００中,在反义ＲＮＡ转染的细胞中,ＭＤＲ１在ｍＲＮＡ和蛋白水平的表达都有下降,细胞内药物的浓度有所提高,对长春新碱、阿霉素的耐药性分别下降了６５％和４７％．实验结果表明,反义ＲＮＡ对ＭＤＲ１的表达有抑制作用,从而使肿瘤细胞内的药物浓度升高,其耐药程度下降．     

Change in expression of MGMT during maturation of human monocytes into dendritic cells

Dendritic cells (DCs) maturated from monocytes play an important role in the immune system, not only in defense against conventional infections but also in cancer rejection. Because of the central role of DCs in tumor host defense it is highly important that DCs as well as the progenitor cell population are protected during cancer therapy. Since most anticancer drugs target DNA, the DNA repair capacity is most importance for the response of DCs and their precursor cells. Here, we studied the expression of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) in monocytes obtained from peripheral blood of healthy donors and DCs maturated from monocytes (moDCs). We show that MGMT is expressed at high level in monocytes, comparable to peripheral lymphocytes. The MGMT expression level declines, however, during DC maturation reaching the low level of CD34+ haematopoetic stem cells. Decline of MGMT was observed on activity, protein and RNA level. It is not related to MGMT promoter methylation, suggesting silencing of the MGMT gene in moDCs occurs by other means. Since maturation of monocytes into DCs is provoked by IL-4 and GM-CSF, the data indicate that MGMT is subject to cytokine-mediated regulation. Despite of the high MGMT level, monocytes were more sensitive to methylating agents (MNNG, temozolomide) and equally sensitive to the chloroethylating agent fotemustine than moDCs, undergoing apoptosis upon treatment. The data provide an example that high MGMT expression level does not necessarily implicate a higher level of resistance against O6-alkylating agents.     

反义RNA抑制人黑色素瘤细胞表面粘附分子CD44的表达及抑瘤效应

将细胞表面粘附分子ＣＤ４４Ｓ的ｃＤＮＡ反向插入到真核细胞表达载体ｐＭＡＭｎｅｏ－ＣＡＴ和ＭＭＴＶ－ＬＴＲ启动子下游,构成ＣＤ４４Ｓ的反义ＲＮＡ载体．将其用电击法导入ＣＤ４４＋的人黑色素瘤细胞系ＨＭＭ２３９,转录出的反义ＲＮＡ能不同程度地抑制ＨＭＭ２３９表面ＣＤ４４的表达．ＣＤ４４的表达被抑制后,瘤细胞与透明质酸的结合力下降,细胞的体外生长速率不受影响．将其接种裸鼠皮下,发现其致瘤性明显降低     

Inhibition of P-glycoprotein expression and reversal of drug resistance of human hepatoma HepG2 cells by multidrug resistance gene (mdr1) antisense RNA

The development of multiple drug resistance in tumor cells is a significant problem in cancer therapy. In human, one of the reasons causing the resistance is due to the overexpression of the mdr1 gene product, P-glycoprotein. In our study, we had developed multiple drug resistant HepG2 cell line (HepG2/DR). To reverse the resistance, HepG2-DR cells were treated with antisense RNA against mdr1 gene. Total RNA and protein were extracted from the transfected cells. Northern analysis showed that mRNA level of mdr1 was decreased whereas a reduction in P-glycoprotein was detected by Western blot. By using flow cytometry, the ability of intracellular doxorubicin retention increased and drug efflux decreased in the treated cells. The result also showed that the cellular sensitivity to doxorubicin, vincristine and methotrexate measured in IC50 increased 83.3% 84.6% and 50% respectively. All these findings suggested that the expression of p-glycoprotein was successfully inhibited by antisense RNA and the drug resistance was reduced.     

中国人肿瘤细胞中O~6-甲基鸟嘌呤DNA甲基转移酶活性与对嘧啶亚硝脲敏感性的关系

 分析了20株中国人肿瘤细胞的O~6-甲基鸟嘌呤DNA甲基转移酶(O~6-MT)活性及对嘧啶亚硝脲ACNU的敏感性,发现两者间有较好的线性关系,O~6-MT低,对ACNU敏感,提示O~6-MT可作为使用ACNU对肿瘤化疗时的一项予见性指标。     

FANCD1/BRCA2 plays predominant role in the repair of DNA damage induced by ACNU or TMZ

Nimustine (ACNU) and temozolomide (TMZ) are DNA alkylating agents which are commonly used in chemotherapy for glioblastomas. ACNU is a DNA cross-linking agent and TMZ is a methylating agent. The therapeutic efficacy of these agents is limited by the development of resistance. In this work, the role of the Fanconi anemia (FA) repair pathway for DNA damage induced by ACNU or TMZ was examined. Cultured mouse embryonic fibroblasts were used: FANCA(-/-), FANCC(-/-), FANCA(-/-)C(-/-), FANCD2(-/-) cells and their parental cells, and Chinese hamster ovary and lung fibroblast cells were used: FANCD1/BRCA2mt, FANCG(-/-) and their parental cells. Cell survival was examined after a 3 h ACNU or TMZ treatment by using colony formation assays. All FA repair pathways were involved in ACNU-induced DNA damage. However, FANCG and FANCD1/BRCA2 played notably important roles in the repair of TMZ-induced DNA damage. The most effective molecular target correlating with cellular sensitivity to both ACNU and TMZ was FANCD1/BRCA2. In addition, it was found that FANCD1/BRCA2 small interference RNA efficiently enhanced cellular sensitivity toward ACNU and TMZ in human glioblastoma A172 cells. These findings suggest that the down-regulation of FANCD1/BRCA2 might be an effective strategy to increase cellular chemo-sensitization towards ACNU and TMZ.