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。     

O^6—甲基鸟嘌呤—DNA—甲基转移酶的基因表达调控

DNA损伤修复是生命科学研究的重要课题。DNA修复在防止基因突变,保证遗传信息稳定性的过程中起着极其重要的作用。近20年来,人们通过大量研究发现细胞内0~6-甲基鸟嘌呤-DNA-甲基转移酶(O~6-metnylguanine-DNA-methyltransferase,MGMT)能专一性很强地修复DNA烷化损伤,防止细胞癌变和死亡;大量试验还证明细胞内MGMT活性的高低是决定肿瘤细胞对亚硝脲类药物产生耐药性的分子基础。详尽了解MGMT基因表达调控机理对DNA损伤修复的理论研究和克服肿瘤细胞耐药性的实践都具有重要的意义。     

DNA烷化损伤及修复的分子基础

烷化剂可以造成细胞DNA分子的烷化损伤。其中鸟嘌呤第6位氧原子的甲基化(O~6-MeG)会造成碱基错误配对,引起细胞致突致死。O~6-甲基鸟嘌呤DNA-甲基转移酶(O~6-MT)可以修复O~6-MeG损伤。原核细胞中编码O~6-MT的烷化损伤修复酶基因ada已经克隆成功。哺乳动物细胞的烷化损伤修复基因的克隆工作正在研究中。根据O~6-MT含量可以把人肿瘤细胞分为两类,Mer~ 和Mer~-。Mer~-不含O~6-MT,约占1/5左右。细胞学及裸鼠实验证明使用双功能烷化剂ACNU可以特异性地杀死Mer~-类肿瘤。提示以DNA烷化损伤修复研究为基础,可以开拓出一条肿瘤选择性化疗的新途径。     

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．     

O~6-甲基鸟嘌呤受体蛋白的定量测定

本文介绍了一种细胞提取液O~6—甲基鸟嘌呤(O~6—MeGua)受体蛋白测定及其底物O~6-[~8H-Me]Gua DNA的制备方法。废物与受体蛋白反应后,甲基从O~6-[~3H-Me]Gua DNA转移到受体蛋白,生成甲基-S-半胱氨酸(Me-S-Cys)。经盐酸水解后,直接测定酸不溶部分的受体蛋白沉淀。方法简便、快速、准确。     

去叶对不同生长习性大豆固氮作用的影响

生殖生长期开始去叶后 ,(1)有限型、亚有限型和无限型 3种类型的大豆根瘤固氮酶活性都降低 ,而根瘤中酰脲含量则不同程度地增加 ;(2 )有限型大豆幼茎中酰脲含量明显增加 ,但亚有限型和无限型大豆变化不大 ;(3) 3种类型大豆幼茎中硝态氮含量增加明显     

应用反义RNA技术降低肿瘤细胞的耐药性

细胞内的Ｏ６－甲基鸟嘌呤－ＤＮＡ甲基转移酶（ＭＧＭＴ）能够修复亚硝脲药物造成的ＤＮＡ损伤,阻止亚硝脲药物对肿瘤的杀伤作用,使细胞对亚硝脲药物产生耐药性．我们曾经构建了三个表达ＭＧＭＴ反义ＲＮＡ的逆转录病毒载体,导入对亚硝脲呈抗性的ＨｅＬａＳ３细胞,并...     

细胞和组织中的O~6-甲基脱氧鸟嘌呤核苷的脱甲基作用

为了探讨细胞和组织中的O~6-甲基脱氧鸟嘌呤核苷的脱甲基作用,我们合成了[~3H]O~6-mGua DNA、O~6-mdGuo、O~6-mdGMP、O~6-mdGTP等底物,在体外利用高压液相色谱分析细胞和组织提取物去除鸟嘌呤基团第六位氧原子上甲基的能力。结果表明,在细胞和组织提取物中存在一种去甲基酶,它能去除O~6-甲基脱氧鸟嘌呤核苷、O~6-甲基脱氧鸟嘌呤核苷-磷酸上的甲基,生成脱氧鸟嘌呤核苷或脱氧鸟嘌呤核苷-磷酸,并伴随着甲醇的释放。它不同于DNA甲基转移酶。没有观察到它对O~6-甲基鸟嘌呤DNA、O~6-甲基脱氧鸟嘌呤核苷三磷酸、O~4-甲基胸腺嘧啶核苷以及O~6-甲基鸟嘌呤的去甲基作用。     

Zn掺杂TiO_2薄膜制备及其光催化降解亚甲基蓝

采用溶胶-凝胶浸渍提拉法在普通载玻片上制备了Zn掺杂TiO2薄膜,利用XRD、SEM对薄膜的结构、形貌进行了分析,并通过对亚甲基蓝(MB)溶液的降解率来评价其光催化活性。XRD结果表明:当掺入的锌含量达到1wt%时,除了锐钛矿型的TiO2,还出现了一种新相ZnTiO3的衍射峰,其结构为钙钛矿型,通过薄膜的SEM图像也能观察到ZnTiO3。通过对亚甲基蓝降解率的分析发现,掺锌量为1wt%的TiO2薄膜光催化活性较高,光照40min后对MB溶液的降解率高达98.7%。     

Pol kappa partially rescues MMR-dependent cytotoxicity of O6-methylguanine

To maintain genomic integrity cells have to respond properly to a variety of exogenous and endogenous sources of DNA damage. DNA integrity is maintained by the coordinated action of DNA damage response mechanisms and DNA repair. In addition, there are also mechanisms of damage tolerance, such as translesion synthesis (TLS), which are important for survival after DNA damage but are potentially error-prone. Here, we investigate the role of DNA polymerase κ (pol κ) in TLS across alkylated lesions by silencing this polymerase (pol) in human cells using transient small RNA interference. We show that human pol κ has a significant protective role against methyl nitrosourea (MNU)-associated cytotoxicity without affecting significantly mutagenicity. The increase in MNU-induced cytotoxicity when pol κ is down-regulated was affected by the levels of O6-methylguanine DNA methyltransferase and fully abolished when mismatch repair (MMR) was defective. Following MNU treatment, the cell cycle profile was unaffected by the pol κ status. The downregulation of pol κ caused a severe delay in the onset of the second mitosis that was fully dependent on the presence of O6-methylguanine ( O6-meGua) lesions. After MNU exposure, in the absence of pol κ, the frequency of sister chromatid exchanges was unaffected whereas the induction of RAD 51 foci increased. We propose that pol κ partially protects human cells from the MMR-dependent cytotoxicity of O6-meGua lesions by restoring the integrity of replicated duplexes containing single-stranded gaps generated opposite O6-meGua facilitated by RAD 51 binding.     

Exogenous O6-methylguanine inhibits adduct removal and sensitizes human cells to killing by the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine

We partially depleted the O6-methylguanine-DNA methyltransferase activity in four O6-methylguanine (O6-mGua) repair-proficient (Mer+) human cell strains with exogenous O6-mGua (2 mM for 3 h, a non-toxic regimen) and then challenged them with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). MT-partially depleted HT29 cells removed O6-mGua from DNA at about half the rate of control cells, while removal of 3-methyladenine was unaffected. In spite of partial depletion of MT, however, cell killing by MNNG in a colony-forming assay with HT29, A549, A498 or KD cells was not greatly affected. (This is in contrast to the dramatic potentiation of CNU cytotoxicity observed previously.) In an attempt to sensitize Mer+ strains to killing by MNNG, we treated cells with O6-mGua following MNNG exposure (0.4 mM for 4 days), in addition to the pre-MNNG treatment of 2 mM O6-mGua for 3 h. This sensitized KD and HT29 cells 2-fold to killing by MNNG, based on the dose at 10% survival, but did not sensitive Mer- A1336. However, post-treatment alone was as effective as combined pre- and post-treatment in sensitizing KD cells to killing. Thus, when the O6-mGua post-treatment was begun, greater than 50% of O6-mGua was already removed from cell DNA. Our findings may be accounted for by at least two schemes, one in which nonlethal O6-mGua are removed from DNA rapidly, while potentially lethal O6-mGua are repaired later. The other scheme proposes that exogenous O6-mGua increases the lethality of a non-O6-mGua lesion by reducing its repair both in Mer+ and Mer- cells. Both schemes are consistent with the hypothesis that O6-mGua may be a lethal DNA lesion in human cells.     

Relationship of methyl purines produced by MNNG in adenovirus 5 DNA to viral inactivation in repair-deficient (Mer-) human tumor cell strains

Adenovirus 5 treated with MNNG (N-methyl-N'-nitro-N-nitrosoguanidine) has greater plaque-forming ability in cell strains having the Mer+ phenotype than in strains having the Mer- phenotype. MNNG-treated Mer- strains repair the N3-methyladenine (N3MeA) but not the O6-methylguanine (O6MeG) produced in their DNA, while MNNG-treated Mer+ strains repair both of these adducts. The fate of N7-methylguanine (another DNA adduct produced by MNNG) is similar in Mer+ and Mer- strains. We show in this paper that 2.3 +/- 0.4 O6MeG and 1.4 N3MeA per adenovirus genome correlate with one lethal hit when the survival assay is done using Mer- strains as viral hosts. We suggest that O6MeG is the lesion lethal to the virus.     

Comparison of geno- and cytotoxicity of methylnitrosourea on MMR-proficient and MMR-deficient human tumor cell lines

Deficient mismatch repair (MMR) is identified as a mutation of one of four major MMR genes and(or) microsatellite instability. These genomic changes are used as markers of MMR status of the heredity nonpolyposis colorectal cancer (HNPCC) spectrum tumors--familial and sporadic tumors of colon and extracolonic cancers fulfilling Amsterdam clinical criteria II. MMR-deficiency results in mutator phenotype and resistance to geno- and cytotoxicity of alkylating agents. The main cytotoxic damage to DNA in response to chemical methylation is O6-methylguanine (O6-mG). The secondary DNA strand breaks, which are formed during the MMR functioning, are proposed to be required for methylation induced cytotoxicity. We have assumed that the secondary double stand breaks (DSB) upon DNA methylation are able to represent functional efficiency of MMR in cells. The purpose of the paper was to test this assumption on human tumor cells differing in MMR-status and pulse-treated with methylnitrosourea (MNU). We used 3 cell lines: HeLa (MMR-competent endometrial tumor cells), HCT116 (MMR-deficient colorectal carcinoma cells), and Colo320 (sigmoid intestine tumor cells with uncharacterized MMR status). DSBs were evaluated with neutral comet assay. Cytotoxicity/viability was evaluated with MTT-asay and apoptotic index (frequency of morphologically determined apoptotic cells). We show that 1) cytotoxic effect of MNU (250 microM) on HeLa cells was exhibited 3 days after pulse-treatment of cells with MNU; 2) DSBs occurred 48 h after the drug treatment but prior to the onset of apoptosis of HeLa cells; 3) MMR-deficient HCT116 cells were resistant to the drug: no decreased viability, DSBs and apoptosis were observed during 3 days after cell treatment. Both cell lines exhibited high sensitivity to etoposide, classical inductor of unrepairable DSBs and p53. Etoposide has been found to induce DSBs in 6-12 h, which was followed by apoptosis (in 24 h). Colo320 cells exhibited intermediate position between HeLa and HCT116 cell lines in regard to sensitivity to MNU according to MTT-assay and the number of secondary DSBs formed in MNU-treated cells. Nevertheless, in contrast to HeLa cells, these breaks did not induce apoptosis in Colo320 cells. Our data confirm the assumption about case/effect relationship between secondary DNA double strand breaks, induced by monofunctional methylating agent MNU, and functioning of MMR in human tumor cells.     

Role of the carbamoylation reaction in the biological activity of methyl nitrosourea

Study of the mutagenic action of methyl nitrosourea (MNU) on the CHO-AT3-2 Chinese hamster cell at 2 regimes of cell treatment (a short-term regime and prolonged 1-h treatment) revealed that increase in the duration of treatment enhanced both cell lethality and clastogenic and mutagenic effects at the TK locus and did not influence the mutation frequency at the OUAr locus. On the basis of kinetic considerations it can be concluded that the base-pair substitution-type mutants (e.g., OUAr) appear as a result of DNA alkylation and the mutants at loci with a wide spectrum of registered mutants (the TK locus) are related to a greater extent to the carbamoylating activity of MNU. This conclusion is confirmed by measurements of the effects of sequential treatment with MNU (7 min) and KNCO (1 h). A synergistic increase in lethality, clastogenicity and mutagenicity at the TK locus was found in experiments with the combined treatment of cells with ethyl methanesulfonate (EMS) and KNCO. Besides, pretreatment of cells with potassium cyanate and subsequent exposure to MNU, EMS and benzopyrene (BP) produced synergistic effects in all the tests: lethality, clastogenicity and mutation frequency at the OUAr and TK loci. Posttreatment of cells with KNCO also led to a synergistic increase in the effects of MNU, EMS and BP treatment in several tests, but not in the OUAr locus. The possible mechanism and levels of interactions between alkylation and carbamoylation and the possibility that potassium cyanate causes supramolecular lesions are discussed.     

Purification to homogeneity and partial amino acid sequence of a fragment which includes the methyl acceptor site of the human DNA repair protein for O6-methylguanine.

DNA repair by O6-methylguanine-DNA methyltransferase (O6-MT) is accomplished by removal by the enzyme of the methyl group from premutagenic O6-methylguanine-DNA, thereby restoring native guanine in DNA. The methyl group is transferred to an acceptor site cysteine thiol group in the enzyme, which causes the irreversible inactivation of O6-MT. We detected a variety of different forms of the methylated, inactivated enzyme in crude extracts of human spleen of molecular weights higher and lower than the usually observed 21-24kDa for the human O6-MT. Several apparent fragments of the methylated form of the protein were purified to homogeneity following reaction of partially-purified extract enzyme with O6-[3H-CH3]methylguanine-DNA substrate. One of these fragments yielded amino acid sequence information spanning fifteen residues, which was identified as probably belonging to human methyltransferase by virtue of both its significant sequence homology to three procaryote forms of O6-MT encoded by the ada, ogt (both from E. coli) and dat (B. subtilis) genes, and sequence position of the radiolabelled methyl group which matched the position of the conserved procaryote methyl acceptor site cysteine residue. Statistical prediction of secondary structure indicated good homologies between the human fragment and corresponding regions of the constitutive form of O6-MT in procaryotes (ogt and dat gene products), but not with the inducible ada protein, indicating the possibility that we had obtained partial amino acid sequence for a non-inducible form of the human enzyme. The identity of the fragment sequence as belonging to human methyltransferase was more recently confirmed by comparison with cDNA-derived amino acid sequence from the cloned human O6-MT gene from HeLa cells (1). The two sequences compared well, with only three out of fifteen amino acids being different (and two of them by only one nucleotide in each codon).     

Comparative mutagenicity of chemicals selected for test in the International Program on Chemical Safety''s collaborative study on plant systems for the detection of environmental mutagens

A review has been made of the four compounds (maleic hydrazide, methyl nitrosourea, sodium azide, azidoglycerol) tested in the International Program on Chemical Safety's collaborative study systems. Maleic hydrazide (MH) is a weak cytotoxic/mutagenic chemical in mammalian tissues and is classified as a class 4 chemical. In contrast, with few exceptions such as Arabidopsis, MH is a potent mutagen/clastogen in plant systems. The difference in its response between plant and animal tissue is likely due to differences in the way MH is metabolized. MH appears to be noncarcinogenic and has been given a negative NCI/NTP carcinogen rating.

Methyl nitrosourea (MNU) is a toxic, mutagenic, radiomimetic, carcinogenic, and teratogenic chemical. It has been shown to be a mutagen in bacteria, fungi, Drosophila, higher plants, and animal cells both in vitro and in vivo. MNU is a clastogen in both animal and human cell cultures, plant root tips and cell cultures inducing both chromosomes and chromatid aberrations as well as sister-chromatid exchanges. Carcinogenicity has been confirmed in numerous studies and involves the nervous system, intestine, kidney, stomach, bladder and uterus, in the rat, mouse, and hamster. MNU produces stage-specific teratogenic effects and also interferes with embryonic development. The experimental evidence that strongly indicates the mutagenic effects of MNU underlines the possible hazard of this compound to human beings. The experimental evidence for the stringent handling of this compound is clear.

Sodium azide (NaN₃) is cytotoxic in several animal and plant systems and functions by inhibiting protein synthesis and replicative DNA synthesis at low dosages. It is mutagenic in bacteria, higher plants and human cells and has been used as a positive control in some systems. In general, tests for clastogenicity have been negative or weakly positive. No evidence of carcinogenicity has been reported in a 2-year study seeking carcinogenic activity in male and female rats. Its advantages in comparison to other efficient mutagens are claimed to be a high production of gene mutations accompanied by a low frequency of chromosomal rearrangements and safer handling because of its nonclastogenic and noncarcinogenic action on humans.     

DNA damage, cytotoxicity and free radical formation by mitomycin C in human cells

Mitomycin C (MMC), a quinone-containing antitumor drug, has been shown to alkylate DNA and to form DNA cross-links. The ability of MMC to alkylate O6-guanine and to form interstrand cross-links (ISC) has been studied using Mer+ and Mer- human embryonic cells. Mer+ (IMR-90) cells have been reported to contain an O6-alkylguanine transferase enzyme and are, in general, more resistant to alkylating agents than the Mer- (VA-13) cell line, which is deficient in the repair of O6-lesions in DNA. Studies reported here show that MMC is more cytotoxic to VA-13 cells compared to IMR-90 cells. The alkaline elution technique was used to quantify MMC-induced ISC, and double strand breaks (DSB) in these cells. The drug-dependent formation of DSB was significantly lower in IMR-90 cells than in VA-13 cells. In contrast, no significant difference in cross-linking could be detected at the end of 2-h drug treatment. Although a small increase in cross-link frequency was observed in the VA-13 cell line relative to the IMR-90 cell line 6 h post drug treatment, it is not clear whether monoalkylated adducts at the O6-position are formed, and contribute to cross-link formation for differential cytotoxicity in VA-13 cells. Electron spin resonance and spin-trapping technique were used to detect the formation of hydroxyl radical from MMC-treated cells. Our studies show that MMC significantly stimulated the formation of hydroxyl radical in VA-13 cells, but not in the IMR-90 cells. The formation of the hydroxyl radical was inhibited by superoxide dismutase (SOD) and catalase. In addition, the presence of these enzymes partially protected VA-13 cells from MMC toxicity but not IMR-90 cells. Further studies indicated that the decreased free radical formation and resistance to MMC may be due to the increased activities of catalase and glutathione transferase in the IMR-90 cell line. These results suggest that MMC-dependent DNA damage (alkylation and DNA DSB) and the stimulation of oxy-radical formation may play critical roles in the determination of MMC-induced cell killing.