S-腺苷酰L-甲硫氨酸 (SAM)对 HL-60细胞 DNA甲基化酶活力和甲基化水平的影响(英文)

 以 S-腺苷酰 - L-甲硫氨酸 (SAM)为诱导物 ,在 1 0 μmol/L最佳浓度下造成 1 6%的 HL- 60细胞分化 .HPLC检测结果表明 ,细胞基因组 DNA甲基化水平升高 .通过3H甲基同位素参入法研究细胞 DNA甲基化酶活力 ,则发现在细胞分化过程中酶活力未见升高 .说明细胞基因组甲基化水平升高并不是胞内 DNA甲基化酶催化能力改变的结果 ,而是由于 SAM进入细胞提供过量甲基造成的 .     

被子植物DNA去甲基化酶基因的进化分析

DNA甲基化模式和水平取决于DNA甲基转移酶和去甲基化酶的作用,而DNA去甲基化酶在DNA主动去甲基化过程中起到关键作用。文章以已知的10个DNA去甲基化酶基因为参照,鉴定出两种单子叶植物(水稻和高粱)、两种双子叶植物(拟南芥和毛果杨)中所有的DNA去甲基化酶同源基因,其中新鉴定出两类DNA去甲基化酶类似基因DML4和DML5。基于保守的糖苷酶结构域序列的系统进化分析以及基因在染色体上的位置分析表明,植物中DNA去甲基化酶基因存在串联复制、染色体区段复制和全基因组复制而导致基因的新功能化和亚功能化。文章还进一步分析了DNA去甲基化酶基因在不同组织中的表达情况,旨在理解DNA去甲基化酶基因的功能与进化的关系,以期为DNA去甲基化酶基因在植物中的利用提供参考。     

甲基化作用对M.L.DMA大分子构象的影响

本文用Hha I DNA甲基化酶为工具酶,制备了不同甲基化水平的Micrococcus IuteusDNA,再用Sephadex G-100凝胶过滤将甲基化了的M.L.DNA分离出来.以园二色差光谱和紫外差光谱等手段检测了不同甲基化水平给M.L.DNA大分子造成的构象变化:随着DNA甲基化水平的的升高,其CD差光谱的值,及紫外差光谱的值均呈增加趋势.这说明DNA大分子的甲基化,使其分子在溶液中变得更为缩拢,而这种缩拢的程度与DNA大分子的甲基化水平呈正相关.     

TET酶的生物学功能及相关机制的研究进展

DNA甲基化（DNA methylation）及去甲基化属于常见的表观遗传修饰,可介导多种生理和病理过程。DNA甲基化及去甲基化修饰参与基因的表达调控,且二者的动态平衡可以维持遗传表达稳定性。DNA甲基转移酶（DNA methyltransferase,DNMT）主要包括DNMT1、DNMT3A、DNMT3B、DNMT3L,DNA去甲基化酶（DNA demethylase）主要指10-11易位蛋白（ten-eleven-translocation protein,TET）家族,包括TET1、TET2、TET3,是调节DNA甲基化和去甲基化的重要酶类。TET酶是目前发现的调节DNA去甲基化（DNA demethylation）过程中最重要的酶。综述了TET酶在DNA去甲基化修饰中的作用机制,探讨了DNA去甲基化酶在生长发育和疾病中的关键作用,以期为今后表观遗传学的相关研究提供新思路。     

抑制mTOR信号通路可增强5-aza-dC对胃癌细胞生长的抑制作用

本实验主要研究哺乳动物雷帕霉素靶蛋白（mTOR）信号通路与DNA甲基化在人胃癌细胞生存活力、细胞周期、相关基因及蛋白表达方面的相互作用．分别单独或联合DNA甲基化酶抑制剂5-氮杂-2’-脱氧胞苷（5-aza-dC）、mTOR抑制剂雷帕霉素（rapamycin,RAPA）和P13K抑制剂LY294002干预人胃癌MKN45和SGC7901细胞,以MTT检测细胞生存活力,流式细胞术检测细胞周期,real-timePCR检测PTEN,p27^Kip1基因表达情况,亚硫酸氢盐修饰后测序检测DNA甲基化改变,蛋白免疫印迹检测相关蛋白表达情况．结果发现单独应用5-aza-dC对胃癌细胞的抑制作用不明显,当其联合mTOR信号通路抑制剂时则显著抑制胃癌细胞生长（P〈0．01）;抑制mTOR信号通路可增强5-aza-dC使细胞阻滞在G2期的作用;联合用药还能提高抑癌基因PTEN,p27^Kip1的表达,但不影响DNA甲基化状态;LY294002及RAPA使Akt,p70S6K及4E-BPl磷酸化表达显著下降（P〈0．01）,但5-aza-dE并不增强这一效应．以上研究提示mTOR信号通路抑制剂可间接促进DNA甲基化酶抑制剂对胃癌细胞的抑制作用,为肿瘤的治疗提供新思路．     

DNA甲基化作用与鼠肝细胞的老化

本文比较了不同年龄的鼠肝ＤＮＡ甲基化酶活力及ＤＮＡ甲基化水平,发现它们均与鼠龄呈反相关。又以不同年龄的鼠肝ＤＮＡ为模板,检验了其体外转录活力,发现其与鼠龄呈正相关。     

DNA甲基化作用与鼠肝细胞的老化

本文比较了不同年龄的鼠肝ＤＮＡ甲基化酶活力及ＤＮＡ甲基化水平,发现它们均与鼠龄呈反相关。又以不同年龄的鼠肝ＤＮＡ为模板,检验了其体外转录活力,发现其与鼠龄呈正相关。     

转化细胞及腹水癌细胞对生长调节因子敏感性的研究

 本文~3H-TdR参入细胞DNA为指标研究了EGF等生长调节因子对小鼠腹水癌细胞DNA合成的影响,发现不同癌细胞对EGF等生长因子的敏感性有所差异,考虑到这也许与肿瘤细胞自身特性如恶性度有关。为了进一步探讨恶性度与这一敏感性是否相关,我们观察并比较了C_3H10T1/2CL_8(一种来源于鼠胚的正常成纤维细胞,简称NC_3H_(10)及转化的C_3H_(10)T1/2CL_8(用~3H-TdR转化的上述细胞,简称TC_3H_(10))对EGF等生长因子的敏感性。实验证明,细胞恶性转化后,对EGF的敏感性明显降低,~3H-TdR参入率降至原先的1/4以下。用DBcAMP作用于NC_3H_(10)和TC_3H_(10)均能抑制~3H-TdR参入DNA并可抑制EGF诱导的~3H-TdR参入作用。因此,我们认为,有关物理的致癌因素如放射性同位素,像生物、化学的致癌因素一样,亦能引起其转化细胞对外源性生长调节因子敏感性的改变。     

正常小鼠肝和腹水型肝癌细胞核内酸溶性蛋白质磷酸化的比较研究

染色质的组成成分,组蛋白和非组蛋白在特异的蛋白激酶作用下可以发生磷酸化修饰,组蛋白和非组蛋白的磷酸化和脱磷酸化可能在染色质的结构,基因表达以及DNA复制中起着重要的作用。本文比较是小鼠腹水型肝癌细胞核和正常小鼠肝细胞核内酸溶性蛋白质及其磷酸化的差异。正常小鼠肝细胞核酸溶性蛋白质的电泳染色图谱有一条明显可见的组蛋白H_1~0蛋白带,而对小鼠腹水型肝癌来说,此带极浅,但在腹水型肝癌细胞核酸溶性蛋白质的电泳染色图谱上可见到表观分子量约为68K的一条蛋白带,而正常小鼠肝未见此带。此外,从电泳胶片~(32)P放射自显影图谱可见腹水型肝癌组蛋白H_1,H_2A和非组蛋白带Ⅱ(MW43K),带Ⅲ(MW.67K)带Ⅳ(M.w.97K)磷酸化程度明显高于正常小鼠肝。     

鼠肝细胞癌变中DNA甲基化作用的研究

Activity of DNA methylase and DNA methylation level were measured from normal mouse liver, mouse liver charged with H22a ascitic hepatoma and H22a ascitic hepatoma cell by measuring incorporation of H3-methyl. S-Adenosyl-3H-methyl-methionine (3H-SAM) was used as methyl donor. DNA methylation level of different cells were measured by HP-LC. DNA methylase activity and DNA methylation level of H22a ascitic hepatoma, mouse liver charged with H22a ascitic hepatoma are lower than normal mouse liver. Treatments of antitumor drugs lead to a rising of DNA methylase activity of tumor cell, however, the DNA methylation level of tumor cell has not rised after such treatments.     

Cell cycle-dependent regulation of eukaryotic DNA methylase level

DNA methylase activity in the nuclei of somatic cells arrested at G0 increased markedly when the cells were subjected to a mitogenic stimulus. Treatment of mouse splenocytes with Concanavalin A resulted in about 20-fold increase in methylase activity within 20 h starting 12-15 h after Concanavalin A addition. The methylase level in rat liver was elevated approximately 3-fold at about 20-h posthepatectomy. A detailed time course of the increase in methylase activity with respect to the cell cycle revealed that the onset of this event coincided with the entry of the cells into S phase. In both systems, the extent of methylation in CpG sequences is not altered significantly even under conditions of active DNA synthesis which is induced by the mitogenic effect. These results suggest that the cell responds to the mitogenic stimulus by adjusting the DNA methylase activity to enable conservation of the methylation level in DNA.     

Nicotinamide methylation tissue distribution,developmental and neoplastic changes

The distribution of cytosolic activity of nicotinamide:S-adenosylmethionine methyltransferase (nicotinamide methylase, EC 2.1.1.1) in normal tissues from adult rat and mouse and in tumors and the change in the enzyme activity during the the development of rat tissues were studied. (1) Rat liver exhibited the highest nicotinamide methylase activity among all adult tissues tested; other rat tissues, like adrenal, pancreas, kidney, brain and mouse tissues, had only less than 15% of the adult rat liver activity. (2) 3 days before birth, fetal liver showed a very low nicotinamide methylase activity (2% of adult rat liver), which, however, increased already 1 day before birth and reached the adult level on the day 28 after birth. (3) In a variety of hepatomas and ascites tumors, an inverse correlation, with some exceptions, between tumor growth rate and nicotinamide methylase activity could be seen. In all hepatomas, with the exception of Morris hepatoma 5123tc, nicotinamide methylase activity was significantly decreased in comparison to normal adult rat liver. The highly malignant Zajdela hepatoma, Yoshida sarcoma, sarcoma 180 and Ehrlich ascites tumor methylated nicotinamide only at a negligibly low rate. (4) Cultured RLC cells (an established rat liver cell line) from the stationary growth phase or G₁-arrested RLC cells had about half of the adult rat liver activity, yet the activity was 70% higher than that of the logarithmically growing RLC cells.     

Effect of 5-azacytidine on DNA methylation in Ehrlich's ascites tumor cells

5-Azacytidine inhibited in vivo DNA methylation in Ehrlich's ascites tumor cells depending upon the dose at which 5-azacytidine did not inhibit DNA synthesis significantly. This drug did not inhibit DNA methylation in vitro. The DNA methylase activity in ascitic cells decreased with the increasing dose of 5-azacytidine. Hypomethylated DNA was obtained from the 5-azacytidine treated ascitic cells.     

Correlation of the increase in DNA methylation and antioxidant activity of mouse liver nuclear lipids after administration of antioxidant and in Ehrlich ascites carcinoma

The content of 5'-methylcytosine in total DNA of mouse liver increases 2--2,5-fold 3 hrs after a single intraperitoneal injection of antioxidant (4-methyl-2,6-ditretbutylphenol) (20 or 60 mg per 1 kg of body weight) and makes up to 2--2.4 mol.%. The methylation of liver DNA is also increased more than 2-fold in Ehrlich ascite carcinoma. The DNA isolated from mouse liver after administration of antioxidant or during cancer growth markedly differs from liver DNA of intact animals in its CH3-accepting ability under in vitro methylation by the methylase complex from Enterobacter cloacea. The changes in DNA methylation in mouse liver under the effects of antioxidant and in Ehrlich ascite carcinoma are correlated with the changes in the antioxidant activity of liver nuclear lipids.     

Comparative study of nucleosome particles in chromatin from normal and tumor cells. I. Structural parameters

The composition and structure of nucleosomic fragments isolated from the ascitic hepatoma 22A cells, liver and from cells of C3HA mice in norm and after partial hepatectomy were investigated. Via electrophoresis in 1.5% agarose gel with the emplogment of reperic restrictive DNA fragments and with the help of mathematical processing, the value of the nucleosomic DNA repeat in ascitic hepatoma 22A was calculated to be 187 b.p., and in regenerating liver--196 b.p. The absence of the H1 degree subfraction in chromatin of ascitic hepatoma 22A cells was found. Lower electrophoretic mobility in 5% polyacrylamid gel of nucleosomic chromatin fragments of ascitic hepatoma 22A as compared with their counterparts from healthy mice liver was established. The method of circular dichroism allowed to reveal differences in the RNA and protein structural state in nucleosomes of normal and tumour cells. The structure of nucleosomes of regenerating mice liver of the C3HA strain did not differ from that of normal liver of the same mice.