异靛甲体外抑制DNA和RNA生物合成的抗癌作用机理

以同位素参入法及光谱法观察异靛甲对核酸合成的抑制作用和药物与DNA相互作用。结果表明,异靛甲对DNA,RNA合成有较强的抑制作用,其IC_(50)分别为9和15μmol/L,药物作用非常迅速。30μmol/L异靛甲作用15μmin后DNA,RNA合成被抑制95％以上。实验还表明,异靛甲不能损伤DNA模板,也不抑制DNA拓扑异构酶及DNA多聚酶Ⅰ,但能明显抑制T7 RNA多聚酶,在’100μmol/L浓度下对mRNA的合成抑制达70％以上。     

Nucleic acid binding of arylamines during the respiratory burst of human granulocytes

Following stimulation with phorbol myristate acetate, human granulocytes were found to incorporate a series of arylamines into cellular nucleic acid. No such binding occurred if the granulocytes were not induced to undergo the respiratory burst. The relative amount of covalent binding to cellular DNA and RNA was found to depend strongly on the chemical structure of the arylamine. 2-Aminofluorene gave the highest ratio of DNA/RNA binding, while 4-nitroaniline showed a very low ratio of DNA/RNA binding. 4-Nitroaniline may bind only to RNA, since the degree of binding to DNA was at the level of detectability. Two other substrates, 4-chloroaniline and 4-methylaniline, gave intermediary ratios of DNA/RNA binding. Studies on the possible role of the granulocyte enzyme myeloperoxidase in the activation and binding of these arylamines were conducted in vitro and also through the use of azide, an inhibitor of myeloperoxidase activity in cells. The results indicate that myeloperoxidase probably plays only a limited role in causing the covalent binding of arylamines to nucleic acid in human granulocytes. It is probable that other reactive oxygen species, which are not dependent upon myeloperoxidase for their production, are necessary for the bioactivation of some arylamines, especially for substrates such as 4-nitroaniline. A free-radical mechanism for arylamine bioactivation, and its potential role in arylamine toxicity, was presented in the context of the current scientific literature.     

靶向诱导DNA甲基化与肿瘤治疗研究进展

DNA甲基化是表观遗传调控的重要方式之一,它能在转录水平上抑制相关基因的表达,从而引起基因沉默。近年来的研究表明,DNA的甲基化不仅在生物体内自然发生,而且可通过特异的RNA、DNA等靶向诱导产生。肿瘤的细胞中存在癌基因的异常低甲基化,而且这种异常被认为是肿瘤的发病机制之一。因此,通过靶向诱导癌基因甲基化来抑制癌基因的表达、抑制肿瘤生长的方法,为肿瘤治疗提供了一种新的思路。本文就靶向诱导DNA甲基化与肿瘤治疗的临床前研究进展作一简述。     

The in vitro study of influence of antioxidant enzymes level and repair capacity on cytotoxic bleomycin activity in human and mouse cells

Bleomycin, an antitumour antibiotic was used to study the possible relationship between DNA single strand breaks repair capacity, antioxidant enzymes level and cytotoxic activity of the drug in mouse cells: AKR and BALB/c and in human cells: CRL 2088 and CRL 1307 (xeroderma pigmentosum). The BALB/c and CRL 1307 cells were used because of having defects in DNA repair capacity. A positive correlation was shown to exist between IC50 values and repair ability which suggested that DNA single strand breaks could be responsible for cytotoxic effects of bleomycin in human and mouse cells. Also antioxidant enzymes level have occurred as, at least partly, participating in bleomycin cytotoxic efficiency. About 10-fold higher resistance of AKR cells to bleomycin in comparison with the other cells, as appeared here, did not exhibit the straight correlation with antioxidant status of the cells. It prompts participation of the other mechanism in bleomycin cytotoxic action than that based on free radical generation. Also drug distribution and metabolism should be considered as a possible factor needed in bleomycin efficacy evaluation.     

Bleomycin inhibition of DNA synthesis in isolated enzyme systems and in intact cell systems.

Blcomycin (BLM) inhibits DNA and RNA synthesis in different isolated enzyme systems. The inhibition effect can be reduced by adcling RNA to the reaction mixture. The activity of the RNA dependent DNA polymerase and of a cell-free protein synthesizing system is not affected by BLM. The antibiotic reduces cell proliferation (L5178y mouse lymphoma cells) in vitro at low concentrations by cytostatis and at higher concentrations by cytotoxicity. In BLM-treated L5178y cells DNA synthesis is strongly reduced, while RNA and protein synthesis are not affected. In vivo, using growing quail oviducts, cell proliferation and cytodifferentiation are markedly inhibited after BLM treatment. This is attributed to the observed inhibition of DNA synthesis. RNA and protein synthesis as well as gene expression are not influenced by BLM under the conditions used.     

Self-amplifying vectors for gene delivery

Current methods of gene transfer have a rather low efficiency, especially in vivo. Therefore, one tries to achieve the highest possible levels of expression in the few cells that do take up foreign DNA. One approach is to use self-amplifying expression vectors. These vectors are based on the (+)-strand RNA viruses (alphaviruses) Sindbis virus and Semliki Forest virus. In these vectors, the viral capsid protein coding sequences are replaced with the gene of interest. After introduction into the target cells, the viral replication proteins will replicate the recombinant genome. The increased levels of mRNA generate very high transgene expression levels. Furthermore, spread throughout large cells (muscle, neurons) is much better compared to conventional expression cassettes. Self-amplifying vectors can be introduced into target cells as RNA, DNA or virions.     

Comparison of protein, RNA, and DNA binding and cell-cycle-specific growth inhibitory effects of nickel compounds in cultured cells

Crystalline NiS particles are potent inducers of morphological transformation and are actively phagocytosed by cells. Water-soluble nickel compounds are less potent, possibly because the total amount of nickel that enters cells is less, and its subcellular distribution differs in a number of ways from that following the entry of NiS in the form of an internalized particle. To further study this problem, we have examined the binding of ⁶³Ni to DNA, RNA, and protein isolated from cultured Chinese hamster ovary cells treated with either crystalline ⁶³NiS or ⁶³NiCl₂. Treatment of cultured cells with ⁶³NiS at 10 μg/ml for 3 days resulted in binding of nickel to DNA, RNA, or protein in the range of 1 μg of nickel bound per milligram macromolecule. However, similar treatment of cells with ⁶³NiCl₂ at 10 μg/ml for 1 to 5 days resulted in approximately one to several orders of magnitude less nickel bound to DNA, RNA, and protein. In the case of ⁶³NiCl₂ treatment, cellular proteins contained about 100 times more ⁶³Ni bound than the respective RNA or DNA fractions. However, the protein fraction obtained from cells treated with crystalline NiS contained about 15 times more nickel bound than the same fraction isolated from cells that were similarly treated with ⁶³NiCl₂. RNA or DNA had 300 to 2000 times more bound nickel following crystalline NiS treatment compared to cells treated similarly with NiCl₂. In contrast to the selective binding of ⁶³NiCl₂ to protein, cultured cells treated with crystalline ⁶³NiS had equivalent levels of nickel associated with RNA, DNA, and protein. Since the interaction of ⁶³Ni with these macromolecules following crystalline ⁶³NiS treatment was not due to the binding of the actual particles, the dissolution of intracellular ⁶³NiS particles probably plays an important role in governing the distribution of ⁶³Ni ions available for binding. The greater intracellular macromolecular binding of ⁶³Ni resulting from treatment of cells with ⁶³NiS compared to cultures similarly exposed to ⁶³NiCl₂ paralleled the more potent effects of crystalline NiS in slowing and arresting cell proliferation. Crystalline NiS caused pronounced cell cycle specific blockage at a considerably lower concentration than was required for NiCl₂ to similarly arrest cell growth. Flow cytometry analysis showed that both compounds selectively blocked cell cycle progression in S phase (DNA synthetic stage). These results are suggestive of a common mechanism and site of toxicity inherent to both compounds and related to the cell growth phase during which DNA is replicated.     

粉防已甲素、乙素对L7712和S180细胞DNA、RNA和蛋白质合成的抑制作用

粉防已甲素和乙素对L7712和S180癌细胞DNA合成的ID_(50)分别为2.6,3.5和27.5,24.5mg/L;它们对L7712细胞DNA合成的抑制是由DNA复制模板受到损伤而引起的。它们对两种癌细胞DNA和RNA的合成有很强的抑制作用,对蛋白质合成的抑制作用较弱。两种药物对癌细胞抑制程度和作用方式相似。     

Inhibition of protein synthesis reduces the cytotoxicity of 4'-(9-acridinylamino)methanesulfon-m-anisidide without affecting DNA breakage and DNA topoisomerase II in a murine mastocytoma cell line

Stimulation of cleavable complex formation by 4'-(9-acridinylamino)methanesulfon-m-anisidide (mAMSA) and related anticancer drugs is an important initial event in drug action which correlates with cytotoxicity. However, it was recently suggested that factors in addition to cleavable complex formation are needed to express lethality. Therefore we investigated the effects of inhibitors of DNA replication and RNA and protein synthesis on mAMSA-induced cell killing in the K21 subline of the P815 murine mastocytoma cell line. This showed that RNA and protein synthesis, but not DNA replication, was necessary for maximal mAMSA cytotoxicity. Moreover, inhibition of RNA synthesis with cordycepin or protein synthesis with cycloheximide protected cells from the cytotoxic action of mAMSA without reducing DNA breakage or cleavable complex formation and there was no decrease in DNA topoisomerase II activity in nuclear extracts from cells treated with cordycepin or cycloheximide. We conclude that cleavable complex formation is independent of RNA and/or protein synthesis and we propose that the subsequent conversion into a lethal event requires an additional labile protein factor.     

Modulation of 5-fluorouracil toxicity by uridine, deoxyuridine, orotate and dipyridamole in normal tissues of rats with liver adenocarcinoma

The cytotoxicity of 5-FU, given by the hepatic artery, was measured by its incorporation into the acid soluble fraction, RNA and DNA in normal tissues and an adenocarcinoma transplanted into the liver in rats. Other substances were simultaneously administered by the portal vein to modulate the cytotoxicity. None of them had any significant influence on the incorporation of 5-FU into tumor. Dipyridamole decreased the incorporation of 5-FU into liver RNA and increased the nucleotide/DNA and RNA/DNA ratios so that the incorporation into mg RNA per liver cell was unchanged. Dipyridamole decreased the incorporation into the acid soluble fraction, RNA and DNA of the small intestine and also into RNA per mg DNA. It increased the nucleotide/RNA and RNA/DNA ratios in the bone marrow. Orotate decreased the incorporation into liver and intestinal RNA. Uridine increased the incorporation into liver RNA. The results obtained with dipyridamole were the most pronounced. Studies are continuing with this and other membrane transport inhibitors.     

Combined action of citrinin and ochratoxin A on hepatoma tissue culture cells

Citrinin (CTN) and ochratoxin A (OT-A) may occur simultaneously in mould-contaminated commodities. Both are cytotoxic to hepatoma tissue culture (HTC) cells. The effect of both mycotoxins, either alone or in association on cellular protein, RNA and DNA synthesis was tested. In the presence of ochratoxin A protein synthesis is first inhibited 30 min after the addition of the toxin and RNA synthesis after 150 min. No inhibition of DNA synthesis occurs for at least 5 h, whereas citrinin inhibits first the RNA synthesis after 10 min, second protein synthesis after 20 min and third DNA synthesis after 120 min.When both mycotoxins are added simultaneously to HTC cells the inhibition of RNA and protein synthesis occurs immediately, that of DNA synthesis after a short lag time. This suggests a cooperative effect of both mycotoxins.     

7β-羟基胆固醇-3,7-双琥珀酸单酯钠(RS034)对 Ehrlich 腹水癌细胞DNA、RNA及蛋白质合成的影响

以同位素体外掺入方法研究了7β-羟基胆固醇-3,7-双琥珀酸单酯钠(RS034)对小鼠 Ehrlich 腹水癌细胞 DNA、RNA 及蛋白质合成的影响。加药50μg/ml 和5μg/ml 后半小时,即对 DNA 合成产生显著的抑制作用,抑制率分别达到66.8%和52.9%;50μg/ml 剂量组对 RNA 合成也有显著的抑制作用;但上述两个剂量对蛋白质合成不仅无抑制作用,相反能显著地促进合成。RS034对肿瘤细胞核酸合成的抑制作用可能是其抗癌作用机制之一。     

Effects of lead acetate on DNA and RNA synthesis by intact HeLa cells, isolated nuclei and purified polymerases

The effects of lead acetate on DNA and RNA synthesis have been investigated with intact HeLa cells, isolated nuclei, and purified DNA and RNA polymerases. No inhibition of DNA or RNA synthesis in intact cells was found even after exposure to 0.5 mM lead acetate for 18 hr. In contrast, both DNA and RNA synthesis in isolated nuclei were inhibited by lead (with 50% inhibition at approximately 150 and 80 microM respectively). Similarly, both HeLa DNA polymerase alpha and RNA polymerase II were inhibited, with 50% inhibition obtained at approximately 150 and 20 microM lead acetate respectively. The inhibition of nucleic acid synthesis in isolated nuclei can thus be accounted for by inhibition of the polymerases. The sensitivity of Escherichia coli DNA polymerase I to lead acetate was found to be significantly greater than the HeLa DNA polymerase alpha (50% inhibition at only 10 microM), but the sensitivity of the E. coli RNA polymerase was the same as that of the HeLa enzyme.     

A comparative study of the effects of mercury compounds on cell viability and nucleic acid synthesis in HeLa cells

The effects of various mercury compounds on HeLa cell viability and DNA and RNA syntheses in intact cells and in isolated nuclei have been studied. The compounds examined were: methylmercuric chloride, ethylmercuric chloride, dimethylmercury, phenylmercuric acetate, p-hydroxymercuribenzoate, p-hydroxymercuribenzenesulfonate, HgCl2, HgSO4 , Hg(ClO4)2 and Hg2(ClO4)2. All of the compounds except dimethylmercury inhibited colony formation as well as DNA synthesis in intact cells and in isolated nuclei. RNA synthesis in intact cells was inhibited by all the compounds except dimethylmercury, p-hydroxymercuribenzoate and Hg(ClO4)2. In isolated nuclei, alpha-amanitin-resistant RNA synthesis was inhibited by all the compounds except dimethylmercury, alpha-Amanitin-sensitive RNA synthesis was stimulated by some compounds, inhibited by some, and unaffected by others. The effects of two non-mercurial sulfhydryl reagents, N-ethylmaleimide and iodoacetic acid, were also examined. These compounds showed a pattern of effects on nucleic acid synthesis which differed considerably from that of the mercury compounds. Neither compound significantly inhibited alpha-amanitin-resistant RNA synthesis in isolated nuclei, although both inhibited RNA synthesis in intact cells. Iodoacetic acid had no inhibitory effect on DNA synthesis in isolated nuclei but strongly inhibited DNA synthesis in intact cells.     

5-Chloro-7-iodo-8-hydroxyquinoline (clioquinol) inhibits the nerve growth factor-induced stimulation of RNA synthesis in neonatal rat superior cervical ganglion, in vitro--comparison with effects of methylmercuric chloride and 4-hydroxyaminoquinoline-N-oxide

The inhibitory effects of 5-chloro-7-iodo-8-hydroxy-quinoline (clioquinol), methylmercuric chloride and 4-hydroxyaminoquinoline-N-oxide(4-HAQO) on DNA, RNA and protein syntheses in the neonatal rat superior cervical ganglion (SCG) were studied in relation to the action of mouse 2.5S nerve growth factor (NGF), using organ cultures. RNA and protein syntheses in SCG were stimulated approximately 3- and 2-fold, respectively, by NGF (1 microgram/ml), but the DNA synthesis was only slightly or not at all stimulated. Methylmercuric chloride and 4-HAQO dose-dependently inhibited DNA, RNA and protein syntheses, either in the presence or in the absence of NGF. On the other hand, clioquinol (up to 100 microM) slightly or not at all inhibited RNA synthesis in the absence of NGF; however, it did abolish the NGF-induced stimulation of RNA synthesis in the presence of NGF. The DNA and protein syntheses were dose-dependently inhibited by clioquinol, either in the presence or in the absence of NGF. We conclude from this study that the interaction between clioquinol and the functions of NGF raises the question of a possible toxicity of the drug on specific neurons.