STUDIES RELATED TO ANTITUMOR ANTIBIOTICS .5. REACTIONS OF MITOMYCIN-C WITH DNA EXAMINED BY ETHIDIUM FLUORESCENCE ASSAY
STUDIES RELATED TO ANTITUMOR ANTIBIOTICS .5. REACTIONS OF MITOMYCIN-C WITH DNA EXAMINED BY ETHIDIUM FLUORESCENCE ASSAY
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DOI：
10.1139/o76-018
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发表时间：
1976-01-01
期刊：
影响因子：
--
通讯作者：
MORGAN, AR
中科院分区：
文献类型：
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作者：
LOWN, JW;BEGLEITER, A;MORGAN, AR
抗肿瘤抗生素丝裂霉素C的细胞毒性作用主要发生在DNA水平。使用高灵敏度的荧光分析，依赖于增强的乙锭荧光只有当它插入DNA的双链体区域，丝裂霉素C对DNA的作用的3个方面进行了研究：交联事件，烷基化，而不一定交联和链断裂。DNA的交联通过在碱性pH下热变性步骤后返回荧光来确定。在这些条件下，变性DNA不产生荧光。交联通过S1-内切核酸酶（EC 3.1.4.-）独立地证实。消化.在相对高浓度的丝裂霉素的抑制乙锭荧光增强不是由于脱嘌呤，而是烷基化，作为潜在的嵌入位点的损失的结果。丝裂霉素的结合率与荧光损失之间存在线性关系。荧光随pH的成比例降低强烈表明，在这些条件下烷基化是由于抗生素的氮丙啶部分。一个平行的增加的速度和整体效率的共价交联的DNA与较低的pH值表明，交联事件，其中的主要细胞毒性作用是连接，发生顺序与烷基化氮丙啶，然后由氨基甲酸酯。丝裂霉素C，化学还原，诱导单链切割以及单烷基化和共价交联PM2共价闭合环状DNA。超氧化物歧化酶（EC 1.15.1.1）和过氧化氢酶（EC 1.11.1.6）以及自由基清除剂对这种切割的抑制表明，观察到的伴随丝裂霉素C细胞毒性作用的DNA降解主要是由于自由基O2 -。与抗生素链黑菌素的行为相反，丝裂霉素C没有抑制保护酶超氧化物歧化酶或过氧化氢酶。最后，丝裂霉素C能够在pH 4下在不存在还原的情况下交联DNA。这与假定的交联机制一致。
The cytotoxic action of the antitumor antibiotic mitomycin C occurs primarily at the level of DNA. Using highly sensitive fluorescence assays which depend on the enhancement of ethidium fluorescence only when it intercalates duplex regions of DNA, 3 aspects of mitomycin C action on DNA were studied: cross-linking events; alkylation without necessarily cross-linking and strand breakage. Cross-linking of DNA was determined by the return of fluorescence after a heat denaturation step at alkaline pH''s. Under these conditions denatured DNA gave no fluorescence. The cross-linking was independently confirmed by S1-endonuclease (EC 3.1.4.-) digestion. At relatively high concentrations of mitomycin the suppression of ethidium fluorescence enhancement was not due to depurination but rather to alkylation, as a result of losses in potential intercalation sites. A linear relationship existed between binding ratio for mitomycin and loss of fluorescence. The proportional decrease in fluorescence with pH strongly suggests that the alkylation was due to the aziridine moiety of the antibiotic under these conditions. A parallel increase in the rate and overall efficiency of covalent cross-linking of DNA with lower pH suggested that the cross-linking event, to which the primary cytotoxic action was linked, occured sequentially with alkylation by aziridine and then by carbamate. Mitomycin C, reduced chemically, induced single strand cleavage as well as monoalkylation and covalent cross-linking in PM2 covalently closed circular DNA. The inhbition of this cleavage by superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6), and by free radical scavengers suggested that the degradation of DNA observed to accompany the cytotoxic action of mitomycin C was largely due to the free radical O2 -. In contrast to the behavior of the antibiotic streptonigrin, mitomycin C did not inactivate the protective enzymes superoxide dismutase or catalase. Lastly, mitomycin C was able to cross-link DNA in the absence of reduction at pH 4. This was consistent with the postulated cross-linking mechanisms.