Bizanek R, Chowdary D, Arai H, Kasai M, Hughes C S, Sartorelli A C, Rockwell S, Tomasz M
Department of Chemistry, Hunter College, City University of New York, New York 10021.
Cancer Res. 1993 Nov 1;53(21):5127-34.
6-CH3-3H-Mitomycin C (MC) was used to identify MC-DNA adducts formed in EMT6 mouse mammary tumor cells. DNA was isolated from cells treated with 3H-MC. The DNA was enzymatically digested, and the digest was analyzed for 3H-labeled adducts by high performance liquid chromatography. All four major adducts previously isolated and characterized in cell-free systems were detected: two different monoadducts and two bisadducts forming DNA-interstrand and DNA-intrastrand cross-links, respectively. No MC-DNA adducts other than the DNA interstrand cross-link had been shown previously to be formed in living cells. A MC-deoxyguanosine adduct of unknown structure was also detected in DNA from EMT6 cells; this adduct was also formed with purified EMT6 DNA. High performance liquid chromatography analysis was further applied to study the relationship between DNA adducts and cytotoxicity. The number of adducts increased with the concentration of MC in both aerobic and hypoxic cells. At a constant drug level, more adducts were observed in cells treated under hypoxic conditions than in cells treated aerobically; at 2 microM MC, 4.8 x 10(-7) and 3.1 x 10(-7) adducts/nucleotide were observed under hypoxic and aerobic conditions, respectively. The increased adduct frequency under hypoxia correlates with the known increased cytotoxicity of MC to EMT6 cells under hypoxic conditions. In addition, a higher ratio of cross-linked adducts to monoadducts was observed in hypoxic cells. The high performance liquid chromatography techniques were also used to examine the effects of dicumarol (DIC) on adduct patterns in cells treated simultaneously with 3H-MC. The MC-DNA adduct frequencies in DIC-treated cells were increased 1.5-fold under hypoxia and decreased 1.6-fold under aerobic conditions from those observed without DIC. This finding correlates with the known DIC-induced increase and decrease in the cytotoxicity of MC in hypoxic and aerobic EMT6 cells, respectively. The monoadduct resulting from monofunctionally activated MC was suppressed by DIC under both hypoxic and aerobic conditions. In addition, DIC induced the selective formation of an unknown DNA-associated radiolabeled substance in hypoxic cells; this is hypothesized to be a cytotoxic DNA lesion produced by a DIC-stimulated oxido-reductase. The methodology developed to measure MC adduct patterns may be useful as an indicator of distinct enzymatic activation processes for this drug.
6 - 甲基 - 3H - 丝裂霉素C（MC）被用于鉴定在EMT6小鼠乳腺肿瘤细胞中形成的MC - DNA加合物。从用3H - MC处理的细胞中分离出DNA。对DNA进行酶切消化，然后通过高效液相色谱分析消化产物中的3H标记加合物。检测到了先前在无细胞系统中分离并表征的所有四种主要加合物：两种不同的单加合物和两种双加合物，分别形成DNA链间和DNA链内交联。此前尚未证明在活细胞中除DNA链间交联外还会形成其他MC - DNA加合物。在EMT6细胞的DNA中还检测到一种结构未知的MC - 脱氧鸟苷加合物；用纯化的EMT6 DNA也能形成这种加合物。进一步应用高效液相色谱分析来研究DNA加合物与细胞毒性之间的关系。在有氧和缺氧细胞中，加合物的数量均随MC浓度的增加而增加。在药物浓度恒定的情况下，缺氧处理的细胞中观察到的加合物比有氧处理的细胞更多；在2 microM MC时，缺氧和有氧条件下分别观察到4.8×10⁻⁷和3.1×10⁻⁷个加合物/核苷酸。缺氧条件下加合物频率的增加与已知的MC在缺氧条件下对EMT6细胞细胞毒性增加相关。此外，在缺氧细胞中观察到交联加合物与单加合物的比例更高。高效液相色谱技术还用于研究双香豆素（DIC）对同时用3H - MC处理的细胞中加合物模式的影响。与未用DIC处理时相比，DIC处理的细胞中，缺氧条件下MC - DNA加合物频率增加了1.5倍，有氧条件下降低了1.6倍。这一发现分别与已知的DIC在缺氧和有氧的EMT6细胞中诱导MC细胞毒性增加和降低相关。在缺氧和有氧条件下，DIC均抑制了单功能活化MC产生的单加合物。此外，DIC在缺氧细胞中诱导选择性形成一种未知的与DNA相关的放射性标记物质；据推测这是由DIC刺激的氧化还原酶产生的一种细胞毒性DNA损伤。所开发的用于测量MC加合物模式的方法可能有助于作为该药物不同酶促活化过程的指标。
