Berridge M V, Tan A S
Malaghan Institute of Medical Research, Wellington School of Medicine, New Zealand.
Arch Biochem Biophys. 1993 Jun;303(2):474-82. doi: 10.1006/abbi.1993.1311.
The MTT assay, which is widely used to measure cell proliferation and to screen for anticancer drugs, is based on reduction of the tetrazolium salt, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) by actively growing cells to produce a blue formazan product. Despite broad acceptance of this assay, neither the subcellular localization, nor the biochemical events involved in MTT reduction are known. Mitochondrial involvement in MTT reduction has been inferred from studies with respiratory inhibitors using succinate as a substrate, but the contribution of this activity to overall cellular MTT reduction is unknown. Using the bone marrow-derived cell line, 32D, we investigated the subcellular localization of MTT reduction using succinate, NADH, and NADPH as substrates. At optimum substrate concentrations, MTT reduction by whole cell homogenates was greatest with NADH and least with succinate, which accounted for less than 10% of the combined activities. Using succinate, 96% of recoverable MTT reducing activity was in particulate fractions of the cell and 77% in the mitochondrial and light mitochondrial/lysosomal fractions. When NADH and NADPH were used as substrates, increased amounts of MTT reducing activity were associated with soluble fractions of the cell and association with mitochondrial fractions was less pronounced. To further characterize MTT reduction by the mitochondrial fraction, respiratory chain inhibitors were used to explore involvement of electron transport in MTT reduction. Succinate-dependent mitochondrial MTT reduction was inhibited by 80% with chlorpromazine, 70% by antimycin A, and 85-90% by thenoyltrifluoracetone (TTFA), but inhibition was not observed with rotenone at < or = 2 microM, Amytal, or azide. These results suggest that when succinate is used as an electron donor, 70-80% of mitochondrial MTT reduction occurs subsequent to transfer of electrons from cytochrome c to cytochrome oxidase, but prior to the point of azide inhibition. In contrast to succinate, NADPH-dependent mitochondrial MTT reduction was not affected by any of the respiratory inhibitors tested, and NADH-dependent reduction was only inhibited by chlorpromazine (40-50% at plateau concentrations). These results suggest that most cellular MTT reduction occurs outside the mitochondrial inner membrane and involves NADH and NADPH-dependent mechanisms that are insensitive to respiratory chain inhibitors. This interpretation is supported by whole cell studies in which rotenone failed to affect basal and interleukin-3-stimulated MTT reduction at times up to 4 h but strongly inhibited DNA synthesis. We conclude that most cellular reduction of MTT occurs extramitochondrially and probably involves the pyridine nucleotide cofactors NADH and NADPH.
MTT 法被广泛用于测量细胞增殖和筛选抗癌药物，其原理是活细胞将四唑盐 MTT（3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐）还原，生成蓝色的甲臜产物。尽管该检测方法被广泛接受，但MTT 还原过程中涉及的亚细胞定位和生化事件均不明确。通过以琥珀酸为底物的呼吸抑制剂研究推测线粒体参与了 MTT 还原过程，但该活性对细胞整体 MTT 还原的贡献尚不清楚。我们使用骨髓来源的细胞系 32D，以琥珀酸、NADH 和 NADPH 为底物，研究了 MTT 还原的亚细胞定位。在最佳底物浓度下，全细胞匀浆以 NADH 进行 MTT 还原的能力最强，以琥珀酸进行还原的能力最弱，琥珀酸还原能力占总还原活性的比例不到 10%。以琥珀酸为底物时，96%的可回收 MTT 还原活性存在于细胞的颗粒组分中，77%存在于线粒体和轻线粒体/溶酶体组分中。当使用 NADH 和 NADPH 作为底物时，MTT 还原活性的增加与细胞的可溶组分相关，与线粒体组分的相关性较弱。为了进一步表征线粒体组分对 MTT 的还原作用，我们使用呼吸链抑制剂来探究电子传递在 MTT 还原中的作用。氯丙嗪可抑制 80%的琥珀酸依赖性线粒体 MTT 还原，抗霉素 A 抑制 70%，噻吩甲酰三氟丙酮（TTFA）抑制 85 - 90%，但在≤2 μM 的鱼藤酮、阿米妥或叠氮化物作用下未观察到抑制作用。这些结果表明，当以琥珀酸作为电子供体时，70 - 80%的线粒体 MTT 还原发生在电子从细胞色素 c 转移至细胞色素氧化酶之后，但在叠氮化物抑制点之前。与琥珀酸不同，NADPH 依赖性线粒体 MTT 还原不受所测试的任何呼吸抑制剂的影响，NADH 依赖性还原仅受氯丙嗪抑制（平台浓度下抑制 40 - 50%）。这些结果表明，大多数细胞内的 MTT 还原发生在线粒体内膜之外，涉及对呼吸链抑制剂不敏感的 NADH 和 NADPH 依赖性机制。这一解释得到了全细胞研究的支持，在该研究中，鱼藤酮在长达 4 小时内未能影响基础状态和白细胞介素 - 3 刺激后的 MTT 还原，但强烈抑制了 DNA 合成。我们得出结论，大多数细胞内的 MTT 还原发生在线粒体之外，可能涉及吡啶核苷酸辅酶 NADH 和 NADPH。
