Mulcahy R T
Int J Radiat Oncol Biol Phys. 1986 Aug;12(8):1389-92. doi: 10.1016/0360-3016(86)90178-1.
Experiments were designed to measure cross-link formation following combined treatment of EMT-6/Ro tumor cells with 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) and misonidazole (MISO) in vitro. To avoid MISO-induced glutathione (GSH) depletion, which might contribute to enhanced monoadduct formation by reducing the protective GSH pools, a post-incubation (i.e. treatment with CCNU for one hour in air followed by MISO treatment in hypoxia) protocol was adopted. Utilizing this treatment scheme, it was possible to significantly enhance CCNU toxicity by post-treating with MISO immediately after exposure to CCNU. Enhanced cross-link formation detected by alkaline elution, at this time, correlated well with the magnitude of cell-kill enhancement, thereby implicating enhanced cross-link formation in the mechanism of potentiation. However, if the cells were allowed to incubate for various intervals between CCNU and MISO treatments, the magnitude of potentiation progressively diminished. Beyond approximately 8-10 hours (corresponding to the time required for maximal cross-link formation after CCNU treatment), treatment with MISO was ineffective at potentiating CCNU cytotoxicity. These experiments suggest that chemopotentiation can be produced by treating with MISO after treatment with CCNU (post-incubation) and that enhanced cross-link formation is involved in the mechanism of MISO chemopotentiation of CCNU activity. The kinetic studies, using the post-incubation protocol, further suggest that the chemopotentiating effect of MISO is exerted subsequent to monoadduct formation and probably does not involve inhibition of DNA-DNA cross-link repair.
实验旨在体外测量1-(2-氯乙基)-3-环己基-1-亚硝基脲（CCNU）和米索硝唑（MISO）联合处理EMT-6/Ro肿瘤细胞后交联形成的情况。为避免MISO诱导的谷胱甘肽（GSH）耗竭，这可能通过减少保护性GSH池而导致单加合物形成增加，采用了孵育后方案（即在空气中用CCNU处理1小时，然后在缺氧条件下用MISO处理）。利用这种处理方案，在暴露于CCNU后立即用MISO进行后处理，可以显著增强CCNU的毒性。此时，通过碱性洗脱检测到的交联形成增强与细胞杀伤增强的程度密切相关，从而表明交联形成增强参与了增效机制。然而，如果在CCNU和MISO处理之间让细胞孵育不同的时间间隔，增效程度会逐渐降低。超过大约8-10小时（对应于CCNU处理后最大交联形成所需的时间），用MISO处理对增强CCNU细胞毒性无效。这些实验表明，CCNU处理后用MISO处理（孵育后）可产生化学增效作用，且交联形成增强参与了MISO对CCNU活性的化学增效机制。使用孵育后方案的动力学研究进一步表明，MISO的化学增效作用在单加合物形成之后发挥，可能不涉及对DNA-DNA交联修复的抑制。
