Lagarde P, Gheuens E E, De Pooter C M, De Bruijn E A, van der Heyden S, Chomy F, Van Oosterom A T, Scalliet P G
Laboratoire de cancérologie expérimentale, université d'Anvers (UIA), Wilrijk, France.
Bull Cancer. 1995 Nov;82(11):929-38.
This paper describes the adaptation of the MTT assay to hypoxic conditions in order to test the in vitro effect of piracetam on hypoxic cells and particularly on the radiosensitivity of hypoxic cells since this drug has shown clinical effect on acute and chronic hypoxia. The V79 cell line was selected by reference to preliminary hypoxic experiments using clonogenic assay and euoxic experiments using clonogenic and MTT assays. Cell growth and survival in our hypoxic conditions were assessed using MTT assay with an enclosure and special 48-well plates both made of glass. Growth curves on glass versus reference polystyrene plates were comparable and confirm the validity of using special glass plates. Growth curves on glass plates after 1-hour exposure to nitrogen versus air were comparable, so there is no bias effect due to gas composition. Survival curves using MTT versus reference clonogenic assay were comparable after radiation exposure in eu- and hypoxic conditions, and confirm the validity of our original technique for creating hypoxia. The Oxygen Enhancement Ratio was of about 3 for 1-hour hypoxic exposure. Piracetam gave no cytotoxic effect up to 10 mM of piracetam. Growth curves after continuous drug exposure and 1-hour euoxic versus hypoxic exposure gave no cytotoxic effect up to 10 mM of piracetam. Survival curves after continuous drug exposure to 10 mM of piracetam gave no significant effect on the radiosensitivity of hypoxic V79 cells using MTT or clonogenic assay. However, this does not preclude a potential in vivo effect of piracetam on the radiosensitivity owing to its action on microcirculation and its rheologic properties. The adaptation of the MTT assay to hypoxic irradiation conditions yields the easy screening of radiosensitizing drugs: shorter incubation, semi-automatic method and simultaneous analysis with different serial concentrations thanks to the special 48-well glass plates.
本文描述了将MTT法应用于缺氧条件，以测试吡拉西坦对缺氧细胞的体外作用，特别是对缺氧细胞放射敏感性的影响，因为该药物已显示出对急慢性缺氧的临床疗效。通过参考使用克隆形成试验的初步缺氧实验以及使用克隆形成试验和MTT试验的常氧实验，选择了V79细胞系。在我们的缺氧条件下，使用由玻璃制成的封闭装置和特殊的48孔板，通过MTT法评估细胞生长和存活情况。玻璃板与参考聚苯乙烯板上的生长曲线具有可比性，证实了使用特殊玻璃板的有效性。在暴露于氮气1小时与暴露于空气后的玻璃板上的生长曲线具有可比性，因此气体成分没有偏差影响。在常氧和缺氧条件下辐射后，使用MTT法与参考克隆形成试验得到的存活曲线具有可比性，证实了我们创建缺氧的原始技术的有效性。1小时缺氧暴露的氧增强比约为3。高达10 mM的吡拉西坦没有细胞毒性作用。连续药物暴露以及1小时常氧与缺氧暴露后的生长曲线显示，高达10 mM的吡拉西坦没有细胞毒性作用。连续药物暴露于10 mM吡拉西坦后，使用MTT或克隆形成试验，对缺氧V79细胞的放射敏感性没有显著影响。然而，这并不排除吡拉西坦因其对微循环及其流变学特性的作用而在体内对放射敏感性产生潜在影响。将MTT法应用于缺氧照射条件可轻松筛选放射增敏药物：孵育时间更短、半自动方法以及由于特殊的48孔玻璃板可同时分析不同系列浓度。
