LONG RECOVERY TIMES IMPROVE THE DETECTION OF CELLULAR-RESISTANCE IN-VITRO

In vitro cytotoxicity testing is used increasingly during the developm ent of clinical treatment protocols. These tests are influenced by man y variables, not all of which have been assessed systematically yet. W e analyzed the influence of the recovery time between the end of treat ments and measurements on the detection of cellular resistance. The de velopment of resistance to cisplatin and radiation was chosen as a mod el since the schedule of these treatments is the objective of several ongoing clinical trials. C6 rat glioma, T98G, 86HG-39, A172 human glio ma and TE671 human rhabdomyosarcoma cells were pretreated with radiati on or cisplatin. The cellular resistance was then compared in pretreat ed and wild-type cells using the (4,5-dimethylthiazol-2-yl)-2,5-diphen yltetrazolium bromide (MTT) test. In all cell lines, apparent drug con centrations killing 50% of the cells were dependent on the recovery ti me. In A172 cells this concentration was 10.3 +/- 02.1 mu M after 48 h but decreased to 3.56 +/- 0.44 mu M after 120 h recovery time (P < 0. 001). After recovery times of more than 168 h, 53% of all pretreated c ell lines were resistant to cisplatin or radiation, 25% were unchanged and 22% were more sensitive. However, only half the resistant cells c ould be identified when the MTT test was done with only 48 h recovery time. The sensitivity of detection increased from 0.46 to 0.83 when th e recovery time of the test system was extended from 48 h to 168 h. Th e specificity was not dependent on the recovery time. Experiments show ing resistance after short recovery times are reliable, but lack of re sistance can only be shown in experiments with long recovery times. Ci splatin treatment can result in resistance to radiation in glioma cell s.