Podophyllotoxin-resistant Mutants of Chinese Hamster Ovary Cells- Cross-Resistance Studies with Various Microtubule Inhibitors and Podophyllotoxin Analogues1

The cross-resistances of several mutants of Chinese hamster ovary cells which have been obtained after one and two selection steps in the presence of the microtubule inhibitor podophyllotoxin ( P o d R~ and P o d R" mutants, respectively) towards various other inhibitors of microtubule assembly (e .g . , colchicine, Colcemid, vinblastine, griseofulvin, maytansine, steganacin, nocodazole, and taxol) have been examined. Based upon their specific patterns of cross-resistance/sensit ivi ty to various microtubule inhibitors, both the P o d RI and P o d ~" classes of mutants appear to be of more than one kind. Studies on the binding of [3H]podophyllotoxin to cytoplasmic extracts indicate that one of the P o d R" mutants which has been shown previously to be affected in a Mr 66,000 to 68,000 microtubule-associated protein shows reduced binding of the drug in comparison to the parental P o d s and P o d R~ cells. The different P o d R~ and P o d R" mutants exhibited proportionally increased cross-resistances to various podophyllotoxin analogues (e .g . , deoxypodophyl lotoxin, epipodophyllotoxin, ,8-peltatin, 4 ' -demethylpodophyl lotoxin, o~-peltatin, podophyllotoxin-/t-D-glucoside, /Y-peltatin-/Yo-glucoside, picropodophyllotoxin, and podophyll ic acid) which possess microtubule-inhibitory activity. However, with the exception of one P o d R~ class of mutant, none of the mutants exhibited any cross-resistance to 4'-demethylepipodophyl lotoxin thenylidine-/~-D-glucoside and 4'-demethylepipodophylIotoxin ethylidine-/t-D-glucoside, the 2 podophyllotoxin analogues which lack microtubule-inhibitory activity. The crossresistance studies with these mutants, which, based upon the biochemical studies and their highly specific patterns of crossresistance, are presumably affected in microtubules, provide some very novel insights into the mechanisms of action of various microtubule inhibitors. The results presented in this paper also show that the cross-resistance studies with the set of podophyllotoxin-resistant mutants provide a sensitive and highly specific screening procedure for identifying compounds which possess podophyllotoxin-l ike activity and for investigating the structure-activity relationship among them. The results of structure-activity relationship studies for the various podophyllotoxin analogues examined are discussed.