Podophyllotoxin-resistant Mutants of Chinese Hamster Ovary Cells

Mutants of Chinese hamster ovary cells which have been obtained after one or two selection steps in the presence of the microtubule inhibitor podophyllotoxin (PodR1 and Podm' mutants, respectively) and which exhibit very specific types of cross resistance (and/or collateral sensitivity) towards various other inhibitors of microtubule assembly (e.g. colchicine, colcemid, stegnacine, vinblastine, nocodazole, griseofulvin, maytansine, taxol, and no cross-resistance to V"26 and puromycin) have been analyzed by two-dimensional gel electrophoresis. In two of the four Pod" mutants, a new protein spot, designated M (Mr = 66,000), is observed. Analyses of peptides obtained after partial protease digestion suggest that protein M has arisen by a charge alteration in a neighboring, more basic protein (P) found in the wild type cells. In the two Podm mutant cells, the P and M proteins were present in a ratio of 1: 0.8, whereas in cell hybrids formed between PodR" X Pod' cells, their relative amounts were only about 1: 0.25. This effect of gene dosage on the relative amounts of P and M proteins is consistent with the co-dominant nature of the PodRn mutation and indicates that in the mutant cells, only one of the two copies of the P gene is altered. The protein affected in these mutants appears to represent one of the microtubule-associated proteins by two criteria: (i) this protein (Pin the case of sensitive cells or both P and M in the mutant), along with tubulin, is released from microtubules upon cold treatment; and (ii) this protein is released by Ca2+ treatment of the detergent-extracted cytoskeletons prepared from normal cells but not from colchicine-pretreated cells (Duerr, A., Pallas, D., and Solomon, F. (1981) CeZZ 24, 203-211). The relative intensities of different spots in gel patterns further suggest that protein P is a major cellular constituent present in cell extracts in amounts comparable to tubulin.