Effects of Microtubule Inhibitors on Etoposide Accumulation and DNA Damage in Human K562 Cells in Vitro1

The effects of microtubule inhibitors on cellular accumulation of 4'demethylepipodophyIlotoxin-9-(4,6-0-ethyIidine-/î-D-glucopyranoside) (VP-16) and subsequent epipodophyllotoxin-induced DNA single-strand breaks were investigated in human leukemia K562 cells. At concentra tions of 0.05-20 MM.vinblastine, vincristine, and maytansine similarly increased the steady-state cell concentration of VP-16 (2.5 MM)up to 2fold. Following removal of extracellular vinblastine, the elevation of cell VP-16 was maintained through an additional 55-min incubation period. Washing cells free of extracellular VP-16 resulted in a IKunexchangeable (or bound) component comprising 15-17% of the VP-16 concentration found before removal of extracellular drug. In cells incubated with VP16 alone, removal of extracellular drug resulted in less than 5% cell retention of drug. At vinblastine concentrations of 0.05-0.2 UM, the increase in cell VP-16 was due to a progressive increase in nonexchangeable VP-16. At greater vinblastine concentrations, up to 10 MM,there was no further increase in nonexchangeable VP-16 but there was a 1.6fold increase in the exchangeable (or free) concentration of VP-16. Similar elevation of both nonexchangeable and exchangeable VP-16 by 10 MMvincristine and maytansine was observed; however, 50-100 MM podophyllotoxin or taxol was required for comparable elevation of ex changeable drug with no increase of nonexchangeable VP-16. Elevation of exchangeable VP-16 in the presence of vinblastine was due to inhibition of the unidirectional efflux of this epipodophyllotoxin with a 69% decline in the rate constant for efflux. There were no effects of vinblastine on VP-16 influx. There was no enhancement of DNA single-strand break frequency when cells were incubated with 2.5 MMVP-16 and 0.2 MM vinblastine, a concentration of the Vinca alkaloid that increased only nonexchangeable VP-16. VP-16-induced DNA damage was enhanced by vinblastine concentrations above 0.5 MM,concentrations that elevated exchangeable VP-16, with a maximum doubling of radiation equivalent single-strand break frequency observed with 20 MMvinblastine, consistent with the maximum elevation of cell VP-16 with 20 MMVinca alkaloid. These results indicate that vinblastine and other microtubule inhibitors elevate cell VP-16 by inhibition of the efflux of exchangeable drug and by increasing the level of nonexchangeable drug. Potentiation of VP-16induced DNA damage is observed only at microtubule inhibitor concen trations which elevate exchangeable VP-16. Studies are under way to identify the cellular locus of elevated nonexchangeable VP-16 and to characterize the cytotoxic role of this bound component.