Cell fusion studies to examine the mechanism for etoposide resistance in Chinese hamster V79 spheroids.

When exposed to etoposide, the outer cells from Chinese hamster V79 spheroids are about 10 times more resistant to DNA strand breaks and cell killing than V79 cells grown as monolayers. Previous results have shown that the outer cells of both spheroids and monolayers grow at the same rate and contain the same amount and activity of the target enzyme, topoisomerase II. In order to examine possible mechanisms for this resistance, cell fusion studies were conducted with fluorescent dye-tagged monolayer and spheroid cells. Fused cells were exposed for 30 min to 1.2 microg/ml etoposide and then separated using fluorescence-activated cell sorting into binucleate cells consisting of two monolayer cells, two spheroid cells, or a mixed doublet consisting of one cell of each type. Individual sorted cell doublets were examined for the presence of etoposide-induced DNA strand breaks using the alkaline comet assay. As expected, doublets of monolayer cells were sensitive to etoposide and doublets of spheroid cells were resistant. However, mixed doublets were as resistant to DNA damage by etoposide as spheroid doublets. In comparison, when etoposide- or adriamycin-resistant V79 monolayer cells were fused to the parent monolayer cells, the expected intermediate sensitivity to etoposide was observed for the mixed doublets. We conclude that etoposide resistance associated with the outer cells of spheroids can be "transferred" to produce resistance in monolayer cells. Rapid changes in phosphorylation that can affect topoisomerase II activity or localization, or that can alter chromatin structure, are suggested as possible mechanisms of resistance. In support of this hypothesis, topo IIalpha phosphorylation was at least 10 times greater in monolayers than in the outer cell layer of spheroids.