In the field of multidrug resistance mediated by P-glycoprotein, our efforts continue to have a major focus on translational research, while trying to pursue basic investigations that have the potential for future clinical correlations. We have identified gene rearrangements as the mechanism responsible for the activation of MDR-1 in an increasing number of cell lines. These rearrangements occur randomly and are characterized by the juxtaposition of a transcriptionally active gene 5' to MDR-1, thus avoiding significant disruption of MDR-1 structure. While the occurrence of this phenomenon in clinical samples remains to be expanded, its demonstration in two samples from patients with refractory ALL, indicates this may be important in a defined group of patients. Our current investigations with MDR-1 arose out of studies which revealed a low frequency of acquired mutations in MDR-1. This prompted us to look at a second mechanism of drug resistance in which mutations had been described as a model for comparison. Similar studies with topoisomerase II-alpha have succeeded in isolating a larger number of acquired mutations, and characterization of these is underway. In one cell line, the identification of impaired nuclear translocation as the mechanism of resistance, led to the identification of a basic pentamer in the C terminal region of the gene as the motiff responsible for nuclear localization. In addition to the above studies, we have been able to show in the majority of single step isolates moderate to marked reductions in the level of topoisomerase II-alpha occurs as a mechanism of resistance, and are currently pursuing the molecular changes responsible for this decreased expression. A third active field of investigation began with our attempts to identify non-Pgp mechanisms of paclitaxel resistance. Selections performed with paclitaxel in the presence of verapamil succeeded in isolating a large number of cell lines with acquired resistance to paclitaxel that did not overexpress MDR-1. While the characterization of these cells is still underway, high resolution isoelectric focusing using purified tubulin has demonstrated the existence of new alpha and beta tubulin isoforms in the resistant cells, consistent with the occurrence of acquired mutations as the mechanism responsible for the resistant phenotype. Identification of the latter is ongoing. We have also used these models to confirm a possible pathway for cell death shared by tubulin active agents: activation of Raf-1 and phosphorylation of bcl-2.
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