In the field of multidrug resistance mediated by the multidrug transporter, P glycoprotein, which is encoded by the MDR-1 gene, 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 a large 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 disruption of MDR-1 structure. Our current efforts are directed at identifying the sites and mechanisms of gene rearrangement and to date we have succeeded in identifying the breakpoint in two cells lines. Our understanding of this process should be very valuable in furthering our understanding of the acquisition of drug resistance. While the occurrence of this phenomenon in clinical samples remains to be expanded, its demonstration in several samples from patients with refractory ALL, indicates this may be important in a defined group of patients, and our efforts are increasingly focused in this direction. Our efforts in this regard will be directed not only at identifying the frequency with which this phenomenon occurs clinically, but also efforts at understanding how this occurs and how it might be prevented. With regard to the latter we have preliminary studies ongoing examining the frequency with which this occurs as a function of the mode of drug administration. Specifically, we are seeking to answer whether administration as a bolus or as a continuous infusion can significantly affect the occurrence of chromosomal aberrations. These studies are being conducted in a primate model by looking at the frequency of chromosomal damage in normal bone marrow following the administration of either bolus or infusional drug. The drugs selected include VP-16, thiotepa and paclitaxel. To date data has been gathered using VP-16 and thiotepa, and this shows a significant difference with less chromosomal damage seen following infusionalpossible therapy than following bolus administration. Finally, we have also directed some of our efforts to further understanding other mechanisms of MDR-1 activation. These studies have characterized the use of other promoters and the involvement of alternate transcription factors in these processes.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 other mechanisms of drug resistance for comparison. Similar studies with topoisomerase II alpha succeeded in isolating a larger number of acquired mutations, and characterization of these has been performed. In addition, we began to actively pursue studies aimed at identifying 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. Characterization of these cells using high resolution isoelectric focusing with purified tubulin 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. In two of the cell lines, mutations in the M40 beta tubulin isoform were confirmed, helping us to localize the site of binding for taxanes to beta tubulin, findings which have been substantiated by crystallographic data, and are currently undergoing further analysis and refinement. In additon, with a desire to understand drugs as extensively as possible before their use clinically, we began similar studies using the epothilones, which are novel microtubule polymerizing agents. These studies have succeeded in identifying additional mutations in beta tubulin, and together with the data obatined with the paclitaxel resistant cell lines, have allowed us to dock both the taxanes and the epothilones onto tubulin, at the same time that we have succeded in identifying a common pharmacophore for these and other microtubule active drugs. The identification of these sites as distinct from those of other microtubule active agents, supports abundant data, and provides excellent models to study the mechanism of paclitaxel and epothilone resistance, and the potential synergism of other agents in drug activity. 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. These studies also stimulated additional work to examine the pathways of cell death following treatment with microtubule active agents, and also motivated studies to examine what other bcl-2 family members might serve a similar function. The observation from our extensive experience with the cell lines of the NCIs anticancer drug screen had indicated that in a large majority of cell lines, expression of bcl-2 and bcl-Xl were inversely correlated, and more recent studies have identified bcl-Xl as a potential bcl 2 surrogate in cells with low to undetectable levels of bcl-2. In addition, we have also examined the role of p53 mutations in the acquisition of paclitaxel and epothilone resistance. Our studies indicate that the occurrence of a non-functional p53 early in the selection process is a universal finding, although the mechanism by which this occurs differ. These mechanisms are under study and the significance of these changes are also being investigated, as well as exploited to understand the normal pathways of p53 trafficking in cells and how this can be impacted by microtubule disrupting agents.We are also involved in collaborative studies with Dr. Susan Bates of the Medicine Branch, and Michael Dean of the Laboratory of Genomic Diversity, examining additional mechanisms of resistance. Molecular cloning of a novel ABC transporter, designated MXR/ABCP1 has recently been accomplished and a significant effort is now being directed to further characterize this (see Susan Bates). Finally, we have been actively studying the expression of cell specific genes in endocrine neoplasms and how this might be potentially exploited in a gene therapy strategy. Studies with adrenal cancers and thyroid cancers are underway, with preliminary results indicating that expression of specific genes occurs in these cells, and that the expression of these genes can be modulated by the addition of differentiating agents which target the cAMP pathway. These studies which arose out of our clinical trails in patients with adrenocortical cancer, are designed to find more specific and effective alternatives for the treatment of endocrine cancers, most of which are composed of very unique cells.
Showing the most recent 10 out of 15 publications
