The study of multidrug resistance continues to be an important part of our laboratory emphasis. Efforts this past year represented a continuation of a study begun six years ago, whose initial goal was the identification of point mutations in drug selected cell lines. For this effort, the technique of RNase protection was utilized to sequence/screen the gene from over 100 sources, including normal tissues, unselected and drug selected dell lines. Few point mutations were identified in the coding sequence of the mdr-1 gene. However, those identified, together with two sites of genetic polymorphism have provided tools for investigations of the mechanisms of mdr-1 activation. These observations served as the starting point for investigations currently being pursued, which attempt to understand how activation of mdr-1 occurs, recognizing that while some mechanisms for overexpression of mdr-1 have been proposed, actual mechanisms remain to be elucidated. The identification of genetic polymorphism allowed for a careful evaluation of the patterns of allelic expression in normal tissues and during the course of drug selection. These studies have demonstrated the co-expression of both alleles at nearly equal levels in the majority of normal tissues and unselected cell lines, with a more pleiotropic pattern observed during the course of drug selection. In the latter, overexpression of one or both alleles occurs during the course of drug selection, with subsequent overexpression of one allele observed in the majority of cell lines preceding the onset of gene amplification. These observations led to the hypothesis that overexpression of a single allele most likely occurs as a result of a mutation in a regulatory element, alterations in the chromosomal structure, or as a consequence of a genetic change such as a rearrangement or translocation. Either of these changes would result in cis activation of an individual allele. The evidence accumulated to date indicates that chromosomal rearrangements or translocations are responsible for activation of individual alleles in cells which do not constitutively express mdr-1. Ongoing studies suggest that in cells which express mdr-1, increased expression occurs as a result of other changes, most likely, changes in the chromatin structure.
