The study of multidrug resistance continues to be a part of our laboratory. Efforts this past year centered on a continuation of a study begun 5 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 cell 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. This field of investigation is currently being actively pursued recognizing that the mechanisms responsible for the overexpression of mdr-1 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 demonstrate co-expression of both alleles at nearly equal levels in the majority of normal tissues and unselected cell lines, with a more pleotropic 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 have led to the working hypothesis that overexpression of one allele most likely occurs as a result of a mutation in a regulatory element, or alternately 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. Pursuing these possibilities has led initially to an in-depth study of a multidrug resistant cell line which in previous studies had been demonstrated to lack the N-terminal 68- 70 residues. Sequence analysis of the 5' region of this gene demonstrated a novel sequence which was identified as belonging to chromosome 4. FISH (fluorescent in situ hybridization) analysis confirmed the occurrence of a 4;7 translocation, t(4q;7q). The translocation results in the appearance of a novel mRNA representing a hybrid gene and provides a model for overexpression of mdr-1 during the course of drug selection that is actively being pursued in other models. The potential significance of understanding the mechanisms of mdr-1 activation are underscored by the results in tumor samples obtained from patients enrolled in the EPOCH trial in the Medicine Branch.
