Multidrug resistance Mediated by P-glycoprotein

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Background: 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.Since its original description nearly 20 years ago, increased expression of P-glycoprotein (Pgp) has been frequently observed in cell culture models of multidrug resistance and in clinical samples obtained from refractory patients. But while progress has been made, the regulation of Pgp expression is not fully understood. Is MDR-1/Pgp expression in drug selected cells and refractory tumors under similar regulatory control as that in normal tissues, or drug sensitive cells? Our results suggest the answer is no. In all drug resistant cell lines derived from parental cells that do not normally express MDR-1 or express MDR-1 at low levels, the mechanisms regulating MDR-1 expression are acquired and abnormal. Expression from an unrelated, active promoter, proceeding in a normal or an aberrant direction, can control transcription. This occurs principally as a result of a gene rearrangement that leads to capture of MDR-1 by an unrelated promoter. Alternately, aberrant transcription can begin in a region 112 kb 5 prime of MDR-1. Following drug selection this region functions as a promoter. Evidence suggests that an HERV LTR is involved in this aberrant transcription and that acetylation of a nearby sequence may be an important epigenetic event in the activation of this aberrant promoter. Our research goals are to (1) understand the molecular basis of acquired MDR-1 expression; (2) comprehend how/why these changes occur; (3) search for them in clinical samples and (4) devise strategies to reduce or prevent their occurrence. In the clinic, in collaborative studies with Susan Bates, M.D. we continue to conduct trials examining the use of Pgp antagonists as modulators of drug sensitivity.Project Description and Plans: We have identified gene rearrangements as the mechanism responsible for the activation of MDR-1 in a large number of cell lines, and in patient samples. These rearrangements occur randomly and are characterized by the juxtaposition of a transcriptionally active gene 5 prime to MDR-1, thus avoiding disruption of MDR-1 structure. These gene rearrangements leading to activation of MDR-1 represent a mechanism of resistance with the following characteristics: (i) the rearrangement is an acquired phenotype, not detected in parental cells, and (ii) the rearrangement provides a mechanism for activation of MDR-1 in cells that do not express MDR-1 or express MDR-1 at very low levels; this is not a mechanism for over-expression of MDR-1 in a cell that expresses MDR-1 endogenously at significant levels. Additional characteristics include the following: (1) The majority of MDR-1 transcripts in these cells are hybrid mRNAs. (2) Activation occurs by juxtaposing an active promoter 5 prime to MDR-1, and initiating transcription at this promoter. Expression of the non-MDR-1 gene can be readily detected in a variety of cells suggesting the non-MDR-1 gene is constitutively active and has widespread expression. Furthermore, where information has been available for the non-MDR-1 sequences, the residues fused to MDR-1 have been from the 5 prime UTR of the respective genes (3) The rearrangements appear to occur randomly and involve genes found in chromosome 7 and in chromosomes other than 7. The sequences within 7 are found either centromeric or telomeric of MDR-1 (i.e. inversions occur). The breakpoints have been characterized in eight drug resistant cell lines. Rearrangements occurred as a result of either homologous recombination or non-homologous end joining.