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? of MDR-1. Following drug selection this region functions as a 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' 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? 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? 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. While the breakpoints appear to be unique, Alu repeats or other commonly occurring repetitive sequences appear to have been involved in the majority of rearrangements. In addition to gene rearrangements that lead to the capture of MDR-1, we have identified a second mechanism of acquired MDR-1 expression: Aberrant transcription from an aberrant promoter located 112 kb 5? to the normal start of MDR-1. Early studies examining MDR-1/Pgp expression in cell culture concluded MDR-1 expression was under the control of two promoters designated the ?upstream? and ?downstream? promoters. Transcripts containing additional sequences 5? of the ?downstream? promoter start residues were assumed to originate at the ?upstream? promoter. We discovered that in many of these cases the ?upstream? promoter is actually the promoter of another unrelated gene as described above. However, in several drug resistant cell lines 5? RACE found similar 5? sequences proximal to residue -194 indicating transcripts in these cell lines shared a similar start site. A GENBANK search found that the 251 bp shared by these resistant cell lines were 112,276 bp 5? of the normal start site of MDR-1 transcription. Expression of the 251 bp could not be detected in any parental cell with the exception of ZR-75B cells, nor in 15 normal tissues suggesting expression does not occur under normal circumstances. Further studies have shown that these transcripts are aberrant and that their expression is regulated by nearby genomic sequences that may include a human endogenous retroviral LTR. Expression of this LTR occurs in all cells. However, following drug selection, MDR-1 transcripts begin near this retroviral LTR with transcription in the direction opposite of the usual LTR transcription. Because expression of these aberrant MDR-1 transcripts is found only in drug-resistant cell lines, we conclude that the development of drug resistance or the attendant drug exposure has a role in the activation of this phenomenon.Do MDR-1 transcripts containing the 251 bp occur clinically? Yes. In 12 of 23 samples from patients with refractory lymphomas, PCR amplification documented hybrid messages containing the 251 bp 5? to MDR-1; similar transcripts were not found in 18 untreated lymphomas. Furthermore, in a subset of the refractory lymphomas, 5? RACE found the 5? sequences matched the 251 bp, differing only in length. These differences are consistent with multiple start sites, a finding that may be explained by the aberrant nature of transcription. Since this is an acquired aberrancy, the start site may not be as well defined.Our observations that gene rearrangements and aberrant transcripts lead to activation of MDR-1 will only be important if similar observations can be made in a substantial number of clinical samples obtained from patients with chemotherapy refractory disease. Therefore, our current efforts are directed at understanding the frequency with which these rearrangements and aberrant transcripts occur in clinical samples. Demonstration in samples from patients with refractory ALL and lymphoma, indicates these mechanisms of over-expression 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 completed studies examining the frequency with which this occurs as a function of the mode of drug administration. Specifically, we sought to answer whether administration as a bolus or as a continuous infusion can significantly affect the occurrence of chromosomal aberrations. These studies were 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. The data gathered using these three drugs shows a significant difference with less chromosomal damage seen following infusional therapy than following bolus administration.
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