Multidrug resistance Mediated by P-glycoprotein

背景：在由MDR-1基因编码的多药转运体P糖蛋白介导的多药耐药领域，我们的工作继续主要集中在转化研究上，同时努力追求具有未来临床相关性潜力的基础研究。p -糖蛋白（Pgp）自近20年前首次被描述以来，在多药耐药的细胞培养模型和从难治性患者获得的临床样本中经常观察到p -糖蛋白（Pgp）的表达增加。但是，虽然取得了进展，但Pgp表达的调控尚不完全清楚。MDR-1/Pgp在药物选择细胞和难治性肿瘤中的表达是否受到与正常组织或药物敏感细胞类似的调控？我们的研究结果表明，答案是否定的。在所有来自不正常表达MDR-1或低水平表达MDR-1的亲本细胞的耐药细胞系中，调节MDR-1表达的机制是获得性和异常的。来自一个不相关的、活跃的启动子的表达，以正常或异常的方向进行，可以控制转录。这主要是由于基因重排导致MDR-1被不相关的启动子捕获。另一种情况是，异常转录可以从112 kb 5？MDR-1。在药物选择之后，这个区域起启动子的作用。我们的研究目标是：(1)了解获得性MDR-1表达的分子基础；理解这些变化是如何/为什么发生的；(3)在临床样本中寻找它们；(4)制定减少或预防它们发生的策略。在临床，在与Susan Bates博士的合作研究中，我们继续进行试验，检查Pgp拮抗剂作为药物敏感性调节剂的使用。项目描述和计划：我们已经确定基因重排是在大量细胞系和患者样本中激活耐多药-1的机制。这些重排是随机发生的，其特点是转录活性基因5'与耐多药1并置于一起，从而避免了耐多药1结构的破坏。这些导致MDR-1激活的基因重排代表了具有以下特征的耐药机制：(i)重排是获得性表型，在亲本细胞中未检测到；（ii）重排为不表达MDR-1或表达MDR-1水平极低的细胞中的MDR-1激活提供了机制；这不是MDR-1在内源性大量表达MDR-1的细胞中过度表达的机制。其他特征包括：(1)这些细胞中的大多数MDR-1转录本是混合mrna。(2)激活发生在并置一个活性启动子5？到耐多药-1，并在这个启动子上启动转录。非耐多药-1基因的表达可以很容易地在多种细胞中检测到，这表明非耐多药-1基因具有组成性活性并且具有广泛的表达。此外，在已获得非耐多药-1序列信息的地方，融合到耐多药-1的残基来自5？(3)重排似乎是随机发生的，涉及7号染色体和7号染色体以外的染色体上的基因。在7个序列中发现了耐多药-1的着丝粒或端粒（即发生反转）。在8个耐药细胞系中发现了断点。重排是同源重组或非同源末端连接的结果。虽然断点似乎是唯一的，但Alu重复或其他常见的重复序列似乎与大多数重排有关。除了导致MDR-1捕获的基因重排外，我们还确定了获得性MDR-1表达的第二种机制：来自位于112 kb的异常启动子的异常转录5？到MDR-1的正常启动早期在细胞培养中检测MDR-1/Pgp表达的研究得出结论，MDR-1的表达受到两个启动子的控制，这两个启动子被称为上游启动子。和下游?启动子。含有附加序列的转录本5？下游的？假设启动子起始残基起源于上游。启动子。我们发现，在很多情况下，上游？启动子实际上是另一个不相关基因的启动子，如上所述。然而，在一些耐药细胞系中，5？种族发现相似5？在这些细胞系中，残基-194附近的序列表明转录本具有相似的起始位点。GENBANK检索发现，这些耐药细胞系共有的251 bp为112,276 bp 5？MDR-1转录的正常起始位点251 bp在除ZR-75B细胞外的任何亲本细胞中均未检测到表达，在15个正常组织中也未检测到表达，提示在正常情况下不会发生表达。进一步的研究表明，这些转录本是异常的，它们的表达受附近的基因组序列调控，其中可能包括人类内源性逆转录病毒LTR，该LTR的表达在所有细胞中都存在。然而，在药物选择之后，MDR-1转录本在这种逆转录病毒LTR附近开始，转录方向与通常的LTR转录方向相反。由于这些异常的MDR-1转录本的表达仅在耐药细胞系中发现，我们得出结论，耐药的发展或随之而来的药物暴露在这种现象的激活中起作用。含有251 bp的耐多药-1转录本在临床上是否出现？是的。在来自难治性淋巴瘤患者的23个样本中的12个中，PCR扩增记录了包含251 bp 5？MDR-1;在18个未经治疗的淋巴瘤中没有发现类似的转录本。此外，在难治性淋巴瘤的一个子集中，5？赛跑找到了5？序列匹配251 bp，只是长度不同。这些差异与多个起始位点一致，这一发现可以用转录的异常性质来解释。由于这是一种后天的异常，起始位置可能没有很好地定义。我们关于基因重排和异常转录导致MDR-1激活的观察只有在从化疗难治性疾病患者获得的大量临床样本中进行类似观察时才有意义。因此，我们目前的努力是为了了解这些重排和异常转录在临床样本中发生的频率。来自难治性ALL和淋巴瘤患者的样本表明，这些过表达机制可能在特定的患者群体中很重要，我们的努力越来越集中在这个方向上。我们在这方面的努力不仅是为了确定这种现象在临床上发生的频率，而且还要努力了解这种现象是如何发生的以及如何预防它。关于后者，我们已经完成了研究，检查这种情况发生的频率作为药物给药模式的函数。具体来说，我们试图回答是否给药作为一个大剂量或连续输注可以显著影响染色体畸变的发生。这些研究是在灵长类动物模型中进行的，通过观察在给药或输注药物后正常骨髓中染色体损伤的频率。选择的药物包括VP-16、硫替帕和紫杉醇。使用这三种药物收集的数据显示，与大剂量给药相比，输注治疗的染色体损伤更小。

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.