Multidrug resistance Mediated by P-glycoprotein

P-糖蛋白介导的多药耐药性

• 批准号: 7331398
• 负责人: Antonio Fojo
• 金额: --
• 依托单位: DIVISION OF CLINICAL SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7331398
• 关键词: Multidrug; resistance; Mediated; glycoprotein

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.

背景资料：在由MDR-1基因编码的多药转运蛋白P糖蛋白介导的多药耐药领域，我们的努力继续主要集中在转化研究上，同时试图追求具有未来临床相关性潜力的基础研究。自近20年前首次描述以来，在多药耐药性的细胞培养模型和从难治性患者获得的临床样品中经常观察到P-糖蛋白（Pgp）表达的增加。但是，虽然已经取得了进展，Pgp表达的调控并不完全清楚。MDR-1/Pgp在药物选择性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相似的调控？我们的研究结果表明答案是否定的。在所有来自亲本细胞的耐药细胞系中，通常不表达MDR-1或低水平表达MDR-1，调节MDR-1表达的机制是获得性的和异常的。从不相关的活性启动子以正常或异常方向进行的表达可以控制转录。这主要是由于基因重排导致MDR-1被不相关的启动子捕获。或者，异常转录可以开始于MDR-1的112 kb 5 ′端区域。在药物选择后，该区域作为启动子发挥作用。有证据表明，HERV LTR参与这种异常转录，并且附近序列的乙酰化可能是这种异常启动子激活中的重要表观遗传事件。我们的研究目标是：（1）了解获得性MDR-1表达的分子基础;（2）理解这些变化如何/为什么发生;（3）在临床样本中寻找它们;（4）设计策略以减少或预防它们的发生。在临床上，在与医学博士苏珊·贝茨的合作研究中，我们将继续进行试验，检查Pgp拮抗剂作为药物敏感性调节剂的用途。项目描述和计划：我们已经在大量细胞系和患者样本中确定了基因重排作为MDR-1激活的机制。这些重排随机发生，其特征在于转录活性基因5引物与MDR-1并置，从而避免了MDR-1结构的破坏。这些导致MDR-1活化的基因重排代表了具有以下特征的抗性机制：（i）重排是获得性表型，在亲本细胞中未检测到，和（ii）重排提供了在不表达MDR-1或以非常低的水平表达MDR-1的细胞中活化MDR-1的机制;这不是MDR-1在以显著水平内源性表达MDR-1的细胞中过表达的机制。其他特征包括以下：（1）这些细胞中的大多数MDR-1转录物是杂合mRNA。(2)激活通过将活性启动子5引物与MDR-1并置并在该启动子处启动转录而发生。非MDR-1基因的表达可以在多种细胞中容易地检测到，表明非MDR-1基因是组成型活性的并且具有广泛表达。此外，在非MDR-1序列的信息可用的情况下，与MDR-1融合的残基来自相应基因的5 ′ UTR（3）重排似乎随机发生，并涉及在7号染色体和7号以外的染色体中发现的基因。发现7内的序列位于MDR-1的着丝粒或端粒（即发生倒位）。已在8个耐药细胞系中表征了断裂点。发生重排作为同源重组或非同源末端连接的结果。