Multidrug Resistance Mediated by P-glycoprotein

P-糖蛋白介导的多药耐药性

• 批准号: 7969762
• 负责人: Antonio Fojo
• 金额: $ 36.99万
• 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/7969762
• 关键词: ABCB1 gene; ABCG2 gene; Accounting; Acetylation; Acute Lymphocytic Leukemia; Adult; Affect; Area; Biological Assay; Brain; Bypass; Cell Culture Techniques; Cell Line; Cells; Characteristics; Child; Childhood Acute Lymphocytic Leukemia; Chromatin Structure; Chromosomes; Chromosomes, Human, Pair 7; Clinical; Clinical Data; Complex; Cytotoxic agent; DNA Sequence Rearrangement; Data; Development; Drug Exposure; Drug resistance; Drug-sensitive; Educational process of instructing; Epigenetic Process; Etiology; Event; Formalin; Frequencies; Future; Gastrointestinal tract structure; Genbank; Gene Rearrangement; Genes; Genetic; Genetic Transcription; Genomics; Goals; HERVs; Histone Acetylation; Homologous Gene; Human; Hybrid Cells; Impairment; In Vitro; Investigation; Knock-out; Knockout Mice; Knowledge; Lead; Learning; Location; Lymphoma; Mediating; Modeling; Molecular; Multi-Drug Resistance; Mus; Nervous system structure; Neurons; Nonhomologous DNA End Joining; Normal tissue morphology; Oral; Outcome; P-Glycoprotein; P-Glycoproteins; Patients; Pharmaceutical Preparations; Phenotype; Physiological; Race; Refractory; Refractory Disease; Regulation; Repetitive Sequence; Research; Resistance; Role; Sampling; Site; Solid; Structure; Toxic effect; Transcript; Translational Research; Untranslated Regions; Wild Type Mouse; Work; base; chemobrain; chemotherapeutic agent; chemotherapy; design; effective therapy; experience; homologous recombination; in vivo; in vivo Model; interest; leukemia; multi drug transporter; novel; prevent; promoter; research study; resistance mechanism; tissue fixing; tumor

Background: In the field of multidrug resistance mediated by the multidrug transporter, P glycoprotein, which is encoded by the MDR-1 gene, our efforts have been a 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 have been 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. Our efforts are increasingly direct ed at understanding how normal tissue might be affected b y these agents and the extent to which they might or might not be protected by drug transporters such as P-glycoprotein and the half-transporter, ABCG2 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. 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 prime 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. We now recognize the downstream promoter to be the normal MDR1 promoter. Transcripts containing additional sequences 5 prime of the downstream promoter start residues were assumed to originate at the putative 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 prime RACE found similar 5 prime 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 prime 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. We have also identified in our cell lines and in collaborative studies evidence that some of the transcripts originating at this aberrant promoter may be starting at this location because of changes in chromatin structure in this region. Evidence for this includes data showing increased histone acetylation in this region in drug resistant cells. Our current efforts are directed at further understanding this phenomenon and at developing an assay that can assess this accurately in patient samples, with an emphasis on developing an assay that can be performed using formalin fixed tissue. We have also been investigating the role of these transporters in affording the brain protection from chemotherapeutic agents,. Driven in part by the recognition that as we develop more and more agents to be administered orally, we are developing agents that are likely to bypass the mechanisms that protect the brain and confer its status as a sanctuary, since many of the same transporters that protect line the GI tract, and drugs must be designed to bypass them if they are to be administered orally. We are conducting studies to hopefully understand the mechanisms that protect the brain and what might be the consequences of bypassing these barriers. We are doing this by both examining in vitro and in vivo models and through an exhaustive search of existing clinical data with the goal of further understanding this problem.

背景：在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，我们的工作一直集中在转化研究上，同时试图进行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。或者，异常转录可以从MDR-1的112 kb 5 '区开始。在药物选择之后，这个区域起启动子的作用。有证据表明，HERV LTR参与了这种异常转录，并且附近序列的乙酰化可能是激活这种异常启动子的重要表观遗传事件。我们的研究目标是：(1)了解获得性耐多药-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。ABCG2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导致MDR-1激活的机制。我们的努力越来越直接地用于了解正常组织如何受到这些药物的影响，以及它们在多大程度上可能受到p -糖蛋白和半转运蛋白等药物转运体的保护。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并列，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)激活通过将活性启动子5 '与MDR-1并置，并在该启动子上启动转录而发生。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，在非MDR-1序列的信息中，融合到MDR-1的残基来自各自基因的5 ' UTR(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：从位于112 kb 5 '的异常启动子到MDR-1的正常启动子的异常转录。早期在细胞培养中检测MDR-1/Pgp表达的研究表明，MDR-1的表达受上游和下游两个启动子的控制。我们现在认识到下游启动子是正常的MDR1启动子。含有下游启动子起始残基附加序列5 '的转录本被假定起源于假定的上游启动子。我们发现，在许多情况下，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 ‘ RACE发现在残基-194附近有相似的5 ’序列，表明这些细胞系的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是MDR-1转录正常起始位点的112,276 bp 5 '。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。我们还在细胞系和合作研究中发现，由于该区域染色质结构的变化，起源于该异常启动子的一些转录本可能从该位置开始。证据包括数据显示耐药细胞中该区域组蛋白乙酰化增加。我们目前的工作旨在进一步了解这一现象，并开发一种能够在患者样本中准确评估这一现象的检测方法，重点是开发一种可以使用福尔马林固定组织进行的检测方法。我们也一直在研究这些转运蛋白在提供对化疗药物的大脑保护中的作用。部分原因是我们认识到，随着我们开发出越来越多的口服药物，我们正在开发的药物可能会绕过保护大脑的机制，并赋予其作为避难所的地位，因为许多相同的转运蛋白保护胃肠道，如果药物被设计成口服给药，就必须绕过它们。我们正在进行研究，希望能够了解保护大脑的机制，以及绕过这些障碍可能带来的后果。我们正在通过检查体外和体内模型，并通过对现有临床数据的详尽搜索来进一步了解这个问题。