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：在由MDR-1基因编码的多药转运蛋白P糖蛋白介导的多药抗性领域中，我们的努力一直集中在翻译研究上，同时试图进行基本研究，从而具有未来临床相关性的潜力。由于其最初的描述将近20年前，因此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本中经常观察到P-糖蛋白（PGP）的表达增加。但是，尽管取得了进展，但PGP表达的调节尚未完全理解。 MDR-1/PGP在选定的细胞中的表达和在正常组织或药物敏感细胞类似的调节性对照下的难治性肿瘤中是否表达？我们的结果表明答案是否定的。在所有通常不表达MDR-1或表达MDR-1的耐药细胞系中，在低水平上，调节MDR-1表达的机制均获得和异常。从正常或异常方向进行的无关，主动启动子的表达可以控制转录。这主要是由于基因重排导致无关启动子捕获MDR-1的结果。或者，异常转录可以从MDR-1的112 kb 5素数开始。在药物选择之后，该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，而附近序列的乙酰化可能是激活该异常启动子的重要表观遗传事件。我们的研究目标是（1）了解获得的MDR-1表达的分子基础； （2）理解这些变化是如何/为什么发生的； （3）在临床样本中搜索它们，（4）制定策略以减少或防止其发生。我们的努力越来越多地旨在了解这些药物可能如何影响正常的组织，以及它们可能会或可能不会受到药物转运蛋白的保护程度，例如P-糖蛋白和半转运蛋白，ABCG2项目描述和计划：我们已经确定了基因重排的大量机制，该机制是MDR-1在蜂窝中的激活机制，以及在蜂窝中的激活机制。这些重排随机发生，其特征是转录活性基因5素与MDR-1并置，从而避免了MDR-1结构的破坏。 这些基因重排导致MDR-1的激活代表具有以下特征的抗性机制：（i）重排是一种获得的表型，未在亲本细胞中检测到，并且（ii）重排提供了一种在不表达MDR-1或表达MDR-1非常低水平的细胞中MDR-1激活的机制；这不是在显着水平内源表达MDR-1的细胞中MDR-1过表达的机制。其他特征包括以下内容：（1）这些细胞中的大多数MDR-1转录本是杂化mRNA。 （2）激活是通过将Active启动子5 Prime与MDR-1并置并在该启动子处启动转录而发生。在各种细胞中可以很容易地检测到非MDR-1基因的表达，这表明非MDR-1基因具有组成性活性，并且具有广泛的表达。此外，如果已适合非MDR-1序列的信息，则融合到MDR-1的残基是来自相应基因的5个主要UTR（3）重排似乎是随机发生的，涉及在染色体7和7中发现7的染色体中发现的基因。在7中发现了7。这些断点已在八种耐药细胞系中表征。重排是由于同源重组或非同源末端连接而发生的。尽管断点似乎是唯一的，但ALU重复序列或其他常见的重复序列似乎参与了大多数重排。除了导致MDR-1捕获的基因重排外，我们还确定了获得的MDR-1表达的第二种机制：从位于112 kb 5 prime的异常启动子到正常启动MDR-1的异常转录。在细胞培养中检查MDR-1/PGP表达的早期研究得出的MDR-1表达在两个启动子的控制下指定上游和下游启动子。现在，我们认识到下游启动子是正常的MDR1启动子。含有其他序列的转录本假定下游启动子开始残基的素数起源于推定的上游启动子。我们发现，在许多情况下，上游启动子实际上是另一个无关基因的启动子，如上所述。 但是，在几种耐药细胞系5中，Prime Race发现了相似的5个序列-194近距离序列-194，这些序列指示这些细胞系中的转录本具有相似的起始位点。 GenBank搜索发现，这些抗性细胞系共享的251 bp为MDR-1转录正常起始位点的112,276 bp 5。除ZR-75B细胞外，在任何亲本细胞中均无法检测到251 bp的表达，也无法在15个正常组织中表达表达，表明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节，可能包括人体内源性逆转录病毒LTR。该LTR的表达发生在所有细胞中。但是，在选择药物后，MDR-1转录本在该逆转录病毒LTR附近开始，其转录在通常的LTR转录相反的方向上开始。由于这些异常的MDR-1转录本仅在耐药细胞系中发现，因此我们得出结论，耐药性或随之而来的药物暴露的发展在这种现象的激活中起作用。我们还在细胞系和协作研究中确定了证据表明，由于该区域的染色质结构的变化，源自该异常启动子的某些转录本可能始于该位置。证据包括显示该区域耐药细胞中该区域组蛋白乙酰化增加的数据。我们目前的努力旨在进一步了解这种现象，并开发可以在患者样品中准确评估的测定法，重点是开发可以使用福尔马林固定组织进行的测定法。我们还一直在研究这些转运蛋白在从化学治疗剂中提供脑部保护方面的作用。在某种程度上，随着我们开发越来越多的代理人口服管理的代理，我们正在开发绕过保护大脑并赋予其作为庇护所的机制的机制，因为许多相同的转运蛋白可以保护胃肠道，并且必须绕过毒品，并且如果要绕过它们，则必须绕过它们。我们正在进行研究，希望了解保护大脑的机制，以及绕过这些障碍的后果。我们正在通过检查体外和体内模型以及对现有临床数据的详尽搜索，以进一步了解此问题来实现这一目标。