三磷酸腺苷结合盒G2调控肝癌细胞多药耐药的作用

目的探讨三磷酸腺苷结合盒(ABC)G2在肝癌耐药细胞中的表达及其在肝癌细胞耐药中的作用。方法 MTT法检测阿霉素(ADM)作用肝癌SMMC-7721细胞24 h后半数生长抑制浓度(IC50)。利用药物浓度递增细胞培养方法建立肝癌耐药细胞,在SMMC-7721细胞培养液中加入ADM,逐渐提高ADM浓度,浓度从0.001μg/ml提高至0.100μg/ml,使细胞在0.100μg/ml ADM中稳定生长,命名为SMMC-7721/ADM细胞。倒置显微镜下观察SMMC-7721/ADM及其亲代SMMC-7721细胞形态。MTT法检测ADM作用肝癌耐药细胞SMMC-7721/ADM 24 h后的IC50,计算耐药指数。流式细胞术(FCM)检测SMMC-7721/ADM及其亲代SMMC-7721细胞凋亡、周期及细胞中ABCG2蛋白表达水平。结果 ADM对SMMC-7721细胞的IC50=(1.11±0.09)μg/ml。历时3个月时间成功使SMMC-7721/ADM细胞在含0.100μg/ml ADM的细胞培养液中稳定生长,细胞命名为SMMC-7721/ADM。倒置显微镜下观察,SMMC-7721/ADM细胞体积增大,细胞形态变得更不规则。SMMC-7721/ADM与SMMC-7721细胞相比细胞凋亡率无显著差异(P0.05),细胞G0/G1期显著降低(P0.05),细胞S及G2/M期无显著差异(P0.05)。SMMC-7721/ADM与SMMC-7721细胞相比,细胞中ABCG2蛋白表达水平显著增高(P0.05)。结论 ABCG2在肝癌多药耐药细胞中异常增高,高表达的ABCG2参与了肝癌多药耐药的形成。     

ATP-binding cassette transporters in the heart

Members of the ATP-binding cassette (ABC) protein superfamily are integral membrane proteins involved in energy-dependent transport of a wide variety of substrates across biologic membranes. ATP-binding cassette transporters serve as functional barriers against the entry of xenobiotics, for example, in the intestine or at the blood-brain barrier, or contribute to drug excretion, for example, in the kidney or the liver. Many human ABC transporters, such as ABCB1 (P-glycoprotein), ABCC5 (MRP5), or ABCC9 (SUR2), are expressed in the heart, suggesting an important role of these transporters in cardiac drug effects or physiology. Interestingly, mutations in ABCC9, a constituent of cardiac K(ATP) channels, can cause dilated cardiomyopathy in humans, providing evidence that dysfunction of cardiac ABC transporters might have clinical implications. This review aims to give insights into the possible functions of ABC transporters in the heart, their role in drug disposition, as well as control of intracellular cyclic nucleotide levels or regulation of K(ATP) channel conductivity.     

ABC转运蛋白在大鼠肝脏卵圆细胞中的表达

目的研究ABC转运蛋白基因家族的三个主要成员MDR1、MRP1和Bcrp1基因在大鼠肝脏卵圆细胞中的表达及意义。方法建立大鼠2-乙酰氨基芴／三分之二肝切除模型，两步胶原酶灌注结合Percoll密度梯度离心分离大鼠肝脏卯圆细胞和肝细胞，采用免疫组织化学染色检测大鼠肝脏组织中MDR1、MRP1、Bcrp1转运蛋白的表达；采用荧光定量PCR方法检测MDR1、MRP1和Bcrp1基因mRNA在卵圆细胞和肝细胞中的表达水平。结果免疫组织化学染色显示大鼠肝脏组织中MDR1表达位于门静脉区附近，呈放射状分布，Bcrp1表达定位在细胞膜上。大鼠肝脏卵圆细胞MDR1、MRP1和Bcrp1基因mRNA的表达水平分别是肝细胞的9倍、1．5倍和13．8倍。结论卵圆细胞表达高水平的ABC转运蛋白，后者参与卵圆细胞免受外源性化学物质损伤的自我保护机制。     

The structure of Escherichia coli BtuF and binding to its cognate ATP binding cassette transporter

Bacterial binding protein-dependent ATP binding cassette (ABC) transporters facilitate uptake of essential nutrients. The crystal structure of Escherichia coli BtuF, the protein that binds vitamin B12 and delivers it to the periplasmic surface of the ABC transporter BtuCD, reveals a bi-lobed fold resembling that of the ferrichrome binding protein FhuD. B12 is bound in the "base-on" conformation in a deep cleft formed at the interface between the two lobes of BtuF. A stable complex between BtuF and BtuCD (with the stoichiometry BtuC2D2F) is demonstrated to form in vitro and was modeled using the individual crystal structures. Two surface glutamates from BtuF may interact with arginine residues on the periplasmic surface of the BtuCD transporter. These glutamate and arginine residues are conserved among binding proteins and ABC transporters mediating iron and B12 uptake, suggesting that they may have a role in docking and the transmission of conformational changes.     

ATP-binding cassette (ABC) transporters in atherosclerosis

Macrophages play a central role in the initiation and progression of atherosclerotic lesions. In the nascent lesion, macrophages transform into foam cells through the excessive accumulation of cholesteryl esters. Dysfunctional lipid homeostasis in macrophages and foam cells ultimately results in the breakdown of membrane integrity and cell death. Studies within the past 2 years have implicated a defined subset of multispan transmembrane proteins, the ATP-binding cassette (ABC) transporters, in macrophage lipid homeostasis. The recent finding that ABCA1, beyond its function as a major regulator of plasma high-density lipoprotein metabolism, exerts significant antiatherosclerotic activities has provided the first direct evidence for the role of an ABC transporter in the pathogenesis of atherosclerosis.     

内脂素对人单核细胞株源性巨噬细胞ATP结合盒转运蛋白A1的调控

目的:观察过氧化物酶体增殖物激活受体γ(PPARγ)信号转导通路在内脂素调控人单核细胞株(THP-1)源性巨噬细胞ATP结合盒转运蛋白A1(ABCA1)表达中的作用,探讨内脂素诱导泡沫细胞形成的机制和途径。方法:THP-1单核细胞诱导分化为巨噬细胞,随机分组,给予不同浓度和不同作用时间的内脂素进行干预,分别运用油红O染色观察细胞浆脂滴变化,反转录聚合酶链反应(RT-PCR)法和蛋白免疫印迹(Westernblot)法检测各组细胞PPARγ及ABCA1mRNA和蛋白表达,酶荧光学法检测细胞内TC和游离胆固醇(FC)含量,TC与FC之差为胆固醇酯(CE)含量。结果:与对照组比较,内脂素干预组细胞浆脂滴明显增多,细胞内FC和CE含量增加(P<0.05),PPARγ及ABCA1mRNA和蛋白表达降低(P<0.05);相关分析显示,内脂素呈浓度和时间依赖性下调PPARγ及ABCA1mRNA和蛋白的表达。结论:内脂素可能通过PPARγ信号转导通路下调ABCA1表达,减少细胞内FC流出,使细胞内CE合成增加,从而诱导泡沫细胞形成。这可能为内脂素致动脉粥样硬化发病机制的研究提供了一个新的理论依据。     

Bile salt-dependent efflux of cellular phospholipids mediated by ATP binding cassette protein B4

Human ABCB4 (multidrug resistance [MDR]3 P-glycoprotein) is expressed in the canalicular membrane of the hepatocyte. ABCB4 has been shown to be required for phosphatidylcholine (PC) secretion into the bile and to translocate PC across the plasma membrane. To further investigate the function of ABCB4, we established a cell line stably expressing ABCB4 (human embryonic kidney [HEK]/ABCB4). The efflux of phospholipids from HEK/ABCB4 cells was remarkably increased by the addition of taurocholate. In addition, the cholesterol efflux from HEK/ABCB4 cells was also enhanced in the presence of taurocholate. Light scattering measurements suggested that the taurocholate monomer plays an important role in ABCB4-mediated lipid secretion. On the other hand, the efflux of phospholipids and cholesterol was not mediated by ABCB1 (MDR1) even in the presence of taurocholate. Taurocholate promoted the efflux of phospholipids and cholesterol from HEK/ABCB4 cells more efficiently than glycocholate and cholate. ABCB4-K435M and ABCB4-K1075M, Walker A lysine mutants, did not mediate the phospholipid and cholesterol efflux in the presence of taurocholate, suggesting that ATP hydrolysis is essential for the efflux. Verapamil completely inhibited the taurocholate-dependent efflux of phospholipids and cholesterol from HEK/ABCB4 cells. Mass spectrometry revealed that, in the presence of taurocholate, HEK/ABCB4 cells preferentially secreted PC compared to sphingomyelin. PC vesicles induced cholesterol diffusion from cell membrane, but did not accept cholesterol from ABCB4. CONCLUSION: ABCB4 mediates the efflux of phospholipids into the canalicular lumen in the presence of bile salts, and plays a crucial role in bile formation and lipid homeostasis.     

Intracellular trafficking and regulation of canalicular ATP-binding cassette transporters

The bile canaliculus contains at least four ATP-binding cassette (ABC) proteins responsible for ATP-dependent transport of bile acids (spgp), nonbile acid organic anions (mrp2), organic cations (mdr1), and phosphatidylcholine (mdr2). Other ABC transporters (including mrp3) have also been partially localized to the canaliculus; however, their function has not been fully delineated. The specific amount and function of spgp and mrp2 in the canalicular membrane increases in response to taurocholate and cAMP. The mechanism involves increased recruitment of spgp and mrp2 from Golgi to the canalicular membrane by a microtubular and PI3 kinase-dependent vesicular trafficking system. Because the effects of taurocholate and cAMP summate, two distinct pathways are proposed. Mdr family members traffic either directly to the apical plasma membrane or, in the case of spgp, through a separate intracellular pool(s); in either case, there is no direct evidence for transcytosis of ABC transporters from Golgi to basolateral plasma membrane and subsequently to the canalicular plasma membrane. Direct transfer from Golgi to apical membrane was demonstrated by in vivo pulse labeling, in vitro membrane localization, and on-line video microscopy in WIFB9 cells that were stably transfected with mdr1-GFP. A critical role for 3'-phosphoinositide products of PI3 kinase was demonstrated in the intracellular trafficking of canalicular ABC transporters and for optimal transporter activity within the canalicular membrane. These studies suggest that many intracellular components, including ATP, Ca2+, numerous GTPases, microtubules, cytoplasmic motors, and other unknown factors, are required for physiologic regulation of ABC transporter traffic from Golgi to the canalicular membrane. Defects in this complex system are postulated to produce an "intrahepatic traffic jam" that results in defective ABC transporter function in the canalicular membrane and, consequently, in cholestasis.     

ATP结合盒转运子A1基因R219K多态性与冠心病的关系

目的:检测冠心病患者及健康对照者ATP结合盒转运子A1(ABCA1)R219K基因多态性,探讨其与中国人冠心病以及血脂水平的关系。方法:选择234例冠心病患者(冠心病组)和198例正常对照者(对照组)为研究对象,用聚合酶链式反应-限制性片段长度多态性方法研究R219K基因多态性与冠心病和血脂水平的关系。结果:与对照组比较,冠心病组RR基因型频率(46·2%)明显高于对照组(33·8%)(P<0·05),而KK型频率(8·9%)显著低于对照组(15·2%)(P<0·05);与RR型患者相比,KK基因型患者血浆高密度脂蛋白(HDL)水平较高。基因型与血脂水平Logistic回归分析结果表明,三酰甘油、HDL、载脂蛋白A1与基因型相关。结论:ABCA1基因R219K变异可能具有动脉硬化保护作用。     

BCRP基因在乳腺癌组织中的表达及意义

目的　研究乳腺癌耐药蛋白 (BCRP)基因在乳腺癌组织中的表达水平 ,探讨其与肿瘤病理分期、淋巴结转移的关系。方法　采用实时荧光定量 RT- PCR检测 5 1例乳腺癌患者癌组织和癌旁组织中 BCRP基因表达。结果　 BCRP基因在乳腺癌组织中的阳性表达率、表达水平均显著高于癌旁组织 (P<0 .0 1、<0 .0 5 ) ;BCRP基因在 、 、 期乳腺癌组织中的表达水平无显著性差异 (P>0 .0 5 ) ;有淋巴结转移者的 BCRP基因表达水平显著高于无淋巴结转移者 (P<0 .0 5 )。结论　 BCRP基因表达水平与肿瘤病理分期无关 ,与淋巴结转移有关 ,BCRP基因过表达可能在乳腺癌的多药耐药中起较重要的作用。     

ATP binding cassette transporter G1 (ABCG1) is an intracellular sterol transporter

Four members of the mammalian ATP binding cassette (ABC) transporter G subfamily are thought to be involved in transmembrane (TM) transport of sterols. The residues responsible for this transport are unknown. The mechanism of action of ABCG1 is controversial and it has been proposed to act at the plasma membrane to facilitate the efflux of cellular sterols to exogenous high-density lipoprotein (HDL). Here we show that ABCG1 function is dependent on localization to intracellular endosomes. Importantly, localization to the endosome pathway distinguishes ABCG1 and/or ABCG4 from all other mammalian members of this superfamily, including other sterol transporters. We have identified critical residues within the TM domains of ABCG1 that are both essential for sterol transport and conserved in some other members of the ABCG subfamily and/or the insulin-induced gene 2 (INSIG-2). Our conclusions are based on studies in which (i) biotinylation of peritoneal macrophages showed that endogenous ABCG1 is intracellular and undetectable at the cell surface, (ii) a chimeric protein containing the TM of ABCG1 and the cytoplasmic domains of the nonsterol transporter ABCG2 is both targeted to endosomes and functional, and (iii) ABCG1 colocalizes with multiple proteins that mark late endosomes and recycling endosomes. Mutagenesis studies identify critical residues in the TM domains that are important for ABCG1 to alter sterol efflux, induce sterol regulatory element binding protein-2 (SREBP-2) processing, and selectively attenuate the oxysterol-mediated repression of SREBP-2 processing. Our data demonstrate that ABCG1 is an intracellular sterol transporter that localizes to endocytic vesicles to facilitate the redistribution of specific intracellular sterols away from the endoplasmic reticulum (ER).     

不同压力对巨噬细胞ATP结合盒转运子A1表达的影响

目的观察不同压力对巨噬细胞ABCA1 mRNA及蛋白表达的影响。方法将THP—1源巨噬细胞依照不同压力分为对照组、60组、80组、100组、120组、140组、160组和180组，分别在大气压下，60mmHg、80mmHg、100mmHg、120mmHg、140mmHg、160mmHg和180mmHg压力下继续培养48h。观察巨噬细胞ABCA1 mRNA及蛋白表达。结果60组、80组、100组、120组ABCA1 mRNA分别为1．11±0．08、1．31±0．04、1．53±0．11和1．15±0．07，较对照组（0．91±0．10）明显增加（P〈0．05或P〈0．01），以100组增加最显著；140组、160组和180组ABCA1mRNA分别为0．75±0．06，0．46±0．08及0．35±0．05，较对照组明显减少（P〈0．01）。ABCA1蛋白表达与mRNA情况相似：60组（1．09±0．06），80组（1．12±0．09），100组（1．41±0．06），120组（1．11±0．06）较对照组（1．00±0．00）明显增加（P〈0．05或P〈0．01），而140组（0．78±0．07）、160组（0．49±0．09）和180组（0．47±0．10）较对照组明显减少（P〈0．01）。结论压力在80---120mmHg范围时，压力升高促进了巨噬细胞ABCA1 mRNA及蛋白表达，压力〉120mmHg后，随着压力的增高，巨噬细胞ABCA1 mRNA及蛋白表达反而减少。     

大肠杆菌ABC转运体研究进展

ABC转运体是整合膜蛋白,穿过细胞膜转运物质,其广泛分布于细菌、真菌和真核生物体内。最近发表的ATP酶结构域、底物结合蛋白及全长转运体的晶体结构^[1]对我们认识转运体的分子结构提供了新的依据。生物信息学方法可确定大肠杆菌的所有转运体以及它们与其它已知转运体的关系,本文就从已经获得的结构数据及构象推测主要集中对大肠杆菌ABC转运体研究进展做一综述。     

Function of the ABC transporters,P-glycoprotein,multidrug resistance protein and breast cancer resistance protein,in minimal residual disease in acute myeloid leukemia

BACKGROUND AND OBJECTIVES: Relapse is common in acute myeloid leukemia (AML) because of persistence of minimal residual disease (MRD). ABC-transporters P-glycoprotein (Pgp) and multidrug resistance protein (MRP), are thought to contribute to treatment failure, while it is unknown whether breast cancer resistance protein (BCRP) does so. However, whether up-regulation of pump activity or selection of subpopulations with higher pump activity occurs during chemotherapy is unclear. The aim of this study was to elucidate whether ABC-transporter function changes during the course of disease. DESIGN AND METHODS: MRD cells were identified using leukemia-associated phenotypes combined with a fluorescent probe assay with substrate/modulator: Syto16/ PSC833 (Pgp), calcein-AM/probenecid (MRP) and BODIPY-prazosin/Ko143 (BCRP); efflux profiles were directly compared with blasts at diagnosis and relapse from the same patient. RESULTS: At diagnosis BCRP activity was undetectable in AML blasts from 23/26 cases, while Pgp activity was present in 36/45 and MRP activity in 26/44 of the cases. Furthermore, no subpopulations of blasts with considerably higher drug efflux capacities were found. Overall, no consistent changes were observed at follow-up [during chemotherapy (n=20), MRD (n=37), relapse (n=26))] in forty-five patients, the mean activities (as percentages of values at diagnosis) were 97% (Pgp), 103% (MRP) and 102% (BCRP). INTERPRETATION AND CONCLUSIONS: Emergence of MRD is thus not accompanied by either upregulation of ABC-transporter function during or after chemotherapy or by selection of pre-existing highly resistant subpopulations. The prognostic value of Pgp and MRP is, therefore, likely related to drug efflux capacity homogeneously distributed in the whole blast population, while BCRP probably has a limited function in drug efflux-related resistance in AML.     

Modulation of drug resistance transporters as a strategy for treating myelodysplastic syndrome

Resistance to chemotherapy is an obstacle to the successful treatment of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). The failure of therapeutic treatment may be due to the development of multidrug resistance (MDR), mechanisms of which include upregulation of membrane-resident transporters which efflux chemotherapeutic drugs from tumor cells, and failure of the cancer cell to undergo apoptosis in response to chemotherapy. Membrane transporter-based drug efflux transporters have been extensively studied, and agents that block drug efflux have been found and investigated. Presence of P-glycoprotein (Pgp, MDR1, ABCB1), a member of the ATP-binding cassette (ABC) transporter family, has been reported to correlate with poor prognosis in AML and MDS. In MDS, Pgp expression increases as the disease progresses. Overexpression of other transporters, such as the multidrug resistance protein (MRP1, ABCC1), and the vault-associated transporter lung resistance protein have been shown as well in both MDS and AML, but their prognostic relevance is not clear. Recently, a novel ABC half-transporter, the breast cancer resistance protein (ABCG2) has been found in approximately 30% of AML cases, and may play a role in resistance to chemotherapy. In clinical trials in MDS, first-generation Pgp blockers, such as cyclosporin-A and verapamil, were minimally effective, non-specific, and toxic. However, another first-generation blocker, quinine, was used in MDS and may specifically benefit MDS patients overexpressing Pgp. A second-generation drug, the non-immunosuppressive cyclosporine analog valspodar (PSC833), was studied in AML and MDS, and was highly toxic, resulting in the need to reduce the dosage of the chemotherapeutic drugs as a result of valspodar reducing the clearance of the chemotherapeutic agents. Third-generation drugs, which are highly specific for Pgp and which seem to have only modest effects on drug clearance, include tariquidar, zosuquidar, laniquidar, and ONT-093. These are all in phase I/II trials and show promise for future treatment.