A cytoplasmic pathway for gapmer antisense oligonucleotide-mediated gene silencing in mammalian cells.

Antisense oligonucleotides (ASOs) are known to trigger mRNA degradation in the nucleus via an RNase H-dependent mechanism. We have now identified a putative cytoplasmic mechanism through which ASO gapmers silence their targets when transfected or delivered gymnotically (i.e. in the absence of any transfection reagent). We have shown that the ASO gapmers can interact with the Ago-2 PAZ domain and can localize into GW-182 mRNA-degradation bodies (GW-bodies). The degradation products of the targeted mRNA, however, are not generated by Ago-2-directed cleavage. The apparent identification of a cytoplasmic pathway complements the previously known nuclear activity of ASOs and concurrently suggests that nuclear localization is not an absolute requirement for gene silencing.

Mechanisms of cardiac arrhythmias and sudden death in transgenic rabbits with long QT syndrome.

Long QT syndrome (LQTS) is a heritable disease associated with ECG QT interval prolongation, ventricular tachycardia, and sudden cardiac death in young patients. Among genotyped individuals, mutations in genes encoding repolarizing K+ channels (LQT1:KCNQ1; LQT2:KCNH2) are present in approximately 90% of affected individuals. Expression of pore mutants of the human genes KCNQ1 (KvLQT1-Y315S) and KCNH2 (HERG-G628S) in the rabbit heart produced transgenic rabbits with a long QT phenotype. Prolongations of QT intervals and action potential durations were due to the elimination of IKs and IKr currents in cardiomyocytes. LQT2 rabbits showed a high incidence of spontaneous sudden cardiac death (>50% at 1 year) due to polymorphic ventricular tachycardia. Optical mapping revealed increased spatial dispersion of repolarization underlying the arrhythmias. Both transgenes caused downregulation of the remaining complementary IKr and IKs without affecting the steady state levels of the native polypeptides. Thus, the elimination of 1 repolarizing current was associated with downregulation of the reciprocal repolarizing current rather than with the compensatory upregulation observed previously in LQTS mouse models. This suggests that mutant KvLQT1 and HERG interacted with the reciprocal wild-type alpha subunits of rabbit ERG and KvLQT1, respectively. These results have implications for understanding the nature and heterogeneity of cardiac arrhythmias and sudden cardiac death.

Rapid evolution of blood-brain-barrier-penetrating AAV capsids by RNA-driven biopanning.

Therapeutic payload delivery to the central nervous system (CNS) remains a major challenge in gene therapy. Recent studies using function-driven evolution of adeno-associated virus (AAV) vectors have successfully identified engineered capsids with improved blood-brain barrier (BBB) penetration and CNS tropism in mouse. However, these strategies require transgenic animals and thus are limited to rodents. To address this issue, we developed a directed evolution approach based on recovery of capsid library RNA transcribed from CNS-restricted promoters. This RNA-driven screen platform, termed TRACER (Tropism Redirection of AAV by Cell-type-specific Expression of RNA), was tested in the mouse with AAV9 peptide display libraries and showed rapid emergence of dominant sequences. Ten individual variants were characterized and showed up to 400-fold higher brain transduction over AAV9 following systemic administration. Our results demonstrate that the TRACER platform allows rapid selection of AAV capsids with robust BBB penetration and CNS tropism in non-transgenic animals.

Pore mutants of HERG and KvLQT1 downregulate the reciprocal currents in stable cell lines.

We previously reported a transgenic rabbit model of long QT syndrome based on overexpression of pore mutants of repolarizing K(+) channels KvLQT1 (LQT1) and HERG (LQT2).The transgenes in these rabbits eliminated the slow and fast components of the delayed rectifier K(+) current (I(Ks) and I(Kr), respectively), as expected. Interestingly, the expressed pore mutants of HERG and KvLQT1 downregulated the remaining reciprocal repolarizing currents, I(Ks) and I(Kr), without affecting the steady-state levels of the native polypeptides. Here, we sought to further explore the functional interactions between HERG and KvLQT1 in heterologous expression systems. Stable Chinese hamster ovary (CHO) cell lines expressing KvLQT1-minK or HERG were transiently transfected with expression vectors coding for mutant or wild-type HERG or KvLQT1. Transiently expressed pore mutant or wild-type KvLQT1 downregulated I(Kr) in HERG stable CHO cell lines by 70% and 44%, respectively. Immunostaining revealed a severalfold lower surface expression of HERG, which could account for the reduction in I(Kr) upon KvLQT1 expression. Deletion of the KvLQT1 NH(2)-terminus did not abolish the downregulation, suggesting that the interactions between the two channels are mediated through their COOH-termini. Similarly, transiently expressed HERG reduced I(Ks) in KvLQT1-minK stable cells. Coimmunoprecipitations indicated a direct interaction between HERG and KvLQT1, and surface plasmon resonance analysis demonstrated a specific, physical association between the COOH-termini of KvLQT1 and HERG. Here, we present an in vitro model system consistent with the in vivo reciprocal downregulation of repolarizing currents seen in transgenic rabbit models, illustrating the importance of the transfection method when studying heterologous ion channel expression and trafficking. Moreover, our data suggest that interactions between KvLQT1 and HERG are mediated through COOH-termini.

Structural determinants of alpha4beta2 nicotinic acetylcholine receptor trafficking.

The structural determinants of nicotinic acetylcholine receptor (AChR) trafficking have yet to be fully elucidated. Hydrophobic residues occur within short motifs important for endoplasmic reticulum (ER) export or endocytotic trafficking. Hence, we tested whether highly conserved hydrophobic residues, primarily leucines, in the cytoplasmic domain of the alpha4beta2 AChR subunits were required for cell surface expression of alpha4beta2 AChRs. Mutation of F350, L351, L357, and L358 to alanine in the alpha4 AChR subunit attenuates cell surface expression of mutant alpha4beta2 AChRs. Mutation of F342, L343, L349, and L350 to alanine at homologous positions in the beta2 AChR subunit abolishes cell surface expression of mutant alpha4beta2 AChRs. The hydrophobic nature of the leucine residue is a primary determinant of its function because mutation of L343 to another hydrophobic amino acid, phenylalanine, in the beta2 AChR subunit only poorly inhibits trafficking of mutant alpha4beta2 AChR to the cell surface. All mutant alpha4beta2 AChRs exhibit high-affinity binding for [3H]epibatidine. In both tsA201 cells and differentiated SH-SY5Y neural cells, wild-type alpha4beta2 AChRs colocalize with the Golgi marker giantin, whereas mutant alpha4beta2 AChRs fail to do so. The striking difference between mutant alpha4 versus mutant beta2 AChR subunits on cell surface expression of mutant alpha4beta2 AChRs points to a cooperative or regulatory role for the alpha4 AChR subunit and an obligatory role for the beta2 AChR subunit in ER export. Collectively, our results identify, for the first time, residues within AChR subunits that are essential structural determinants of alpha4beta2 AChR ER export.

A functional role of intracellular loops of human multidrug resistance protein 1.

Multidrug resistance protein 1 (MRP1) is a human ATP-binding cassette (ABC) transporter in the plasma membrane. It confers multidrug resistance to tumor cells by actively effluxing intracellular drugs. To examine the functional significance of intracellular loops (ICLs) in MRP1, we determined the effect of mutation of the amino acid sequence EXXXG, which is conserved in ICL5 and ICL7 of human MRP1, 2 and 3, sulfonylurea receptor (SUR) 1 and 2, and mouse MRP1 and 2. E and G in the ICLs of human MRP1 were mutated to L and P, respectively, and the N-terminal (including ICL5) and C-terminal (including ICL7) wild type or mutant halves of MRP1 were co-expressed in insect cells. The mutation of either ICL5 or ICL7 considerably decreased ATP-dependent LTC4 uptake into vesicles of insect cells expressing mutated MRP1. GSH-dependent photolabeling of MRP1 with an 125I-labeled photoaffinity analog of azido agosterol A (azido AG-A) was abolished by the mutations in ICL5 and ICL7. Mutations in ICL5 of MRP1 almost completely inhibited the labeling of NBD2, but not NBD1, by 8-azido-alpha-[32P]ATP. In contrast, mutations in ICL7 of MRP1 abolished the labeling of both NBDs. Mutation of either ICL5 or ICL7 of MRP1 almost completely inhibited vanadate trapping with 8-azido-alpha-[32P]ATP by both NBD1 and NBD2 domains. These findings indicate that the intramolecular signaling between NBD and ICLs in MRP1 is vital for MRP1 function.

Transport of methotrexate, methotrexate polyglutamates, and 17beta-estradiol 17-(beta-D-glucuronide) by ABCG2: effects of acquired mutations at R482 on methotrexate transport.

ABCG2 is a plasma membrane efflux pump that is able to confer resistance to several anticancer agents, including mitoxantrone, camptothecins, anthracyclines, and flavopiridol. The antimetabolite methotrexate (MTX) was inferred recently to be an additional substrate of the pump based on the analysis of ABCG2-overexpressing cell lines. However, the transport characteristics of the pump with regard to this agent have not been determined. In addition, physiological substrates of ABCG2 have not been identified. Here we examine the in vitro transport properties of the pump using membrane vesicles prepared from HEK293 cells transfected with ABCG2 expression vector. In so doing it is shown that MTX is a high capacity low affinity substrate of the pump, with K(m) and V(max) values of 1.34 +/- 0.18 mM and 687 +/- 87 pmol/mg/min, respectively. Unlike previously characterized multidrug resistance protein family members, ABCG2 is also able to transport MTX diglutamate and MTX triglutamate. However, addition of even one more glutamyl residue is sufficient to completely abrogate ABCG2-mediated transport. By contrast with the wild-type protein (ABCG2-R482), two ABCG2 variants that have been identified in drug selected cell lines, R482T and R482G, were unable to transport MTX to any extent. Similarly, folic acid was subject to efflux by the wild-type protein but not by the two mutants. However, transport of the reduced folate leucovorin was not detected for either the wild-type or the mutant proteins. Finally, it is shown that ABCG2 is capable of transporting E(2)17betaG with K(m) and V(max) values of 44.2 +/- 4.3 micro M and 103 +/- 17 pmol/mg/min, respectively. These results indicate that ABCG2 is a component of the energy-dependent efflux system for certain folates and antifolates, but that its transport characteristics with respect to polyglutamates and reduced folates are not identical to those of multidrug resistance protein family members. In addition, it is demonstrated that R482 mutations observed in drug-resistant cell lines have profound effects on the in vitro transport properties of the pump.

MRP1 mutated in the L0 region transports SN-38 but not leukotriene C4 or estradiol-17 (beta-D-glucuronate).

Multidrug resistance protein 1 (MRP1) is an ATP-binding cassette transporter that confers multidrug resistance on tumor cells. Much convincing evidence has accumulated that MRP1 transports most substances in a GSH-dependent manner. On the other hand, several reports have revealed that MRP1 can transport some substrates independently of GSH; however, the importance of GSH-independent transport activity is not well established and the mechanistic differences between GSH-dependent and -independent transport by MRP1 are unclear. We previously demonstrated that the amino acids W261 and K267 in the L0 region of MRP1 were important for leukotriene C4 (LTC4) transport activity of MRP1 and for GSH-dependent photolabeling of MRP1 with azidophenyl agosterol-A (azidoAG-A). In this paper, we further tested the effect of W222L, W223L and R230A mutations in MRP1, designated dmL0MRP1, on MRP1 transport activity. SN-38 is an active metabolic form of CPT-11 that is one of the most promising anti-cancer drugs. Membrane vesicles prepared from cells expressing dmL0MRP1 could transport SN-38, but not LTC4 or estradiol-17 (beta-D-glucuronate), and could not be photolabeled with azidoAG-A. These data suggested that SN-38 was transported by a different mechanism than that of GSH-dependent transport. Understanding the GSH-independent transport mechanism of MRP1, and identification of drugs that are transported by this mechanism, will be critical for combating MRP1-mediated drug resistance. We performed a pairwise comparison of compounds that are transported by MRP1 in a GSH-dependent or -independent manner. These data indicated that it may be possible to predict compounds that are transported by MRP1 in a GSH-independent manner.

Overcoming multidrug drug resistance in P-glycoprotein/MDR1-overexpressing cell lines by ecteinascidin 743.

Ecteinascidin 743 (Et-743) is a novel anticancer agent forming covalent guanine adducts at specific sites in the DNA minor groove. Et-743 has a unique mechanism of action because it kills cancer cells by poisoning transcription-coupled nucleotide excision repair. Recent studies suggested a complex relationship between P-glycoprotein (P-gp)/MDR1 and Et-743. On one hand, Et-743 was reported to down-regulate the MDR1 promoter in vitro. On the other hand, P-gp overexpression was hypothesized to contribute to Et-743 resistance in an ovarian cell line. The present study was performed to further investigate the relationship between P-gp/MDR1 and the activity of Et-743. First, we found no P-gp/MDR1 overexpression (mRNA and protein levels) in two independently generated Et-743-resistant human colon carcinoma cell lines (HCT116/ER5 and SW480/ER0.5). Secondly, we found no cross-resistance to Et-743 in two well-characterized P-gp/MDR1-overexpressing cell lines (KB-8-5 and KB-C-2). Third, Et-743 pretreatment enhanced the cytotoxicity and the cellular accumulation of doxorubicin and vincristine in P-gp/MDR1-overexpressing KB-8-5/KB-C-2 cell lines. Fourth, we observed P-gp/MDR1 down-regulation by Et-743 in KB-C-2 cells. These results indicate that Et-743 does not select for the emergence of a P-gp phenotype in all cell lines made resistant to Et-743 and that P-gp overexpression is not sufficient to confer resistance to Et-743. Furthermore, Et-743 is an effective agent in P-gp-overexpressing cells. Et-743 can potentiate the activity of other chemotherapeutic agents by down-regulating P-gp/MDR1, suggesting that the combination of Et-743 and chemotherapeutic agents that are substrates for P-gp/MDR1 may be valuable in the clinic.

Localization of the GSH-dependent photolabelling site of an agosterol A analog on human MRP1.

1. Human multidrug resistance protein 1 (MRP1) is a 190 kDa membrane glycoprotein that confers multidrug resistance (MDR) to tumor cells. We recently demonstrated that glutathione (GSH) is required for the labelling of the C-terminal half of MRP1 with a photoanalog of agosterol A (azido AG-A). In this study, we further characterized the GSH-dependent photolabelling site of azido AG-A on MRP1. 2. An epitope-inserted MRP1, MRP1 1222HA, which has two hemagglutinin A (HA) epitopes in the extracellular loop between transmembrane segment (TM) 16 and TM17 of the transporter, could bind azido AG-A in a GSH-dependent manner. 3. Protease digestion of the photolabelled MRP1 1222HA, followed by immunoprecipitation with an anti-HA antibody suggested that the GSH-dependent azido AG-A photolabelling site on MRP1 resides in the region within TM14-17 and the cytoplasmic region proximate to the C-terminus of TM17. 4. Arg(1210) in human MRP2 that corresponds to Arg(1202) in human MRP1 has an important role in the transporting activity of MRP2. Therefore, we replaced the Arg residue at position 1202 of MRP1 with Gly. Whereas photolabelling of the mutant MRP1 R1202G was greatly reduced, it retained leukotriene C(4) (LTC(4)) transport activity and conferred Vincristine resistance in LLC-PK1 cells. 5. In summary, this study demonstrated that the GSH-dependent azido AG-A photolabelling site on MRP1 resides in the region within TM14-17 and the cytoplasmic region proximate to the C-terminus of TM17. The charged amino acid Arg(1202) proximate to TM helix 16 is of critical importance for the GSH-dependent photolabelling of MRP1 with azido AG-A. Arg(1202) itself or the region nearby Arg(1202) may be involved in azido AG-A photolabelling.

Reversal of P-glycoprotein mediated multidrug resistance by a newly synthesized 1,4-benzothiazipine derivative, JTV-519.

A newly synthesized 1,4-benzothiazipine derivate, 4-[3-(4-benzylpiperidin-1-yl) propionyl]-7-methoxy-2,3,4,5-tetrahydro-1, 4-benzothiazepine monohydrochloride (JTV-519) was examined for its ability to reverse P-glycoprotein (P-gp) and multidrug resistance protein 1 (MRP1) mediated multidrug resistance (MDR) in K562/MDR and KB/MRP cells, respectively. JTV-519 at 3 microM reversed the resistance of K562/MDR cells to vincristine (VCR), taxol, etoposide (VP16), adriamycin (ADM) and actinomycin D and at 0.5 or 1 microM reversed their resistance to STI571. JTV-519 at 10 microM enhanced the accumulation of ADM in K562/MDR cells to the level in parental K562 cells and inhibited the efflux of ADM from K562/MDR cells. Photoaffinity labeling of P-gp with 3H-azidopine was almost completely inhibited by 500 microM JTV-519. JTV-519 at 3 microM also partially reversed the resistance of KB/MRP cells to VCR and at 500 microM partially inhibited the photoaffinity labeling of MRP1 with (125)I-II-azidophenyl agosterol A (125I-azidoAG-A). These results suggest that JTV-519 reversed the resistance to the anti-cancer agents in P-gp and MRP1 overexpressing multidrug-resistant cells by directly binding to P-gp and MRP1, and competitively inhibiting transport of the anti-cancer agents.

Dioxin exposure down-regulates nitric oxide synthase and NADPH-diaphorase activities in the hypothalamus of Long-Evans rat.

In this study, we investigated the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) gastric administration on the expression of neuronal nitric oxide synthase (nNOS) and the NADPH-diaphorase (NADPH-d) activities in the brain of the Long-Evans rat. A single dose of TCDD (dissolved in olive oil, 50 microg/kg) or olive oil alone was administered to the rats by gavage. nNOS Western blotting experiment indicated a marked decrease in nNOS immunoreactivity at 1 and 2 weeks after TCDD treatment. NADPH-d histochemistry results showed a marked decrease in the number of NADPH-d stained cell bodies in the paraventricular hypothalamic nucleus, lateral hypothalamic area and perifornical nucleus in the TCDD-treated rats. The present study suggests that TCDD administration down-regulates nitric oxide product in the hypothalamus, which may be partially responsible for TCDD-induced feeding inhibition.

Binding site(s) on P-glycoprotein for a newly synthesized photoaffinity analog of agosterol A.

Agosterol A (AG-A) is a novel agent that reverses P-glycoprotein (P-gp) and multidrug resistance protein-1 (MRP1)-meditated multidrug resistance (MDR). We have synthesized [125I]11-azidophenyl agosterol A (azidoAG-A), a photoaffinity analog of AG-A, and characterized its binding to P-gp in membrane vesicles prepared from multidrug-resistant P-gp-overexpressing KB-C2 cells. The photoanalog photolabeled intact P-gp and both the N- and C-terminal fragments of P-gp. [125I]AzidoAG-A is transported by P-gp and the intracellular accumulation of both [125I]azidoAG-A and [3H]AG-A in KB-C2 cells was lower than that in the parental drug-sensitive KB-3-1 cells. [125I]AzidoAG-A bound to the drug binding site(s) on P-gp because photoaffinity labeling of P-gp was inhibited by a variety of known P-gp substrates, including anticancer, reversing, and anti-human immunodeficiency virus (HIV) agents. The binding of [125I]azidoAG-A to P-gp differs from the binding of other photolabeled probes such as iodoaryl-azidoprazosin (IAAP) to P-gp and from the binding of [125I]azidoAG-A to MRP1 based on the differing effects of flupentixol and glutathione (GSH) on their binding. Thus, [125I]azidoAG-A will be a useful tool to elucidate the structure and function of P-gp because it directly binds to the drug binding site(s) on P-gp, is transported by P-gp, and exhibits different P-gp binding characteristics than IAAP.

Presynaptic targeting of alpha4beta 2 nicotinic acetylcholine receptors is regulated by neurexin-1beta.

The mechanisms involved in the targeting of neuronal nicotinic acetylcholine receptors (AChRs), critical for their functional organization at neuronal synapses, are not well understood. We have identified a novel functional association between alpha4beta2 AChRs and the presynaptic cell adhesion molecule, neurexin-1beta. In non-neuronal tsA 201 cells, recombinant neurexin-1beta and mature alpha4beta2 AChRs form complexes. alpha4beta2 AChRs and neurexin-1beta also coimmunoprecipitate from rat brain lysates. When exogenous alpha4beta2 AChRs and neurexin-1beta are coexpressed in hippocampal neurons, they are robustly targeted to hemi-synapses formed between these neurons and cocultured tsA 201 cells expressing neuroligin-1, a postsynaptic binding partner of neurexin-1beta. The extent of synaptic targeting is significantly reduced in similar experiments using a mutant neurexin-1beta lacking the extracellular domain. Additionally, when alpha4beta2 AChRs, alpha7 AChRs, and neurexin-1beta are coexpressed in the same neuron, only the alpha4beta2 AChR colocalizes with neurexin-1beta at presynaptic terminals. Collectively, these data suggest that neurexin-1beta targets alpha4beta2 AChRs to presynaptic terminals, which mature by trans-synaptic interactions between neurexins and neuroligins. Interestingly, human neurexin-1 gene dysfunctions have been implicated in nicotine dependence and in autism spectrum disorders. Our results provide novel insights as to possible mechanisms by which dysfunctional neurexins, through downstream effects on alpha4beta2 AChRs, may contribute to the etiology of these neurological disorders.

GSH inhibits trypsinization of the C-terminal half of human MRP1.

MRP1 is a 190-kDa membrane glycoprotein that confers multidrug resistance to tumor cells. The accumulated evidence has proved that GSH interacts with MRP1 and stimulates drug transport. However, the mechanism of GSH-dependent drug transport by MRP1 remains unclear. In this study, we used limited tryptic digestion of MRP1 in isolated membrane vesicles, in the presence and absence of GSH, to investigate the influence of GSH on MRP1 conformation. We found that GSH inhibited the generation of an approximately 35-kDa C-terminal tryptic fragment (including a C-terminal His tag) termed C2 from MRP1. This effect of GSH was not because of direct inhibition of trypsin activity, and agosterol A enhanced the inhibitory effect of GSH. The main cleavage site in MRP1 for the generation of the C2 fragment by trypsin resided between TMD2 and NBD2 of MRP1. Limited tryptic digestion of membrane vesicles expressing various truncated and co-expressed MRP1 fragments in the presence and absence of GSH revealed that GSH inhibited the production of the C2 fragment only in the presence of the L(0) region of MRP1. Thus the L(0) region is required for the inhibition of trypsinization of the C-terminal half of MRP1 by GSH. These findings, together with previous reports, suggest that GSH induces a conformational change at a site within the MRP1 that is indispensable for the interaction of MRP1 with its substrates.

Function of the ABC signature sequences in the human multidrug resistance protein 1.

Human multidrug resistance protein 1 (MRP1) is a membrane ATP-binding cassette transporter that confers multidrug resistance to tumor cells by effluxing intracellular drugs in an ATP-dependent manner. The mechanisms by which transport occurs and by which ATP hydrolysis is coupled to drug transport are not fully elucidated. In particular, the function of the signature sequences in the nucleotide binding domains (NBDs) of MRP1 is unknown. We therefore investigated the effect of mutation of the signature sequences (G771D and G1433D) and of the Walker A motifs (K684M and K1333M) in the NBDs on the 8-azido-[alpha-32P]ATP photolabeling and 8-azido-[alpha-32P]ADP vanadate trapping of MRP1. Both mutations in the Walker A motif almost completely inhibited the labeling of the mutated NBD with 8-azido-[alpha-32P]ATP but not the labeling of the other intact NBD. In contrast, the G771D mutation in the signature sequence of NBD1 enhanced the labeling of NBD1 but slightly decreased the labeling of NBD2. The G1433D mutation in the signature motif of NBD2 enhanced the labeling of NBD2 but did not affect the labeling of NBD1. These effects were all substrate-independent. Photolabeling of NBD2 and a very slight photolableing of NBD1 were detectable under vanadate trapping conditions with 8-azido-[alpha-32P]ATP. Trapping at both NBD1 and NBD2 was almost completely inhibited by K684M and K1333M mutations and by the K684M/K1333M double mutation. The G771D mutation completely inhibited trapping at NBD2 and considerably inhibited trapping at NBD1. However, whereas the G1433D mutation also considerably inhibited trapping at NBD1, it only partially inhibited trapping of NBD2, and the trapping could still be enhanced by leukotriene C4. Our findings suggest that both signature sequences of MRP1 are involved in ATP hydrolysis and must be intact for the ATP hydrolysis and the transport by MRP1.

A positively charged amino acid proximal to the C-terminus of TM17 of MRP1 is indispensable for GSH-dependent binding of substrates and for transport of LTC4.

MRP1 is a 190 kDa membrane glycoprotein that confers multidrug resistance (MDR) to tumor cells. Our recent study demonstrated that GSH is required for the labeling of MRP1(932)(-)(1531) with a photoanalogue of agosterol A (AG-A) and suggested that GSH interacts with the L(0) region of MRP1. In this study, we further characterized the GSH-dependent binding site of azido AG-A on MRP1. Coexpression of the N- and C-terminal halves of MRP1 (residues 1-1222, TM1-16, and 1223-1531, TM17, respectively) in Sf21 insect cells reconstituted a functional drug transporter with a K(m) for LTC(4) (97 nM) similar to that of intact MRP1. In membrane vesicles from those cells, GSH-dependent photolabeling of the MRP1 fragment (1-1222) required the coexpression of the C-terminal MRP1 fragment (1223-1531). An MRP1 fragment extending from residue 1 to 1295 however could be photolabeled by azido AG-A in a GSH-dependent manner. These data indicate that amino acids 1223-1295 of MRP1 are required for AG-A binding to MRP1 in a GSH-dependent manner. However, cross-linking of the photolabel to MRP1 occurs at a more upstream site. An arginine residue at position 1249 of MRP1 was shown to be important for the GSH-dependent binding of AG-A to MRP1. Mutation of this arginine to alanine (R1249A) resulted in a decreased level of GSH-dependent azido AG-A photolabeling of MRP1. Furthermore, this mutant attenuated MRP1 function by decreasing the level of LTC(4) substrate transport and impairing resistance to the drug vincristine (VCR). In summary, this study demonstrates that a region of MRP1 (amino acids 1223-1295), which includes TM helix 17, is required for azido AG-A binding to MRP1 in a GSH-dependent manner. A GSH-dependent drug binding site may exist in this region. Furthermore, our findings suggest that the charged amino acid Arg(1249) proximal to the C-terminus of TM helix 17 is indispensable for MRP1-substrate interaction and the function of MRP1.

Reversal of the resistance to STI571 in human chronic myelogenous leukemia K562 cells.

STI571, an Abl-specific tyrosine kinase inhibitor, selectively kills Bcr-Abl-containing cells in vitro and in vivo. However, some chronic myelogenous leukemia (CML) cell lines are resistant to STI571. We evaluated whether STI571 interacts with P-glycoprotein (P-gp) and multidrug resistance protein 1 (MRP1), and examined the effect of agents that reverse multidrug resistance (MDR) on the resistance to SI571 in MDR cells. STI571 inhibited the [(125)I]azidoagosterol A-photolabeling of P-gp, but not that of MRP1. K562/MDR cells that overexpress P-gp were 3.67 times more resistant to STI571 than the parental Philadelphia-chromosome-positive (Ph +) CML K562 cells, and this resistance was most effectively reversed by cepharanthine among the tested reversing agents. The concentration of STI571 required to completely inhibit tyrosine phosphorylation in K562/MDR cells was about 3 times higher than that in K562 cells, and cepharanthine abolished the difference. In KB-G2 cells that overexpress P-gp, but not Bcr-Abl, 2.5 micro M STI571 partly reversed the resistance to vincristine (VCR), paclitaxel, etoposide (VP-16) and actinomycin D (ACD) but not to Adriamycin (ADM) or colchicine. STI571 increased the accumulation of VCR, but not that of ADM in KB-G2 cells. STI571 did not reverse resistance to any agent in KB/MRP cells that overexpress MRP1. These findings suggest that STI571 is a substrate for P-gp, but is less efficiently transported by P-gp than VCR, and STI571 is not a substrate for MRP1. Among the tested reversing agents that interact with P-gp, cepharanthine was the most effective agent for the reversal of the resistance to STI571 in K562/MDR cells. Furthermore, STI571 itself was a potent reversing agent for MDR in P-gp-expressing KB-G2 cells.

Molecular basis for the inhibition of hypoxia-induced apoptosis by 2-deoxy-D-ribose.

An angiogenic factor, platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP), stimulates the chemotaxis of endothelial cells and confers resistance to apoptosis induced by hypoxia. 2-deoxy-D-ribose, a degradation product of thymidine generated by TP enzymatic activity partially prevented hypoxia-induced apoptosis. 2-Deoxy-D-ribose inhibits a number of components of the caspase-mediated hypoxia-induced apoptotic pathway. It inhibits hypoxia-induced caspase 3 activation, mitochondrial cytochrome c release, downregulation of Bcl-2 and Bcl-x(L), upregulation of hypoxia-inducible factor (HIF)-1 alpha, and loss of mitochondrial transmembrane potential in human leukemia HL-60 cell line. These findings suggest a molecular mechanism by which 2-deoxy-d-ribose confers the resistance to apoptosis. Thus 2-deoxy-D-ribose-modulated suppression of HIF-1 alpha expression could prevent the hypoxia-induced decrease of the anti-apoptotic Bcl-2 and Bcl-x(L) on the mitochondria. 2-Deoxy-L-ribose and its analogs may enhance apoptosis and suppress the growth of tumors by competitively inhibiting the activities of 2-deoxy-d-ribose and thus these analogs show promise for anti-tumor therapy.

Thymidine phosphorylase inhibits apoptosis induced by cisplatin.

An angiogenic factor, platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP), stimulates the chemotaxis of endothelial cells and confers resistance to apoptosis induced by hypoxia. 2-Deoxy-D-ribose, a degradation product of thymidine generated by TP, partially prevents hypoxia-induced apoptosis. TP is expressed at higher levels in tumor tissues compared to the adjacent non-neoplastic tissues in a variety of human carcinomas. High expression of TP is associated with an unfavorable prognosis. To investigate the effect of TP on cisplatin-induced apoptosis, human leukemia Jurkat cells were transfected with wild-type or mutant (L148R) TP cDNA. TP inhibited a number of steps in the cisplatin-induced apoptotic pathway, activation of caspases 3 and 9 and mitochondrial cytochrome c release. These findings suggest a mechanism by which TP confers resistance to apoptosis induced by cisplatin. Moreover, mutant TP that has no enzymatic activity also suppressed cisplatin-induced apoptosis. These findings indicate that TP has cytoprotective functions against cytotoxic agents which are independent of its enzymatic activity.