Simultaneous quantitation of oxidized and reduced glutathione via LC-MS/MS: An insight into the redox state of hematopoietic stem cells.

Cellular redox balance plays a significant role in the regulation of hematopoietic stem-progenitor cell (HSC/MPP) self-renewal and differentiation. Unregulated changes in cellular redox homeostasis are associated with the onset of most hematological disorders. However, accurate measurement of the redox state in stem cells is difficult because of the scarcity of HSC/MPPs. Glutathione (GSH) constitutes the most abundant pool of cellular antioxidants. Thus, GSH metabolism may play a critical role in hematological disease onset and progression. A major limitation to studying GSH metabolism in HSC/MPPs has been the inability to measure quantitatively GSH concentrations in small numbers of HSC/MPPs. Current methods used to measure GSH levels not only rely on large numbers of cells, but also rely on the chemical/structural modification or enzymatic recycling of GSH and therefore are likely to measure only total glutathione content accurately. Here, we describe the validation of a sensitive method used for the direct and simultaneous quantitation of both oxidized and reduced GSH via liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) in HSC/MPPs isolated from bone marrow. The lower limit of quantitation (LLOQ) was determined to be 5.0ng/mL for GSH and 1.0ng/mL for GSSG with lower limits of detection at 0.5ng/mL for both glutathione species. Standard addition analysis utilizing mouse bone marrow shows that this method is both sensitive and accurate with reproducible analyte recovery. This method combines a simple extraction with a platform for the high-throughput analysis, allows for efficient determination of GSH/GSSG concentrations within the HSC/MPP populations in mouse, chemotherapeutic treatment conditions within cell culture, and human normal/leukemia patient samples. The data implicate the importance of the modulation of GSH/GSSG redox couple in stem cells related diseases.

Genetics of CD33 in Alzheimer's disease and acute myeloid leukemia.

The CD33 single-nucleotide polymorphism (SNP) rs3865444 has been associated with the risk of Alzheimer's disease (AD). Rs3865444 is in linkage disequilibrium with rs12459419 which has been associated with efficacy of an acute myeloid leukemia (AML) chemotherapeutic agent based on a CD33 antibody. We seek to evaluate the extent to which CD33 genetics in AD and AML can inform one another and advance human disease therapy. We have previously shown that these SNPs are associated with skipping of CD33 exon 2 in brain mRNA. Here, we report that these CD33 SNPs are associated with exon 2 skipping in leukocytes from AML patients and with a novel CD33 splice variant that retains CD33 intron 1. Each copy of the minor rs12459419T allele decreases prototypic full-length CD33 expression by ∼ 25% and decreases the AD odds ratio by ∼ 0.10. These results suggest that CD33 antagonists may be useful in reducing AD risk. CD33 inhibitors may include humanized CD33 antibodies such as lintuzumab which was safe but ineffective in AML clinical trials. Here, we report that lintuzumab downregulates cell-surface CD33 by 80% in phorbol-ester differentiated U937 cells, at concentrations as low as 10 ng/ml. Overall, we propose a model wherein a modest effect on RNA splicing is sufficient to mediate the CD33 association with AD risk and suggest the potential for an anti-CD33 antibody as an AD-relevant pharmacologic agent.

Mapping the functional yeast ABC transporter interactome.

ATP-binding cassette (ABC) transporters are a ubiquitous class of integral membrane proteins of immense clinical interest because of their strong association with human disease and pharmacology. To improve our understanding of these proteins, we used membrane yeast two-hybrid technology to map the protein interactome of all of the nonmitochondrial ABC transporters in the model organism Saccharomyces cerevisiae and combined this data with previously reported yeast ABC transporter interactions in the BioGRID database to generate a comprehensive, integrated 'interactome'. We show that ABC transporters physically associate with proteins involved in an unexpectedly diverse range of functions. We specifically examine the importance of the physical interactions of ABC transporters in both the regulation of one another and in the modulation of proteins involved in zinc homeostasis. The interaction network presented here will be a powerful resource for increasing our fundamental understanding of the cellular role and regulation of ABC transporters.

Casein kinase 2α regulates multidrug resistance-associated protein 1 function via phosphorylation of Thr249.

We have shown previously that the function of Ycf1p, yeast ortholog of multidrug resistance-associated protein 1 (MRP1), is regulated by yeast casein kinase 2α (Cka1p) via phosphorylation at Ser251. In this study, we explored whether casein kinase 2α (CK2α), the human homolog of Cka1p, regulates MRP1 by phosphorylation at the semiconserved site Thr249. Knockdown of CK2α in MCF7-derived cells expressing MRP1 [MRP1 CK2α(-)] resulted in increased doxorubicin sensitivity. MRP1-dependent transport of leukotriene C(4) and estradiol-17ß-d-glucuronide into vesicles derived from MRP1 CK2α(-) cells was decreased compared with MRP1 vesicles. Moreover, mutation of Thr249 to alanine (MRP1-T249A) also resulted in decreased MRP1-dependent transport, whereas a phosphomimicking mutation (MRP1-T249E) led to dramatic increase in MRP1-dependent transport. Studies in tissue culture confirmed these findings, showing increased intracellular doxorubicin accumulation in MRP1 CK2α(-) and MRP1-T249A cells compared with MRP1 cells. Inhibition of CK2 kinase by 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole resulted in increased doxorubicin accumulation in MRP1 cells, but not in MRP1 CK2α(-), MRP1-T249A, or MRP1-T249E cells, suggesting that CK2α regulates MRP1 function via phosphorylation of Thr249. Indeed, CK2α and MRP1 interact physically, and recombinant CK2 phosphorylates MRP1-derived peptide in vitro in a Thr249-dependent manner, whereas knockdown of CK2α results in decreased phosphorylation at MRP1-Thr249. The role of CK2 in regulating MRP1 was confirmed in other cancer cell lines where CK2 inhibition decreased MRP1-mediated efflux of doxorubicin and increased doxorubicin cytotoxicity. This study supports a model in which CK2α potentiates MRP1 function via direct phosphorylation of Thr249.

Ycf1p attenuates basal level oxidative stress response in Saccharomyces cerevisiae.

Ycf1p function is regulated by casein kinase 2α, Cka1p, via phosphorylation of Ser251. Cka1p-mediated phosphorylation of Ycf1p is attenuated in response to high salt stress. Previous results from our lab suggest a role for Ycf1p in cellular resistance to salt stress. Here, we show that Ycf1p plays an important role in cellular resistance to salt stress by maintaining the cellular redox balance via glutathione recycling. Our results suggest that during acute salt stress increased Sod1p, Sod2p and Ctt1p activity is the main compensatory for the loss in Ycf1p function that results from reduced Ycf1p-dependent recycling of cellular GSH levels.

The G671V variant of MRP1/ABCC1 links doxorubicin-induced acute cardiac toxicity to disposition of the glutathione conjugate of 4-hydroxy-2-trans-nonenal.

OBJECTIVE: Doxorubicin-induced acute cardiotoxicity is associated with the Gly671Val (G671V; rs45511401) variant of multidrug resistance-associated protein 1 (MRP1). Doxorubicin redox cycling causes lipid peroxidation and generation of the reactive electrophile, 4-hydroxy-2-trans-nonenal (HNE). Glutathione forms conjugates with HNE, yielding an MRP1 substrate, GS-HNE, whose intracellular accumulation can cause toxicity. METHODS: We established stable HEK293 cell lines overexpressing wild-type MRP1 (HEKMRP1), G671V (HEKG671V), and R433S (HEKR433S), a variant not associated with doxorubicin-induced cardiotoxicity and investigated the sensitivity of HEKG671V cells to doxorubicin and transport capacity of G671V toward GS-HNE. RESULTS: In ATP-dependent transport studies using plasma membrane-derived vesicles, the Vmax (pmol/min/mg) for GS-HNE transport was the lowest for G671V (69±4) and the highest for R433S (972±213) compared with wild-type MRP1 (416±22), whereas the Km values were 2.8±0.4, 6.0 or more, and 1.7±0.2 µmol/l, respectively. In cells, the doxorubicin IC50 (48 h) was not different in HEKMRP1 (463 nmol/l) versus HEKR433S (645 nmol/l), but this parameter was significantly lower in HEKG671V (181 nmol/l). HEKG671V retained significantly (approximately 20%) more, whereas HEKR433S retained significantly less intracellular doxorubicin than HEKMRP1. Similarly, HEKG671V cells treated with 1.5 µmol/l of doxorubicin for 24 h retained significantly more GS-HNE. In cells treated with 0.5 µmol/l of doxorubicin for 48 , glutathione and glutathione disulfide levels and the glutathione/glutathione disulfide ratio were significantly decreased in HEKG671V versus HEKMRP1; these values were similar in HEKR433S versus HEKMRP1. CONCLUSION: These data suggest that decreased MRP1-dependent GS-HNE efflux contributes to increased doxorubicin toxicity in HEKG671V and potentially in individuals carrying the G671V variant.

Functional analysis of nonsynonymous single nucleotide polymorphisms of multidrug resistance-associated protein 2 (ABCC2).

BACKGROUND: Multidrug resistance-associated protein 2 (MRP2; ABCC2) mediates the biliary excretion of glutathione, glucuronide, and sulfate conjugates of endobiotics and xenobiotics. Single nucleotide polymorphisms (SNPs) of MRP2 contribute to interindividual variability in drug disposition and ultimately in drug response. OBJECTIVES: To characterize the transport function of human wild-type (WT) MRP2 and four SNP variants, S789F, A1450T, V417I, and T1477M. METHODS: The four SNP variants were expressed in Sf9 cells using recombinant baculovirus infection. The kinetic parameters [Km, (µmol/l); V(max), (pmol/mg/min); the Hill coefficient] of ATP-dependent transport of leukotriene C(4) (LTC(4)), estradiol-3-glucuronide (E(2)3G), estradiol-17ß-glucuronide (E(2)17G), and tauroursodeoxycholic acid (TUDC) were determined in Sf9-derived plasma membrane vesicles. Transport activity was normalized for expression level. RESULTS: The V(max) for transport activity was decreased for all substrates for S789F, and for all substrates except E(2)17G for A1450T. V417I showed decreased apparent affinity for LTC(4), E(2)3G, and E(2)17G, whereas transport was similar between wild-type (WT) and T1477M, except for a modest increase in TUDC transport. Examination of substrate-stimulated MRP2-dependent ATPase activity of S789F and A1450T, SNPs located in MRP2 nucleotide-binding domains (NBDs), demonstrated significantly decreased ATPase activity and only modestly decreased affinity for ATP compared with WT. CONCLUSION: SNPs in the NBDs (S789F in the D-loop of NBD1, or A1450T near the ABC signature motif of NBD2) variably decreased the transport of all substrates. V417I in membrane spanning domain 1 selectively decreased the apparent affinity for the glutathione and glucuronide conjugated substrates, whereas the T1477M SNP in the carboxyl terminus altered only TUDC transport.

The Rho1 GTPase acts together with a vacuolar glutathione S-conjugate transporter to protect yeast cells from oxidative stress.

Maintenance of redox homeostasis is critical for the survival of all aerobic organisms. In the budding yeast Saccharomyces cerevisiae, as in other eukaryotes, reactive oxygen species (ROS) are generated during metabolism and upon exposure to environmental stresses. The abnormal production of ROS triggers defense mechanisms to avoid the deleterious consequence of ROS accumulation. Here, we show that the Rho1 GTPase is necessary to confer resistance to oxidants in budding yeast. Temperature-sensitive rho1 mutants (rho1(ts)) are hypersensitive to oxidants and exhibit high accumulation of ROS even at a semipermissive temperature. Rho1 associates with Ycf1, a vacuolar glutathione S-conjugate transporter, which is important for heavy metal detoxification in yeast. Rho1 and Ycf1 exhibit a two-hybrid interaction with each other and form a bimolecular fluorescent complex on the vacuolar membrane. A fluorescent-based complementation assay suggests that the GTP-bound Rho1 associates with Ycf1 and that their interaction is enhanced upon exposure to hydrogen peroxide. The rho1(ts) mutants also exhibit hypersensitivity to cadmium, while cells carrying a deletion of YCF1 or mutations in a component of the Pkc1-MAP kinase pathway exhibit little or minor sensitivity to oxidants. We thus propose that Rho1 protects yeast cells from oxidative stress by regulating multiple downstream targets including Ycf1. Since both Rho1 and Ycf1 belong to highly conserved families of proteins, similar mechanisms may exist in other eukaryotes.

Regulation of ABC transporter function via phosphorylation by protein kinases.

ATP-binding cassette (ABC) transporters are multispanning membrane proteins that utilize ATP to move a broad range of substrates across cellular membranes. ABC transporters are involved in a number of human disorders and diseases. Overexpression of a subset of the transporters has been closely linked to multidrug resistance in both bacteria and viruses and in cancer. A poorly understood and important aspect of ABC transporter biology is the role of phosphorylation as a mechanism to regulate transporter function. In this review, we summarize the current literature addressing the role of phosphorylation in regulating ABC transporter function. A comprehensive list of all the phosphorylation sites that have been identified for the human ABC transporters is presented, and we discuss the role of individual kinases in regulating transporter function. We address the potential pitfalls and difficulties associated with identifying phosphorylation sites and the corresponding kinase(s), and we discuss novel techniques that may circumvent these problems. We conclude by providing a brief perspective on studying ABC transporter phosphorylation.

Suppression of Ycf1p function by Cka1p-dependent phosphorylation is attenuated in response to salt stress.

The yeast vacuolar membrane protein Ycf1p and its mammalian counterpart, MRP1, belong to the ABCC subfamily of ATP-binding cassette transporters. Genetic evidence suggests that the yeast casein kinase 2α, Cka1p, negatively regulates Ycf1p function via phosphorylation of Ser251 within the N-terminus. In this study, we provide strong evidence that Cka1p regulates Ycf1p function via phosphorylation of Ser251. We show that the CK2 holoenzyme interacts with Ycf1p. However, genetic analysis suggests that only Cka1p is required for Ser251 phosphorylation, as the deletion of CKA1 significantly reduces Ser251 phosphorylation in vivo. Furthermore, purified recombinant Cka1p phosphorylates a Ycf1p-derived peptide containing Ser251. We also demonstrate that Ycf1p function is induced in response to high salt stress. Induction of the Ycf1p function strongly correlates with reduced phosphorylation of Ser251. Importantly, Cka1p activity in vivo is similarly reduced in response to salt stress, consistent with our finding that Cka1p directly phosphorylates Ser251 of Ycf1p. We provide genetic and biochemical evidence that strongly suggests that the induction of Ycf1p function is the result of decreased phosphorylation of Ser251. In conclusion, our work demonstrates a novel biochemical role for Cka1p regulation of Ycf1p function in the cellular response of yeast to salt stress.

ABC transporters in Saccharomyces cerevisiae and their interactors: new technology advances the biology of the ABCC (MRP) subfamily.

Members of the ATP-binding cassette (ABC) transporter superfamily exist in bacteria, fungi, plants, and animals and play key roles in the efflux of xenobiotic compounds, physiological substrates, and toxic intracellular metabolites. Based on sequence relatedness, mammalian ABC proteins have been divided into seven subfamilies, ABC subfamily A (ABCA) to ABCG. This review focuses on recent advances in our understanding of ABC transporters in the model organism Saccharomyces cerevisiae. We propose a revised unified nomenclature for the six yeast ABC subfamilies to reflect the current mammalian designations ABCA to ABCG. In addition, we specifically review the well-studied yeast ABCC subfamily (formerly designated the MRP/CFTR subfamily), which includes six members (Ycf1p, Bpt1p, Ybt1p/Bat1p, Nft1p, Vmr1p, and Yor1p). We focus on Ycf1p, the best-characterized yeast ABCC transporter. Ycf1p is located in the vacuolar membrane in yeast and functions in a manner analogous to that of the human multidrug resistance-related protein (MRP1, also called ABCC1), mediating the transport of glutathione-conjugated toxic compounds. We review what is known about Ycf1p substrates, trafficking, processing, posttranslational modifications, regulation, and interactors. Finally, we discuss a powerful new yeast two-hybrid technology called integrated membrane yeast two-hybrid (iMYTH) technology, which was designed to identify interactors of membrane proteins. iMYTH technology has successfully identified novel interactors of Ycf1p and promises to be an invaluable tool in future efforts to comprehensively define the yeast ABC interactome.

Negative regulation of the yeast ABC transporter Ycf1p by phosphorylation within its N-terminal extension.

The yeast vacuolar membrane protein Ycf1p and its mammalian counterpart, MRP1, belong to the ABCC subfamily of ATP-binding cassette (ABC) transporters that rid cells of toxic endogenous and xenobiotic compounds. Like most members of the ABCC subfamily, Ycf1p contains an N-terminal extension in addition to its ABC "core" domain and transports substrates in the form of glutathione conjugates. Ycf1p is subject to complex regulation to ensure its optimal function. Previous studies showed that Ycf1p activity is stimulated by a guanine nucleotide exchange factor, Tus1p, and is positively regulated by phosphorylation in its ABC core domain at residues Ser-908 and Thr-911. Here we provide evidence that phosphorylation of Ser-251 in the Ycf1p N-terminal extension negatively regulates activity. Mutant Ycf1p-S251A exhibits increased resistance to cadmium in vivo and increased Ycf1p-dependent transport of [(3)H]estradiol-beta-17-glucuronide in vitro as compared with wild-type Ycf1p. Activity is restored to the wild-type level for Ycf1-S251E. To identify kinase(s) that negatively regulate Ycf1p function, we conducted an integrated membrane yeast two-hybrid (iMYTH) screen and identified two kinase genes, CKA1 and HAL5, deletion of which increases Ycf1p function. Genetic evidence suggests that Cka1p may regulate Ycf1p function through phosphorylation of Ser-251 either directly or indirectly. Overall, this study provides compelling evidence that negative, as well as positive, regulation of Ycf1p is mediated by phosphorylation.

Mapping protein-protein interactions for the yeast ABC transporter Ycf1p by integrated split-ubiquitin membrane yeast two-hybrid analysis.

The ATP binding cassette (ABC) transporters are important in human health and disease and represent the largest family of transmembrane proteins; however, their highly hydrophobic nature complicates the use of standard biochemical approaches to identify interacting proteins. Here, we report the development of a modified version of the split-ubiquitin membrane yeast two-hybrid (MYTH) technology using genomically integrated "bait" constructs, hence the designation iMYTH. We used iMYTH in a library-screening format and identified six potential interacting partners of the yeast ABC transporter Ycf1p. Strains deleted for several of these genes result in arsenite sensitivity similar to a Deltaycf1 strain. Transport assays show that one of these, Tus1p, a guanine nucleotide exchange factor (GEF) for the small GTPase Rho1p, is a Rho1p-dependent-positive regulator of Ycf1p. Our study provides proof of principle that iMYTH is an ideal methodology to identify physiological interactors and regulators of ABC transporters and other yeast transmembrane proteins.

5-Oxo-ETE analogs and the proliferation of cancer cells.

MDA-MB-231, MCF7, and SKOV3 cancer cells, but not HEK-293 cells, expressed mRNA for the leukocyte G protein-coupled 5-oxo-eicosatetraenoate (ETE) OXE receptor. 5-Oxo-ETE, 5-oxo-15-OH-ETE, and 5-HETE stimulated the cancer cell lines but not HEK-293 cells to mount pertussis toxin-sensitive proliferation responses. Their potencies in eliciting this response were similar to their known potencies in activating leukocytes and OXE receptor-transfected cells. However, high concentrations of 5-oxo-ETE and 5-oxo-15-OH-ETE, but not 5-HETE, arrested growth and caused apoptosis in all four cell lines; these responses were pertussis toxin-resistant. The same high concentrations of the oxo-ETEs but again not 5-HETE also activated peroxisome proliferator-activated receptor (PPAR)-gamma. Pharmacological studies indicated that this activation did not mediate their effects on proliferation. These results are the first to implicate the OXE receptor in malignant cell growth and to show that 5-oxo-ETEs activate cell death programs as well as PPARgamma independently of this receptor.

Glutathione S-transferases (GSTs) inhibit transcriptional activation by the peroxisomal proliferator-activated receptor gamma (PPAR gamma) ligand, 15-deoxy-delta 12,14prostaglandin J2 (15-d-PGJ2).

15-Deoxy-Delta(12,14)prostaglandin J(2) (15-d-PGJ(2)), a terminal metabolite of the J-series cyclopentenone prostaglandins, influences a variety of cellular processes including gene expression, differentiation, growth, and apoptosis. As a ligand of peroxisomal proliferator-activated receptor gamma (PPAR gamma), 15-d-PGJ(2) can transactivate PPAR gamma-responsive promoters. Previously, we showed that multidrug resistance proteins MRP1 and MRP3 attenuate cytotoxic and transactivating activities of 15-d-PGJ(2) in MCF7 breast cancer cells. Attenuation was glutathione-dependent and was associated with formation of the glutathione conjugate of 15-d-PGJ(2), 15-d-PGJ(2)-SG, and its active efflux by MRP. Here we have investigated whether the glutathione S-transferases (GST) can influence biological activities of 15-d-PGJ(2). MCF7 cells were stably transduced with human cytosolic GST isozymes M1a, A1, or P1a. These GSTs had no effect on 15-d-PGJ(2) cytotoxicity when expressed either alone or in combination with MRP1. However, expression of any of the three GSTs significantly inhibited 15-d-PGJ(2)-dependent transactivation of a PPAR gamma-responsive reporter gene. The degree of inhibition correlated with the level of GST expressed. Under physiologic conditions, the nonenzymatic rate of 15-d-PGJ(2) conjugation with glutathione was significant. Of the three GST isozymes, only GSTM1a-1a further stimulated the rate of 15-d-PGJ(2)-SG formation. Moreover, GSTM1a-1a rate enhancement was only a transient burst that was complete within 15 s. Hence, catalysis plays little, if any, role in GST inhibition of 15-d-PGJ(2)-dependent transactivation. In contrast, inhibition of transactivation was associated with strong GST/15-d-PGJ(2) interactions. Potent inhibition by 15-d-PGJ(2) and 15-d-PGJ(2)-SG of GST activity was observed with K(i) in the 0.15-2.0 microM range for the three GST isozymes, results suggesting avid associations between GST and 15-d-PGJ(2) or 15-d-PGJ(2)-SG. Electrospray ionization mass spectrometry (ESI/MS) studies revealed no stable adducts of GST and 15-d-PGJ(2) indicating that GST/15-d-PGJ(2) interactions are primarily noncovalent. These results are consistent with a mechanism of GST-mediated inhibition of transactivation in which GST binds 15-d-PGJ(2) and 15-d-PGJ(2)-SG thereby sequestering the ligands in the cytosol away from their nuclear target, PPAR gamma.

Multidrug resistance protein (MRP) 1 and MRP3 attenuate cytotoxic and transactivating effects of the cyclopentenone prostaglandin, 15-deoxy-Delta(12,14)prostaglandin J2 in MCF7 breast cancer cells.

One of the most potent cyclopentenone prostaglandins, 15-deoxy-Delta(12,14)prostaglandin J(2) (15-d-PGJ(2)), has been shown to be cytotoxic in some tumor cells and, as a ligand of peroxisome proliferator activated receptor gamma (PPARgamma), to influence the transcriptional regulation of several genes. We examined whether a glutathione conjugate of 15-d-PGJ(2), 15-d-PGJ(2)-SG, is formed and if the glutathione conjugate efflux pumps, MRP1 and MRP3, could transport this conjugate, thereby attenuating the cytotoxicity and transactivating activity of 15-d-PGJ(2) in MCF7 breast cancer cells. Formation of 15-d-PGJ(2)-SG was demonstrated both in vitro and in cells, and its structure was determined by ESI/MS and NMR. Expression of MRP1 and MRP3 was achieved by stable transduction of parental MCF7 cells. Membrane vesicles derived from these cells supported efficient, ATP-dependent transport of 15-d-PGJ(2)-SG (K(M) 1.4 and 2.9 microM for MRP1 and MRP3, respectively). When compared with parental, MRP-minus MCF7 cells, expression of MRP1 and MRP3 conferred approximately 2-fold protection from 15-d-PGJ(2) cytotoxicity. 15-d-PGJ(2)-mediated transcriptional activation was evaluated in cells transiently transfected with a reporter gene under the transcriptional control of a PPAR responsive element. Treatment of parental MCF7 cells with 15-d-PGJ(2) resulted in a time-dependent induction of reporter gene activity-induction that was measurable with concentrations of added 15-d-PGJ(2) as low as 100 nM. In contrast, expression of MRP1 or MRP3 abolished 15-d-PGJ(2)-dependent reporter gene induction. Depletion of intracellular glutathione reversed MRP1- and MRP3-mediated attenuation of 15-d-PGJ(2) cytotoxicity and transactivation. These data indicate that MRP1 and MRP3 can modulate the biological effects of 15-d-PGJ(2), and likely other cyclopentenone prostaglandins, in a glutathione-dependent manner. The results are consistent with a mechanism for the attenuation of the biological activities of 15-d-PGJ(2) that involves the formation and active efflux of its glutathione conjugate, 15-d-PGJ(2)-SG.