To examine the role of multidrug resistance protein 1 (MRP1) and glutathione S-transferases (GSTs) in cellular resistance to antineoplastic drugs, derivatives of MCF7 breast carcinoma cells were developed that express MRP1 in combination with one of three human cytosolic isozymes of GST. Expression of MRP1 alone confers resistance to several drugs representing the multidrug resistance phenotype, drugs including doxorubicin, vincristine, etoposide, and mitoxantrone. However, co-expression with MRP1 of any of the human GST isozymes A1-1, M1-1, or P1-1 failed to augment MRP1-associated resistance to these drugs. In contrast, combined expression of MRP1 and GST A1-1 conferred ϳ4-fold resistance to the anticancer drug chlorambucil. Expression of MRP1 alone failed to confer resistance to chlorambucil, showing that the observed protection from chlorambucil cytotoxicity was absolutely dependent upon GST A1-1 protein. Moreover, using inhibitors of GST (dicumarol) or MRP1 (sulfinpyrazone), it was shown that in MCF7 cells resistance to chlorambucil requires both intact MRP1-dependent efflux pump activity and, for full protection, GST A1-1 catalytic activity. These results are the first demonstration that GST A1-1 and MRP1 can act in synergy to protect cells from the cytotoxicity of a nitrogen mustard, chlorambucil.

The newly defined eicosatetraenoates (ETEs), 5-oxoETE and 5-oxo-15(OH)-ETE, share structural motifs, synthetic origins, and bioactions with leukotriene B 4 (LTB 4 ). All three eicosanoids stimulate Ca 2؉ transients and chemotaxis in human neutrophils (PMN). However, unlike LTB 4 , 5-oxoETE and 5-oxo-15(OH)-ETE alone cause little degranulation and no superoxide anion production. However, we show herein that, in PMN pretreated with granulocyte-macrophage or granulocyte colony-stimulating factor (GM-CSF or G-CSF), the oxoETEs become potent activators of the last responses. The oxoETEs also induce translocation of secretory vesicles from the cytosol to the plasmalemma, an effect not requiring cytokine priming. To study the mechanism of PMN activation in response to the eicosanoids, we examined the activation of mitogen-activated protein kinase (MAPK) and cytosolic phospholipase A 2 (cPLA 2 ). PMN expressed three proteins (40, 42, and 44 kDa) that reacted with anti-MAPK antibodies. The oxoETEs, LTB 4 , GM-CSF, and G-CSF all stimulated PMN to activate the MAPKs and cPLA 2 , as defined by shifts in these proteins' electrophoretic mobility and tyrosine phosphorylation of the MAPKs. However, the speed and duration of the MAPK response varied markedly depending on the stimulus. 5-OxoETE caused a very rapid and transient activation of MAPK. In contrast, the response to the cytokines was rather slow and persistent. PMN pretreated with GM-CSF demonstrated a dramatic increase in the extent of MAPK tyrosine phosphorylation and electrophoretic mobility shift in response to 5-oxoETE. Similarly, 5-oxoETE induced PMN to release some preincorporated [ 14 C]arachidonic acid, while GM-CSF greatly enhanced the extent of this release. Thus, the synergism exhibited by these agents is prominent at the level of MAPK stimulation and phospholipid deacylation. Pertussis toxin, but not Ca 2؉ depletion, inhibited MAPK responses to 5-oxoETE and LTB 4 , indicating that responses to both agents are coupled through G proteins but not dependent upon Ca 2؉ transients. 15-OxoETE and 15(OH)-ETE were inactive while 5-oxo-15(OH)-ETE and 5(OH)-ETE had 3-and 10-fold less potency than 5-oxoETE, indicating a rather strict structural specificity for the 5-keto group. LY 255283, a LTB 4 antagonist, blocked the responses to LTB 4 but not to 5-oxoETE. Therefore, the oxoETEs do not appear to operate through the LTB 4 receptor. In summary, the oxoETEs are potent activators of PMN that share some but not all activities with LTB 4 . The response to the oxoETEs is greatly enhanced by pretreatment with cytokines, indicating that combinations of these mediators may be very important in the pathogenesis of inflammation.

Model cell lines developed from MCF7 breast carcinoma cells were used to examine the roles of glutathione S-transferase P1-1 (GSTP1-1) and multidrug resistance protein (MRP) in the protection of cells from 4-nitroquinoline 1-oxide (4NQO) toxicities. Increased expression of GSTP1-1 alone in MCF7 cells results in limited protection from the formation of 4NQO-derived covalent adducts of nucleic acids but affords no protection from 4NQO-mediated cytotoxicity. Increased expression of MRP alone conferred modest protection while co-expression of GSTP1-1 with MRP produced high-level protection from both 4NQO-derived adduct formation and 4NQO cytotoxicity. This synergistic resistance to 4NQO toxicities (both nucleic acid adduct formation and cytotoxicity) is associated with a GSTP1-1-dependent increase in 4NQO-glutathione (QO-SG) conjugate formation and a MRP-dependent increase in QO-SG efflux. These data indicate that MRP is an important export transporter for the glutathione conjugate of the carcinogen, 4NQO. Moreover, this MRP-dependent efflux activity is necessary to achieve the full protection from 4NQO toxicity-protection that is potentiated by GSTP1-1-mediated QO-SG formation.

Based upon several previous reports, no consistent relationship between multidrug resistance protein 1 (MRP1, ABCC1) expression and cellular sensitivity to mitoxantrone (MX) toxicity can be ascertained; thus, the role of MRP1 in MX resistance remains controversial. The present study, using paired parental, MRP1-poor, and transduced MRP1-overexpressing MCF7 cells, unequivocally demonstrates that MRP1 confers resistance to MX cytotoxicity and that resistance is associated with reduced cellular accumulation of MX. This MRP1-associated reduced accumulation of MX was partially reversed by treatment of cells with 50propionic acid]-an MRP inhibitor that increased MX accumulation in MRP1-expressing MCF7 cells but had no effect on MRP-poor MCF7 cells. Moreover, in vitro experiments using inside-out membrane vesicles show that MRP1 supports ATPdependent, osmotically sensitive uptake of MX. Unlike ABCG2 (breast cancer resistance protein, mitoxantrone-resistant protein), MRP1-mediated MX transport is dependent upon the presence of glutathione or its S-methyl analog. In addition, MX stimulates transport of [ 3 H]glutathione. Together, these data are consistent with the interpretation that MX efflux by MRP1 involves cotransport of MX and glutathione. The results suggest that MRP1-like the alternative MX transporters ABCG2 and ABCB1 (MDR1, P-glycoprotein)-can significantly influence tumor cell sensitivity to and pharmacological disposition of MX.

Cells expressing elevated levels of allelic variants of human glutathione S-transferase P1 (GSTP1) and/or efflux transporters, MRP1 or MRP2, were used to evaluate the role of GSTP1-1 in cisplatin resistance. These studies revealed that GSTP1-1 confers low-level resistance (1.4-to 1.7-fold) to cisplatin-induced cytotoxicity in MCF7 cells. However, expression of MRP1 (MCF7 cells) or MRP2 (HepG2 cells) failed to augment or potentiate GSTP1-1-mediated resistance in either cell line. To understand the mechanism by which variants of GSTP1-1 confer resistance to cisplatin, their relative abilities to catalyze conjugation of cisplatin with glutathione were examined. Enzymes encoded by all three alleles tested, GSTP1a (I 104 A 113 ), GSTP1b (V 104 A 113 ), and GSTP1c (V 104

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.

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