Sensing of latent EBV infection through exosomal transfer of 5'pppRNA.

Complex interactions between DNA herpesviruses and host factors determine the establishment of a life-long asymptomatic latent infection. The lymphotropic Epstein-Barr virus (EBV) seems to avoid recognition by innate sensors despite massive transcription of immunostimulatory small RNAs (EBV-EBERs). Here we demonstrate that in latently infected B cells, EBER1 transcripts interact with the lupus antigen (La) ribonucleoprotein, avoiding cytoplasmic RNA sensors. However, in coculture experiments we observed that latent-infected cells trigger antiviral immunity in dendritic cells (DCs) through selective release and transfer of RNA via exosomes. In ex vivo tonsillar cultures, we observed that EBER1-loaded exosomes are preferentially captured and internalized by human plasmacytoid DCs (pDCs) that express the TIM1 phosphatidylserine receptor, a known viral- and exosomal target. Using an EBER-deficient EBV strain, enzymatic removal of 5'ppp, in vitro transcripts, and coculture experiments, we established that 5'pppEBER1 transfer via exosomes drives antiviral immunity in nonpermissive DCs. Lupus erythematosus patients suffer from elevated EBV load and activated antiviral immunity, in particular in skin lesions that are infiltrated with pDCs. We detected high levels of EBER1 RNA in such skin lesions, as well as EBV-microRNAs, but no intact EBV-DNA, linking non-cell-autonomous EBER1 presence with skin inflammation in predisposed individuals. Collectively, our studies indicate that virus-modified exosomes have a physiological role in the host-pathogen stand-off and may promote inflammatory disease.

Proteasome inhibition and mechanism of resistance to a synthetic, library-based hexapeptide.

Background The hexapeptide 4A6 (Ac-Thr(tBu)-His(Bzl)-Thr(Bzl)-Nle-Glu(OtBu)-Gly-Bza) was isolated from a peptide library constructed to identify peptide-based transport inhibitors of multidrug resistance (MDR) efflux pumps including P-glycoprotein and Multidrug Resistance-associated Protein 1. 4A6 proved to be a substrate but not an inhibitor of these MDR efflux transporters. In fact, 4A6 and related peptides displayed potent cytotoxic activity via an unknown mechanism. Objective To decipher the mode of cytotoxic activity of 4A6. Methods Screening of 4A6 activity was performed against the NCI60 panel of cancer cell lines. Possible interactions of 4A6 with the 26S proteasome were assessed via proteasome activity and affinity labeling, and cell growth inhibition studies with leukemic cells resistant to the proteasome inhibitor bortezomib (BTZ). Results The NCI60 panel COMPARE analysis revealed that 4A6 had an activity profile overlapping with BTZ. Consistently, 4A6 proved to be a selective and reversible inhibitor of ß5 subunit (PSMB5)-associated chymotrypsin-like activity of the 26S proteasome. This conclusion is supported by several lines of evidence: (i) inhibition of chymotrypsin-like proteasome activity by 4A6 and related peptides correlated with their cell growth inhibition potencies; (ii) 4A6 reversibly inhibited functional ß5 active site labeling with the affinity probe BodipyFL-Ahx3L3VS; and (iii) human myeloid THP1 cells with acquired BTZ resistance due to mutated PSMB5 were highly (up to 287-fold) cross-resistant to 4A6 and its related peptides. Conclusion 4A6 is a novel specific inhibitor of the ß5 subunit-associated chymotrypsin-like proteasome activity. Further exploration of 4A6 as a lead compound for development as a novel proteasome-targeted drug is warranted.

Novel Stool-Based Protein Biomarkers for Improved Colorectal Cancer Screening: A Case-Control Study.

BACKGROUND: The fecal immunochemical test (FIT) for detecting hemoglobin is used widely for noninvasive colorectal cancer (CRC) screening, but its sensitivity leaves room for improvement. OBJECTIVE: To identify novel protein biomarkers in stool that outperform or complement hemoglobin in detecting CRC and advanced adenomas. DESIGN: Case-control study. SETTING: Colonoscopy-controlled referral population from several centers. PARTICIPANTS: 315 stool samples from one series of 12 patients with CRC and 10 persons without colorectal neoplasia (control samples) and a second series of 81 patients with CRC, 40 with advanced adenomas, and 43 with nonadvanced adenomas, as well as 129 persons without colorectal neoplasia (control samples); 72 FIT samples from a third independent series of 14 patients with CRC, 16 with advanced adenomas, and 18 with nonadvanced adenomas, as well as 24 persons without colorectal neoplasia (control samples). MEASUREMENTS: Stool samples were analyzed by mass spectrometry. Classification and regression tree (CART) analysis and logistic regression analyses were performed to identify protein combinations that differentiated CRC or advanced adenoma from control samples. Antibody-based assays for 4 selected proteins were done on FIT samples. RESULTS: In total, 834 human proteins were identified, 29 of which were statistically significantly enriched in CRC versus control stool samples in both series. Combinations of 4 proteins reached sensitivities of 80% and 45% for detecting CRC and advanced adenomas, respectively, at 95% specificity, which was higher than that of hemoglobin alone (P < 0.001 and P = 0.003, respectively). Selected proteins could be measured in small sample volumes used in FIT-based screening programs and discriminated between CRC and control samples (P < 0.001). LIMITATION: Lack of availability of antibodies prohibited validation of the top protein combinations in FIT samples. CONCLUSION: Mass spectrometry of stool samples identified novel candidate protein biomarkers for CRC screening. Several protein combinations outperformed hemoglobin in discriminating CRC or advanced adenoma from control samples. Proof of concept that such proteins can be detected with antibody-based assays in small sample volumes indicates the potential of these biomarkers to be applied in population screening. PRIMARY FUNDING SOURCE: Center for Translational Molecular Medicine, International Translational Cancer Research Dream Team, Stand Up to Cancer (American Association for Cancer Research and the Dutch Cancer Society), Dutch Digestive Foundation, and VU University Medical Center.

ABCC5 supports osteoclast formation and promotes breast cancer metastasis to bone.

INTRODUCTION: Bone is the most common site of breast cancer metastasis, and complications associated with bone metastases can lead to a significantly decreased patient quality of life. Thus, it is essential to gain a better understanding of the molecular mechanisms that underlie the emergence and growth of breast cancer skeletal metastases. METHODS: To search for novel molecular mediators that influence breast cancer bone metastasis, we generated gene-expression profiles from laser-capture microdissected trephine biopsies of both breast cancer bone metastases and independent primary breast tumors that metastasized to bone. Bioinformatics analysis identified genes that are differentially expressed in breast cancer bone metastases compared with primary, bone-metastatic breast tumors. RESULTS: ABCC5, an ATP-dependent transporter, was found to be overexpressed in breast cancer osseous metastases relative to primary breast tumors. In addition, ABCC5 was significantly upregulated in human and mouse breast cancer cell lines with high bone-metastatic potential. Stable knockdown of ABCC5 substantially reduced bone metastatic burden and osteolytic bone destruction in mice. The decrease in osteolysis was further associated with diminished osteoclast numbers in vivo. Finally, conditioned media from breast cancer cells with reduced ABCC5 expression failed to induce in vitro osteoclastogenesis to the same extent as conditioned media from breast cancer cells expressing ABCC5. CONCLUSIONS: Our data suggest that ABCC5 functions as a mediator of breast cancer skeletal metastasis. ABCC5 expression in breast cancer cells is important for efficient osteoclast-mediated bone resorption. Hence, ABCC5 may be a potential therapeutic target for breast cancer bone metastasis.

Exposure of CD34+ precursors to cytostatic anthraquinone-derivatives induces rapid dendritic cell differentiation: implications for cancer immunotherapy.

Appropriate activation of dendritic cells (DC) is essential for successful active vaccination and induction of cell-mediated immunity. The scarcity of precursor cells, as well as long culture methods, have hampered wide-scale application of DC vaccines derived from CD34(+) precursors, despite their suggested superior efficacy over the more commonly applied monocyte-derived DC (MoDC). Here, employing the CD34(+)/CD14(+) AML-derived human DC progenitor cell line MUTZ3, we show that cytostatic anthraquinone-derivatives (i.e., the anthracenedione mitoxantrone and the related anthracyclin doxorubicin) induce rapid differentiation of CD34(+) DC precursors into functional antigen-presenting cells (APC) in a three-day protocol. The drugs were found to act specifically on CD34(+), and not on CD14(+) DC precursors. Importantly, these observations were confirmed for primary CD34(+) and CD14(+) DC precursors from peripheral blood. Mitoxantrone-generated DC were fully differentiated within three days and after an additional 24 h of maturation, were as capable as standard 9-day differentiated and matured DC to migrate toward the lymph node-homing chemokines CCL19 and CCL21, to induce primary allogeneic T cell proliferation, and to prime functional MART1-specific CD8(+) T lymphocytes. Our finding that anthraquinone-derivatives like mitoxantrone support rapid high-efficiency differentiation of DC precursors may have consequences for in vitro production of DC vaccines as well as for novel immunochemotherapy strategies.

Preferential Langerhans cell differentiation from CD34(+) precursors upon introduction of ABCG2 (BCRP).

Epidermal Langerhans cells (LC) and dermal interstitial dendritic cells (IDC) were found to express the ATP-binding cassette (ABC) transporter breast cancer resistance protein (BCRP; ABCG2). Also, low BCRP expression was present on CD34(+) blood DC precursors and expression was increased upon their differentiation to LC. The CD34(+) acute myeloid leukemia-derived DC cell line MUTZ3 can be cultured into LC or IDC, depending on the cytokine cocktail used. Introduction of functional BCRP in MUTZ3 progenitor cells through retroviral transduction resulted in the emergence of typical LC-characteristics in IDC cultures; the majority of cells remained negative for the IDC-specific C-type lectin DC-SIGN, but rather displayed enhanced expression of the LC-specific C-type lectin Langerin and characteristic high expression levels of CD1a. BCRP-induced skewing toward LC-like differentiation coincided with early RelB expression in 'IDC', derived from MUTZ3-BCRP, and depended on endogenous transforming growth factor beta (TGF-ß) production. Intriguingly, cellular BCRP localization differed between skin LC and IDC, and a more cytoplasmic BCRP localization, as observed in primary skin LC, seemed to relate to LC-like differentiation in IDC cultures upon BCRP introduction in MUTZ3 progenitors. Together these data support a role for BCRP in preferential LC differentiation from CD34(+) myeloid DC progenitors.

Inactivating PSMB5 mutations and P-glycoprotein (multidrug resistance-associated protein/ATP-binding cassette B1) mediate resistance to proteasome inhibitors: ex vivo efficacy of (immuno)proteasome inhibitors in mononuclear blood cells from patients with rheumatoid arthritis.

Bortezomib (BTZ), a registered proteasome inhibitor (PI) for multiple myeloma, has also been proposed as a potential antirheumatic agent. Its reported side effects, however, make it unappealing for long-term administration, and resistance may also develop. To overcome this, second-generation PIs became available. Here, we investigated whether a novel class of peptide epoxyketone-based PIs, including carfilzomib, N-((S)-3-methoxy-1-(((S)-3-methoxy-1-(((S)-1-((R)-2-methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)-2-methylthiazole-5-carboxamide (ONX0912), and (S)-3-(4-methoxyphenyl)-N-((S)-1-((S)-2-methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)propanamido)propanamide (ONX0914), might escape two established BTZ-resistance mechanisms: 1) mutations in the proteasome ß5 subunit (PSMB5) targeted by these PIs, and 2) drug efflux mediated by ATP-binding cassette transporters. THP1 myeloid sublines with acquired resistance to BTZ (54- to 235-fold) caused by mutations in the PSMB5 gene displayed marked cross-resistance but less pronounced cross-resistance to carfilzomib (9- to 32-fold), ONX0912 (39- to 62-fold), and ONX0914 (27- to 97-fold). As for ATP-binding cassette transporter-mediated efflux, lymphoid CEM/VLB cells with P-glycoprotein (Pgp)/multidrug resistance 1 overexpression exhibited substantial resistance to carfilzomib (114-fold), ONX0912 (23-fold), and ONX0914 (162-fold), whereas less resistance to BTZ (4.5-fold) was observed. Consistently, ß5 subunit-associated chymotrypsin-like proteasome activity was significantly less inhibited in these CEM/VLB cells. Ex vivo analysis of peripheral blood mononuclear cells from therapy-naive patients with rheumatoid arthritis revealed that, although basal Pgp levels were low, P-glycoprotein expression compromised the inhibitory effect of carfilzomib and ONX0914. However, the use of P121 (reversin 121), a Pgp transport inhibitor, restored parental cell inhibitory levels in both CEM/VLB cells and peripheral blood mononuclear cells. These results indicate that the pharmacologic activity of these PIs may be hindered by drug resistance mechanisms involving PSMB5 mutations and PI extrusion via Pgp.

Altered arsenic disposition in experimental nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is represented by a spectrum of liver pathologies ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). Liver damage sustained in the progressive stages of NAFLD may alter the ability of the liver to properly metabolize and eliminate xenobiotics. The purpose of the current study was to determine whether NAFLD alters the disposition of the environmental toxicant arsenic. C57BL/6 mice were fed either a high-fat or a methionine-choline-deficient diet to model simple steatosis and NASH, respectively. At the conclusion of the dietary regimen, all mice were given a single oral dose of either sodium arsenate or arsenic trioxide. Mice with NASH excreted significantly higher levels of total arsenic in urine (24 h) compared with controls. Total arsenic in the liver and kidneys of NASH mice was not altered; however, NASH liver retained significantly higher levels of the monomethyl arsenic metabolite, whereas dimethyl arsenic was retained significantly less in the kidneys of NASH mice. NASH mice had significantly higher levels of the more toxic trivalent form in their urine, whereas the pentavalent form was preferentially retained in the liver of NASH mice. Moreover, hepatic protein expression of the arsenic biotransformation enzyme arsenic (3+ oxidation state) methyltransferase was not altered in NASH animals, whereas protein expression of the membrane transporter multidrug resistance-associated protein 1 was increased, implicating cellular transport rather than biotransformation as a possible mechanism. These results suggest that NASH alters the disposition of arsenical species, which may have significant implications on the overall toxicity associated with arsenic in NASH.

The ABC of dendritic cell development and function.

ATP-binding cassette (ABC) transporters are known for their involvement in clinical multidrug resistance (MDR) and their physiological defensive functions in barrier organs. More recently, attention has been focused on their possible involvement in the regulation of immune responses following the identification of their substrates as known immunomodulating agents (e.g. prostaglandins, leukotrienes and cyclic nucleotides) and their functional expression in various immune effector cells, most notably in dendritic cells (DCs). This review addresses the possible roles of ABC transporters in DC development and function, as well as the putative immunostimulatory potential of their cytostatic substrates and how this knowledge might benefit DC-based chemo-immunotherapies.

High susceptibility of c-KIT+CD34+ precursors to prolonged doxorubicin exposure interferes with Langerhans cell differentiation in a human cell line model.

As neoadjuvant and adjuvant chemotherapy schedules often consist of multiple treatment cycles over relatively long periods of time, it is important to know what effects protracted drug administration can have on the immune system. Here, we studied the long-term effects of doxorubicin on the capacity of dendritic cell (DC) precursors to differentiate into a particular DC subset, the Langerhans cells (LC). In order to achieve high telomerase activity as detected in hematological stem cells, precursor cells from the acute-myeloid leukemia (AML)-derived cell line MUTZ3 were stably transduced with human telomerase reverse transcriptase (hTERT) to facilitate their growth potential, while preventing growth, and drug-induced senescence, and preserving their unique capacity for cytokine-dependent DC and LC differentiation. The hTERT-MUTZ3 cells were selected with increasing concentrations of the anthracyclin doxorubicin. After 1-2 months of selection with 30-90 nM doxorubicin, the cells completely lost their capacity to differentiate into LC. This inhibition turned out to be reversible, as the cells slowly regained their capacity to differentiate after a 3- to 4-month drug-free period and with this became capable again of priming allogeneic T cells. Of note, the loss and gain of this capacity to differentiate coincided with the loss and gain of a subpopulation within the CD34(+) proliferative compartment with surface expression of the stem cell factor receptor (SCF-R/CD117/c-Kit). These data are in favor of cytostatic drug-free intervals before applying autologous DC-based vaccination protocols, as specific DC precursors may need time to recover from protracted chemotherapy treatment and re-emerge among the circulating CD34(+) hematopoietic stem and precursor cells.

Variations in ATP-binding cassette transporter regulation during the progression of human nonalcoholic fatty liver disease.

Transporters located on the sinusoidal and canalicular membranes of hepatocytes regulate the efflux of drugs and metabolites into blood and bile, respectively. Changes in the expression or function of these transporters during liver disease may lead to a greater risk of adverse drug reactions. Nonalcoholic fatty liver disease (NAFLD) is a progressive condition encompassing the relatively benign steatosis and the more severe, inflammatory state of nonalcoholic steatohepatitis (NASH). Here, we present an analysis of the effect of NAFLD progression on the major ATP-binding cassette (ABC) efflux transport proteins ABCC1-6, ABCB1, and ABCG2. Human liver samples diagnosed as normal, steatotic, NASH (fatty), and NASH (not fatty) were analyzed. Increasing trends in mRNA expression of ABCC1, ABCC4-5, ABCB1, and ABCG2 were found with NAFLD progression, whereas protein levels of all transporters exhibited increasing trends with disease progression. Immunohistochemical staining of ABCC3, ABCB1, and ABCG2 revealed no alterations in cellular localization during NAFLD progression. ABCC2 staining revealed an alternative mechanism of regulation in NASH in which the transporter appears to be internalized away from the canalicular membrane. This correlated with a preferential shift in the molecular mass of ABCC2 from 200 to 180 kDa in NASH, which has been shown to be associated with a loss of glycosylation and internalization of the protein. These data demonstrate increased expression of multiple efflux transporters as well as altered cellular localization of ABCC2 in NASH, which may have profound effects on the ability of patients with NASH to eliminate drugs in an appropriate manner.

A Bispecific Antibody Antagonizes Prosurvival CD40 Signaling and Promotes Vγ9Vδ2 T cell-Mediated Antitumor Responses in Human B-cell Malignancies.

Novel T cell-based therapies for the treatment of B-cell malignancies, such as chronic lymphocytic leukemia (CLL) and multiple myeloma (MM), are thought to have strong potential. Progress, however, has been hampered by low efficacy and high toxicity. Tumor targeting by Vγ9Vδ2 T cells, a conserved T-cell subset with potent intrinsic antitumor properties, mediated by a bispecific antibody represents a novel approach promising high efficacy with limited toxicity. Here, we describe the generation of a bispecific Vγ9Vδ2 T-cell engager directed against CD40, which, due to its overexpression and biological footprint in malignant B cells, represents an attractive target. The CD40-targeting moiety of the bispecific antibody was selected because it can prevent CD40L-induced prosurvival signaling and reduce CD40-mediated resistance of CLL cells to venetoclax. Selective activation of Vγ9Vδ2 T cells in the presence of CD40+ tumor cells induced potent Vγ9Vδ2 T-cell degranulation, cytotoxicity against CLL and MM cells in vitro, and in vivo control of MM in a xenograft model. The CD40-bispecific γδ T-cell engager demonstrated lysis of leukemic cells by autologous Vγ9Vδ2 T cells present in patient-derived samples. Taken together, our CD40 bispecific γδ T-cell engager increased the sensitivity of leukemic cells to apoptosis and induced a potent Vγ9Vδ2 T cell-dependent antileukemic response. It may, therefore, represent a potential candidate for the development of novel treatments for B-cell malignancies.

Protease inhibitors atazanavir, lopinavir and ritonavir are potent blockers, but poor substrates, of ABC transporters in a broad panel of ABC transporter-overexpressing cell lines.

OBJECTIVES: A possible mechanism for HIV therapy failure is the efflux of HIV drugs from viral target cells or certain body compartments by ATP-binding cassette (ABC) transporters, allowing ongoing viral replication. Here, we investigated the interaction between protease inhibitors (PIs) and ABC transporters. METHODS: To explore the potential blocking capacity of PIs, we exposed cells overexpressing multidrug resistance 1 P-glycoprotein (MDR1 P-gp), multidrug resistance protein 1 (MRP1) and breast cancer resistance protein (BCRP) to established cytotoxic substrates with or without one of the PIs atazanavir, lopinavir or ritonavir. Furthermore, to assess whether PIs serve as substrates, cell growth-inhibitory effects of these PIs were evaluated on cells overexpressing 1 of 11 ABC transporters and their parental counterparts. RESULTS: Atazanavir, lopinavir and ritonavir were highly effective in reversing resistance against established substrates in cells overexpressing MDR1 P-gp and MRP1, and, to a lesser extent, BCRP. Concurrently, however, PIs appeared to be relatively poor substrates for ABC transporters. Only a moderate level of resistance to atazanavir was observed in cells overexpressing MRP6 and MRP9 [resistance factor (RF): 2.0-2.6]. Cells overexpressing MDR1 P-gp, MRP3, MRP4 and MRP5 displayed low levels of resistance to atazanavir (RF: 1.3-1.7); MRP7- and MRP9-overexpressing cells to lopinavir (RF: 1.4-1.5); and MRP9-overexpressing cells to ritonavir (RF: 1.4). CONCLUSIONS: PIs can act as potent blockers of MDR1 P-gp, MRP1 and BCRP, but they are poor substrates for 11 ABC transporters. Consequently, ABC transporters are unlikely to play a major role in PI failure, but still may contribute to drug-specific adverse events and drug-drug interactions.

A role for multidrug resistance protein 4 (MRP4; ABCC4) in human dendritic cell migration.

The capacity of dendritic cells (DCs) to migrate from peripheral organs to lymph nodes (LNs) is important in the initiation of a T cell-mediated immune response. The ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp; ABCB1) and the multidrug resistance protein 1 (MRP1; ABCC1) have been shown to play a role in both human and murine DC migration. Here we show that a more recently discovered family member, MRP4 (ABCC4), is expressed on both epidermal and dermal human skin DCs and contributes to the migratory capacity of DCs. Pharmacological inhibition of MRP4 activity or down-regulation through RNAi in DCs resulted in reduced migration of DCs from human skin explants and of in vitro generated Langerhans cells. The responsible MRP4 substrate remains to be identified as exogenous addition of MRP4's known substrates prostaglandin E(2), leukotriene B(4) and D(4), or cyclic nucleotides (all previously implicated in DC migration) could not restore migration. This notwithstanding, our data show that MRP4 is an important protein, significantly contributing to human DC migration toward the draining lymph nodes, and therefore relevant for the initiation of an immune response and a possible target for immunotherapy.

Molecular basis of bortezomib resistance: proteasome subunit beta5 (PSMB5) gene mutation and overexpression of PSMB5 protein.

The proteasome inhibitor bortezomib is a novel anticancer drug that has shown promise in the treatment of refractory multiple myeloma. However, its clinical efficacy has been hampered by the emergence of drug-resistance phenomena, the molecular basis of which remains elusive. Toward this end, we here developed high levels (45- to 129-fold) of acquired resistance to bortezomib in human myelomonocytic THP1 cells by exposure to stepwise increasing (2.5-200 nM) concentrations of bortezomib. Study of the molecular mechanism of bortezomib resistance in these cells revealed (1) an Ala49Thr mutation residing in a highly conserved bortezomib-binding pocket in the proteasome beta5-subunit (PSMB5) protein, (2) a dramatic overexpression (up to 60-fold) of PSMB5 protein but not of other proteasome subunits including PSMB6, PSMB7, and PSMA7, (3) high levels of cross-resistance to beta5 subunit-targeted cytotoxic peptides 4A6, MG132, MG262, and ALLN, but not to a broad spectrum of chemotherapeutic drugs, (4) no marked changes in chymotrypsin-like proteasome activity, and (5) restoration of bortezomib sensitivity in bortezomib-resistant cells by siRNA-mediated silencing of PSMB5 gene expression. Collectively, these findings establish a novel mechanism of bortezomib resistance associated with the selective overexpression of a mutant PSMB5 protein.

Cellular folate status modulates the expression of BCRP and MRP multidrug transporters in cancer cell lines from different origins.

As cellular folate levels seem to have a different effect on cancer cells from different origins, we extended our initial study to a broader panel of cancer cells. BCRP and MRP1-5 expression was determined in KB, OVCAR-3, IGROV-1, ZR75-1/R/MTX, SCC-11B, SCC-22B, and WiDr either grown in standard RPMI 1640 containing 2.3 micromol/L supraphysiologic concentration of folic acid [high folate (HF)] or adapted to more physiologic concentrations [1-5 nmol/L folic acid or leucovorin; low folate (LF)]. Compared with the HF counterparts, KB LF cells displayed 16.1-fold increased MRP3 and OVCAR-3 LF cells showed 4.8-fold increased MRP4 mRNA levels along with increased MRP3 and MRP4 protein expression, respectively. A marked increase on BCRP protein and mRNA expression was observed in WiDr LF cells. These cells acquired approximately 2-fold resistance to mitoxantrone compared with the HF cell line, a phenotype that could be reverted by the BCRP inhibitor Ko143. Of note, WiDr cells expressed BCRP in the intracellular compartment, similarly to what we have described for Caco-2 cells. Our results provide further evidence for an important role of cellular folate status in the modulation of the expression of multidrug resistance transporters in cancer cells. We show that up-regulation of intracellularly localized BCRP in response to adaptation to LF conditions may be a common feature within a panel of colon cancer cell lines. Under these circumstances, folate supplementation might improve the efficacy of chemotherapeutic drugs by decreasing BCRP expression.

Substrate overlap between Mrp4 and Abcg2/Bcrp affects purine analogue drug cytotoxicity and tissue distribution.

The use of probe substrates and combinations of ATP-binding cassette (ABC) transporter knockout (KO) animals may facilitate the identification of common substrates between apparently unrelated ABC transporters. An unexpectedly low concentration of the purine nucleotide analogue, 9-(2-(phosphonomethoxy)ethyl)-adenine (PMEA), and up-regulation of Abcg2 in some tissues of the Mrp4 KO mouse prompted us to evaluate the possibility that Abcg2 might transport purine-derived drugs. Abcg2 transported and conferred resistance to PMEA. Moreover, a specific Abcg2 inhibitor, fumitremorgin C, both increased PMEA accumulation and reversed Abcg2-mediated PMEA resistance. We developed Mrp4 and Abcg2 double KO mice and used both single KOs of Abcg2 and Mrp4 mice to assess the role of these transporters in vivo. Abcg2 contributed to PMEA accumulation in a variety of tissues, but in some tissues, this contribution was only revealed by the concurrent absence of Mrp4. Abcg2 also transported and conferred resistance to additional purine analogues, such as the antineoplastic, 2-chloro-2'-deoxyadenosine (cladribine) and puromycin, a protein synthesis inhibitor that is often used as a dominant selectable marker. Purine analogues interact with ABCG2 by a site distinct from the prazosin binding site as shown by their inability to displace the substrate analogue and photoaffinity tag [(125)I]iodoarylazidoprazosin. These studies show that Abcg2, like Mrp4, transports and confers resistance to purine nucleoside analogues and suggest that these two transporters work in parallel to affect drug cytotoxicity and tissue distribution. This new knowledge will facilitate an understanding of how Abcg2 and Mrp4, separately and in combination, protect against purine analogue host toxicity as well as resistance to chemotherapy.

Folate deprivation induces BCRP (ABCG2) expression and mitoxantrone resistance in Caco-2 cells.

Folates can induce the expression and activity of the breast-cancer-resistance-protein (BCRP) and the multidrug-resistance-protein-1 (MRP1). Our aim was to study the time-dependent effect of folate deprivation/supplementation on (i) BCRP and MRP expression and (ii) on drug resistance mediated by these transporters. Therefore Caco-2 colon cancer cells usually grown in standard RPMI-medium containing supraphysiological folic acid (FA) concentrations (2.3 muM; high-folate, HF) were gradually adapted to more physiological folate concentrations (1 nM leucovorin (LV) or 1 nM FA; low-folate, LF), resulting in the sublines Caco-2-LF/LV and Caco-2-LF/FA. Caco-2-LF/LV and LF/FA cells exhibited a maximal increase of 5.2- and 9.6-fold for BCRP-mRNA and 3.9- and 5.7-fold for BCRP protein expression, respectively, but no major changes on MRP expression. Overexpression of BCRP in the LF-cells resulted in 3.6- to 6.3-fold resistance to mitoxantrone (MR), which was completely reverted by the BCRP inhibitor Ko143. On the other hand, LF-adapted cells were markedly more sensitive to methotrexate than the HF-counterpart, both after 4-hr (9,870- and 23,923-fold for Caco-2-LF/LV and LF/FA, respectively) and 72-hr (11- and 22-fold for Caco-2-LF/LV and LF/FA, respectively) exposure. Immunofluorescent staining observed with a confocal-laser-scan-microscope revealed that in Caco-2 cells (both HF and LF), BCRP is mainly located in the cytoplasm. In conclusion, folate deprivation induces BCRP expression associated with MR resistance in Caco-2 cells. The intracellular localization of BCRP in these cells suggests that this transporter is not primarily extruding its substrates out of the cell, but rather to an intracellular compartment where folates can be kept as storage.

Androgen regulation of multidrug resistance-associated protein 4 (MRP4/ABCC4) in prostate cancer.

BACKGROUND: MRP4/ABCC4 is an ATP-binding cassette transporter expressed in normal prostate. This study aimed to define the pattern of MRP4/ABCC4 expression in normal and malignant prostate tissue and the relationship of MRP4/ABCC4 expression and function in response to androgen signaling. METHODS: Eighty-four radical prostatectomy specimens from patients with localized prostate cancer (PC) (22 neoadjuvant androgen ablation, AA, 62 no AA), 42 non-cancer and 16 advanced PCs were assessed for MRP4/ABCC4 mRNA/protein expression. The effect of DHT and bicalutamide on LNCaP cells was assessed by immunoblotting. HEK293 cells (+/-MRP4/ABCC4) were assessed for the ability to efflux androgens and anti-androgens. RESULTS: MRP4/ABCC4 mRNA/protein levels were higher in localized PC compared to non-cancer (P = 0.006). MRP4/ABCC4 levels were significantly decreased in PCs treated with AA compared to cancers exposed to normal testosterone levels (P < 0.0001). MRP4/ABCC4 expression in normal human tissues was limited to the prostate and the renal tubules. MRP4/ABCC4 protein levels increased in LNCaP cells after DHT which was partially blocked by bicalutamide. However, DHT did not alter the activation of the MRP4/ABCC4 promotor in luciferase reporter assays and testosterone, DHT, flutamide and hydroxy-flutamide were not substrates for MRP4/ABCC4. DISCUSSION: Elevated MRP4/ABCC4 expression is found in malignant compared to benign prostate tissue while lower MRP4/ABCC4 expression is seen after AA. Furthermore, MRP4/ABCC4 is upregulated by androgen and downregulated by anti-androgen treatment in vitro potentially through an indirect mode of action. These data strongly suggest that MRP4/ABCC4 is an androgen-regulated gene important in the progression to PC and may be a potential drug target.

Oxidative and electrophilic stress induces multidrug resistance-associated protein transporters via the nuclear factor-E2-related factor-2 transcriptional pathway.

UNLABELLED: Multidrug resistance-associated proteins (Mrps) are adenosine triphosphate-dependent transporters that efflux chemicals out of cells. In the liver, Mrp2 transports bilirubin-glucuronide, glutathione (GSH), and drug conjugates into bile, whereas Mrp3 and Mrp4 efflux these entities into blood. The purpose of this study was to determine whether oxidative conditions (that is, the disruption of hepatic GSH synthesis) or the administration of nuclear factor-E2-related factor-2 (Nrf2) activators (oltipraz and butylated hydroxyanisole) can induce hepatic Mrp transporters and whether that induction is through the Nrf2 transcriptional pathway. Livers from hepatocyte-specific glutamate-cysteine ligase catalytic subunit-null mice had increased nuclear Nrf2 levels, marked gene and protein induction of the Nrf2 target gene NAD(P)H:quinone oxidoreductase 1, as well as Mrp2, Mrp3, and Mrp4 expression. The treatment of wild-type and Nrf2-null mice with oltipraz and butylated hydroxyanisole demonstrated that the induction of Mrp2, Mrp3, and Mrp4 is Nrf2-dependent. In Hepa1c1c7 cells treated with the Nrf2 activator tert-butyl hydroquinone, chromatin immunoprecipitation with Nrf2 antibodies revealed the binding of Nrf2 to antioxidant response elements in the promoter regions of mouse Mrp2 [-185 base pairs (bp)], Mrp3 (-9919 bp), and Mrp4 (-3767 bp). CONCLUSION: The activation of the Nrf2 regulatory pathway stimulates the coordinated induction of hepatic Mrps.