Multidrug resistance protein 4 protects bone marrow, thymus, spleen, and intestine from nucleotide analogue-induced damage.

Nucleoside-based analogues are mainstays in the treatment of cancer, viral infections, and inflammatory diseases. Recent studies showing that the ATP-binding cassette transporter, multidrug resistance protein 4, is able to efflux nucleoside and nucleotide analogues from transfected cells suggests that the pump may affect the efficacy of this class of agents. However, the in vivo pharmacologic functions of the pump are largely unexplored. Here, using Mrp4(-/-) mice as a model system, and the nucleotide analogue, 9'-(2'-phosphonylmethoxyethyl)-adenine (PMEA) as a probe, we investigate the ability of Mrp4 to function in vivo as an endogenous resistance factor. In the absence of alterations in plasma PMEA levels, Mrp4-null mice treated with PMEA exhibit increased lethality associated with marked toxicity in several tissues. Affected tissues include the bone marrow, spleen, thymus, and gastrointestinal tract. In addition, PMEA penetration into the brain is increased in Mrp4(-/-) mice. These findings indicate that Mrp4 is an endogenous resistance factor, and that the pump may be a component of the blood-brain barrier for nucleoside-based analogues. This is the first demonstration that an ATP-binding cassette transporter can affect in vivo tissue sensitivity towards this class of agents.

Analysis of the drug resistance profile of multidrug resistance protein 7 (ABCC10): resistance to docetaxel.

The multidrug resistance protein (MRP) family consists of nine members that can be categorized according to whether or not a third (NH(2)-terminal) membrane-spanning domain is present. Three (MRP1, MRP2, and MRP3) of the four members that have this structural feature are able to confer resistance to natural product anticancer agents. We previously established that MRP7, the remaining family member that has three membrane-spanning domains, possesses the cardinal biochemical activity of MRPs in that it is able to transport amphipathic anions such as 17beta-estradiol 17-(beta-d-glucuronide). However, the drug resistance profile of the pump has not been determined. In this study, the drug resistance capabilities of MRP7 are evaluated by analyzing the resistance profiles of two clones of HEK293 cells in which the pump was ectopically expressed. MRP7-transfected HEK293 cells exhibited the highest levels of resistance toward docetaxel (9-13-fold). In addition, lower levels of resistance were observed for paclitaxel (3-fold), vincristine (3-fold), and vinblastine (3-4-fold). Consistent with the operation of an ATP-dependent efflux pump, MRP7-transfected cells exhibited reduced accumulation of radiolabeled paclitaxel compared with HEK293 cells transfected with parental plasmid. These results indicate that MRP7, unlike other MRPs, is a resistance factor for taxanes.

Characterization of the drug resistance and transport properties of multidrug resistance protein 6 (MRP6, ABCC6).

Mutations in human multidrug resistance protein 6 (MRP6, ABCC6), a member of the MRP family of drug efflux pumps, are the genetic basis of Pseudoxanthoma elasticum, a disease that affects elastin fibers in the skin, retina, and blood vessels. However, little is known about the functional characteristics of the protein, including its potential activity as a resistance factor for anticancer agents. Here, we report the results of investigations of the in vitro transport properties and drug resistance activity of MRP6. Using membrane vesicles prepared from Chinese hamster ovary cells transfected with MRP6 expression vector, it is shown that expression of MRP6 is specifically associated with the MgATP-dependent transport of the glutathione S-conjugates leukotriene C(4) and S-(2, 4-dinitrophenyl)glutathione and the cyclopentapeptide BQ123 but not glucuronate conjugates such as 17beta-estradiol 17-(beta-D-glucuronide). Analysis of the drug sensitivity of MRP6-transfected cells revealed low levels of resistance to several natural product agents, including etoposide, teniposide, doxorubicin, and daunorubicin. These results indicate that MRP6 is a glutathione conjugate pump that is able to confer low levels of resistance to certain anticancer agents.

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.

Combining anti-ERBB3 antibodies specific for domain I and domain III enhances the anti-tumor activity over the individual monoclonal antibodies.

BACKGROUND: Inappropriate signaling through the epidermal growth factor receptor family (EGFR1/ERBB1, ERBB2/HER2, ERBB3/HER3, and ERBB4/HER4) of receptor tyrosine kinases leads to unregulated activation of multiple downstream signaling pathways that are linked to cancer formation and progression. In particular, ERBB3 plays a critical role in linking ERBB signaling to the phosphoinositide 3-kinase and Akt signaling pathway and increased levels of ERBB3-dependent signaling is also increasingly recognized as a mechanism for acquired resistance to ERBB-targeted therapies. METHODS: We had previously reported the isolation of a panel of anti-ERBB3 single-chain Fv antibodies through use of phage-display technology. In the current study scFv specific for domain I (F4) and domain III (A5) were converted into human IgG1 formats and analyzed for efficacy. RESULTS: Treatment of cells with an oligoclonal mixture of the A5/F4 IgGs appeared more effective at blocking both ligand-induced and ligand-independent signaling through ERBB3 than either single IgG alone. This correlated with improved ability to inhibit the cell growth both as a single agent and in combination with other ERBB-targeted therapies. Treatment of NCI-N87 tumor xenografts with the A5/F4 oligoclonal led to a statistically significant decrease in tumor growth rate that was further enhanced in combination with trastuzumab. CONCLUSION: These results suggest that an oligoclonal antibody mixture may be a more effective approach to downregulate ERBB3-dependent signaling.

Evaluation of the anti-HER2 C6.5 diabody as a PET radiotracer to monitor HER2 status and predict response to trastuzumab treatment.

PURPOSE: The rapid tumor targeting and pharmacokinetic properties of engineered antibodies make them potentially suitable for use in imaging strategies to predict and monitor response to targeted therapies. This study aims to evaluate C6.5 diabody (C6.5 db), a noncovalent anti-HER2 single-chain Fv dimer, as a radiotracer for predicting response to HER2-targeted therapies such as trastuzumab. EXPERIMENTAL DESIGN: Immunodeficient mice bearing established HER2-positive tumor xenografts were injected with radioiodinated C6.5 db and imaged by PET/CT. Radiotracer biodistribution was quantified by biopsied tumor and normal tissues. Potential competition between trastuzumab and C6.5 db was examined in vitro by flow cytometry and coimmunoprecipitations. RESULTS: Biodistribution analysis of mice bearing xenografts with varying HER2 density revealed that the tumor uptake of (125)I-C6.5 db correlates with HER2 tumor density. In vitro competition experiments suggest that the C6.5 db targets an epitope on HER2 that is distinct from that bound by trastuzumab. Treatment of mice affected with SK-OV-3 tumor with trastuzumab for 3 days caused a 42% (P = 0.002) decrease in tumor uptake of (125)I-C6.5 db. This is consistent with a dramatic decrease in the tumor PET signal of (124)I-C6.5 db after trastuzumab treatment. Furthermore, mice affected with BT-474 tumor showed an approximately 60% decrease (P = 0.0026) in C6.5 db uptake after 6 days of trastuzumab treatment. Immunohistochemistry of excised xenograft sections and in vitro flow cytometry revealed that the decreased C6.5 db uptake on trastuzumab treatment is not associated with HER2 downregulation. CONCLUSIONS: These studies suggest that (124)I-C6.5 db-based imaging can be used to evaluate HER2 levels as a predictor of response to HER2-directed therapies.

Analysis of the in vivo functions of Mrp3.

Multidrug resistance protein 3 (MRP3) is an ATP-binding cassette transporter that is able to confer resistance to anticancer agents such as etoposide and to transport lipophilic anions such as bile acids and glucuronides. These capabilities, along with the induction of the MRP3 protein on hepatocyte sinusoidal membranes in cholestasis and the expression of MRP3 in enterocytes, have led to the hypotheses that MRP3 may function in the body to protect normal tissues from etoposide, to protect cholestatic hepatocytes from endobiotics, and to facilitate bile-acid reclamation from the gut. To elucidate the role of Mrp3 in these processes, the Mrp3 gene (Abcc3) was disrupted by homologous recombination. Homozygous null animals were healthy and physically indistinguishable from wild-type mice. Mrp3(-/-) mice did not exhibit enhanced lethality to etoposide phosphate, although an analysis of transfected human embryonic kidney 293 cells indicated that the potency of murine Mrp3 toward etoposide ( approximately 2.0- to 2.5-fold) is comparable with that of human MRP3. After induction of cholestasis by bile duct ligation, Mrp3(-/-) mice had 1.5-fold higher levels of liver bile acids and 3.1-fold lower levels of serum bilirubin glucuronide compared with ligated wild-type mice, whereas significant differences were not observed between the respective sham-operated mice. Bile acid excretion, pool size, and fractional turnover rates were similar in Mrp3(-/-) and wild-type mice. We conclude that Mrp3 functions as an alternative route for the export of bile acids and glucuronides from cholestatic hepatocytes, that the pump does not play a major role in the enterohepatic circulation of bile acids and that the lack of chemosensitivity is probably attributable to functional redundancy with other pumps.

Characterization of the transport properties of human multidrug resistance protein 7 (MRP7, ABCC10).

Human multidrug resistance protein 7 (MRP7, ABCC10) is a recently described member of the C family of ATP binding cassette proteins (Cancer Lett 162:181-191, 2001). However, neither its biochemical activity nor physiological functions have been determined. Here we report the results of investigations of the in vitro transport properties of MRP7 using membrane vesicles prepared from human embryonic kidney 293 cells transfected with MRP7 expression vector. It is shown that expression of MRP7 is specifically associated with the MgATP-dependent transport of 17beta-estradiol-(17-beta-D-glucuronide) (E(2)17betaG). E(2)17betaG transport was saturable, with K(m) and V(max) values of 57.8 +/- 15 microM and 53.1 +/- 20 pmol/mg/min. By contrast, with E(2)17betaG, only modest enhancement of LTC(4) transport was observed and transport of several other established substrates of MRP family transporters was not detectable to any extent. In accord with the notion that MRP7 has a bipartite substrate binding pocket composed of sites for anionic and lipophilic moieties, transport of E(2)17betaG was susceptible to competitive inhibition by both amphiphiles, such as leukotriene C(4) (K(i(app)), 1.5 microM), glycolithocholate 3-sulfate (K(i(app)), 34.2 microM) and MK571 (K(i(app)), 28.5 microM), and lipophilic agents such as cyclosporine A (K(i(app)), 14.4 microM). Of the inhibitors tested, LTC(4) was the most potent, in agreement with the possibility that it is a substrate of the pump. The determination that MRP7 has the facility for mediating the transport of conjugates such as E(2)17betaG indicates that it is a lipophilic anion transporter involved in phase III (cellular extrusion) of detoxification.