Antiviral Chemistry & Chemotherapy 16:213–216
Pointer The role of breast cancer resistance protein (BCRP/ ABCG2) in cellular resistance to HIV-1 nucleoside reverse transcriptase inhibitors Xin Wang* and Masanori Baba Division of Antiviral Chemotherapy, Center for Chronic Viral Diseases, Graduate School of Medical & Dental Sciences, Kagoshima University, Kagoshima, Japan *Corresponding author: Tel: +81 99 275 5931; Fax: +81 99 275 5932; E-mail:
[email protected]
Treatment of HIV-1-infected patients with antiretroviral agents is not always successful due to the emergence of resistant HIV-1 mutants with reduced susceptibility to the agents. However, factors other than viral mutation may also contribute to treatment failure. It has been demonstrated that the ATP-binding cassette (ABC) transporter P-glycoprotein (P-gp/ABCB1) is a key determinant of oral bioavailability of HIV-1 protease inhibitors and their penetration of the central nervous system. More recently, we have found that the expression of breast cancer resistance protein (BCRP/ABCG2) in a CD4+ T-cell line confers cellular resistance to nucleoside reverse
transcriptase inhibitors (NRTIs). The anti-HIV-1 activity of the NRTI zidovudine (AZT) was significantly diminished through the reduction of its metabolite levels in MT-4 cells which express high levels of BCRP. Moreover, the BCRP-specific inhibitor fumitremorgin C could completely restore the cytotoxicity of AZT and intracellular levels of its metabolites in BCRP-expressing cells. Thus, BCRP is considered to be a cellular factor that modulates the anti-HIV-1 activity of NRTIs. Keywords: ABC transporter, BCRP/ABCG2, HIV-1, NRTI-resistance
Introduction Significant progress in the treatment of HIV-1 infection has been achieved with the advent of highly active antiretroviral therapy (HAART), which targets different steps in the viral replication cycle with multiple inhibitors (Yeni et al., 2004). At present, one entry inhibitor, eight nucleoside or nucleotide reverse transcriptase inhibitors (NRTIs), three non-nucleoside reverse transcriptase inhibitors (NNRTIs) and eight protease inhibitors (PIs) are available for the treatment of HIV-1 infection. HAART with these inhibitors has brought about suppression of plasma HIV-1 RNA levels less than 50 copies/ml (Zhang et al., 1999). However, the emergence of drug-resistant HIV-1 mutants often results in the failure of therapy (Hirsch et al., 2003). Investigations on the host cellular factors responsible for antiviral resistance have revealed that several ATP-binding cassette (ABC) transporters also contribute to the reduced efficiency of anti-HIV-1 agents, including NRTIs, NNRTIs and PIs. The ABC transporter P-glycoprotein (P-gp/ABCB1), which was identified in 1976 and encoded by the multidrug resistance (MDR) gene, has been shown to be a key
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determinant of oral bioavailability of PIs and their penetration of the central nervous system ( Juliano et al., 1976; Kim et al., 2003). On the other hand, the role of ABC transporters in the acquisition of drug resistance to NRTIs remains unclear. In initial studies, the NRTI zidovudine (AZT) was found to be less active against HIV-1 replication in P-gp-expressing cells (Antonelli et al., 1992). However, this effect was possibly mediated by other ABC transporters, such as multidrug resistance protein (MRP4/ABCC4) and/or MRP8/ABCC11] (Schuetz et al., 1999; Turriziani et al., 2002). More recently, we found that high expression of breast cancer resistance protein (BCRP/ABCG2), a new member of the ABC transporter superfamily, in a CD4+ T cell line also induced significant resistance to NRTIs (Wang et al., 2003; Wang et al., 2004).
ABC transporter BCRP/ABCG2 BCRP is the second member of the G (white) subfamily of ABC transporters (ABCG2) and is also known as
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MXP/ABCP. BCRP was independently cloned from placenta as well as cell lines selected for resistance to mitoxantrone or anthracyclines (Allikmets et al., 1998; Doyle et al., 1998; Miyake et al., 1999). This glycosylated plasma membrane protein is a half-size transporter. BCRP consists of a nucleotide-binding domain (NBD) at the amino terminus and a transmembrane domain at the carboxyl terminus, and is postulated to form a homodimer to exert its biological functions (Figure 1). Over-expression of BCRP in cell lines confers resistance to a wide variety of anti-cancer drugs, including mitoxantrone, daunorubicin, doxorubicin and topotecan. BCRP has been detected in breast, colon and gastric cancers, and in acute myeloid and lymphoblastic leukaemias (Sauerbrey et al., 2002). BCRP is also expressed in some normal tissues (including placenta, liver, breast and venous), capillary endothelium and other organs and tissues (Maliepaard et al., 2001). High expression of BCRP in placenta and the luminal surface of microvessel endothelium of the blood-brain barrier suggest that it plays a role in limiting the penetration of drugs from the maternal plasma into the fetus and from blood to brain (Cooray et al., 2002). A variety of BCRP-inhibitors including fumitremorgin C may sensitize cancer cells to anticancer agents or alter drug distribution in vivo.
BCRP and HIV-1 NRTI resistance To investigate the effect of BCRP on the cellular resistance to anti-HIV-1 agents, we established a doxorubicin (DOX)-resistant CD4+ T-cell line MT-4/DOX500 by exposing MT-4 cells to increasing concentrations of the DOX, which expressed a high level of BCRP but not other
multidrug-resistant proteins (Wang et al., 2003). Compared with the parental MT-4 cells, MT-4/DOX500 cells showed reduced sensitivity to AZT but not nelfinavir in terms of drug-cytotoxicity, and this reduced cytotoxicity could be completely abolished by addition of fumitremorgin C. The anti-HIV-1 activity of NRTIs was also impaired in MT-4/DOX500 cells. AZT, didanosine (ddI) and stavudine (d4T) proved to be 7.5, 2.7 and 1.6fold less inhibitory to HIV-1 replication in MT-4/DOX500 cells than in MT-4 cells. Furthermore, the anti-HIV-1 activity of lamivudine (3TC) was severely (more than 77fold) impaired in MT-4/DOX500 cells. However, the antiviral activities of NNRTIs and PIs were not affected in MT-4/DOX500 cells. Huisman et al. also demonstrated that BCRP is not an efficient transporter of the PIs saquinavir, ritonavir and indinavir (Huisman et al. 2002). Those results indicate the specific interaction of BCRP with HIV-1 NRTIs but not NNRTIs or PIs. Analysis for intracellular metabolism of AZT in MT-4 and MT-4/DOX500 cells revealed the reduced accumulation of AZT and its metabolites in MT-4/DOX500 cells (Wang et al., 2003). DOX- or mitoxantrone-selection in human cell lines always brought about an amino acid mutation from arginine to glycine or threonine at position 482 (R482G/T) in human BCRP (Honjo et al., 2001). This amino acid mutation plays an important role in determining the substrate specificity to BCRP, possibly by virtue of its tentative strategic location at the cytoplasmic end of transmembrane domain 3 (Figure 1). Sequencing of the full-length BCRP cDNA identified the R482M mutation in MT-4/DOX500 cells (Wang et al, 2003). Therefore, possibly NRTI resistance is conferred by mutant, but not wild type, BCRP.
Figure 1. Proposed membrane topology of the BCRP/ABCG2 multidrug transporter
TM6
TM5
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intracellular
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R482
The arginine at position 482 (R482) was indicated. NBD, nucleotide binding domain; TM, transmembrane regions.
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BCRP and HIV-1 NRTI-resistance
To exclude this possibility, we generated a CD4+ T-cell line over-expressing wild type BCRP (MT-4/BCRP) by transducing MT-4 cells with an HaBCRP retrovirus supernatant and examining whether the wild type also affects the anti-HIV-1 activity and cytotoxicity of NRTIs (Wang et al., 2004). Compared to the parental cells, MT-4/BCRP cells also displayed resistance to NRTIs in terms of drug cytotoxicity as well as antiviral activity. Levels of AZT and its metabolite in MT-4/BCRP cells were completely restored in the presence of fumitremorgin C in MT4/BCRP cells, suggesting the roles of the wild-type BCRP in cellular resistance to NRTIs (Figure 2). Thus, future studies of BCRP inhibitors may be beneficial for improving the efficacy of NRTIs. Although no solid evidence of increased expression and functions of BCRP in HIV-1-infected patients have been demonstrated yet, BCRP mRNA has been detected in bone marrow and peripheral blood mononuclear cells (Sauerbrey et al., 2002). Furthermore, it must be noted that AIDS patients often suffer from malignant diseases and, thereby, have the opportunity to be treated with anticancer agents (Dezube et al., 2002). If anticancer agents could upregulate BCRP expression in the HIV-1 infected cells, the intracellular NRTI concentrations would decrease in these tissues. Thus, reduced intra-cellular levels of active compounds may result in the insufficient suppression of HIV-1 replication an increasing opportunity for the emergence of drug-resistant mutants. Since BCRP was also identified in the blood-brain barrier of both normal and tumourous human brain tissues, and is mainly located at the luminal surface of microvessel endothelium, a possible
role of BCRP in the limited distribution of AZT, ddI and related nucleoside derivatives in the central nervous system should also be considered (Cooray et al., 2002; Masereeuw et al., 1994).
Conclusions NRTIs are still the most important class of anti-HIV-1 agents. In fact, HAART is generally initiated with two NRTIs plus one NNRTI, or two NRTIs plus one PI. If NRTI resistance could be induced by host cellular factors, it would be a more serious impediment to the progress of HAART (Lavie et al., 1997). In this point of view, a more detailed knowledge of BCRP function in the HIV-1 infected patients and its interaction with NRTIs will improve or develop new strategies for anti-HIV-1 chemotherapy.
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Figure 2. Restoration of intracellular AZT metabolism by fumitremorgin C in MT-4/BCRP cells
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The control MT-4 cells (panel A) and the MT-4 cells highly expressing the wild-type BCRP (MT-4/BCRP) (panel B) were incubated with [methyl-3H]AZT in the absence or presence of fumitremorgin C (5 µM). The methanol extracts were separated with HPLC, and each fraction was analysed for radioactivity. Adapted with permission from Wang et al. (2004), Biochemical Pharmacology, 68:1363–1370.
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Received 11 March 2005, accepted 7 April 2005
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