ABCG2 Harboring the Gly482 Mutation Confers High-Level Resistance to Various Hydrophilic Antifolates

Abstract

ABCG2 is an ATP-binding cassette transporter that confers resistance to various chemotherapeutic agents. Recent studies have established that an Arg (wild-type) to Gly mutation at amino acid 482 in ABCG2 alters substrate specificity. Here, we explored the role of this G482 mutation in antifolate resistance using a clinically relevant 4-hour drug exposure. Stable transfectants overexpressing the mutant G482 transporter displayed 120-, 1,000-, and >6,250-fold resistance to the antifolates methotrexate, GW1843, and Tomudex, respectively, relative to parental human embryonic kidney cells. Moreover, although overexpressing equal transporter levels at the plasma membrane, G482-ABCG2 cells were 6-, 23-, and >521-fold more resistant to methotrexate, GW1843, and Tomudex, respectively, than R482-ABCG2 cells. In contrast, upon a continuous (72-hour) drug exposure, both the G482-and R482-ABCG2 cells lost almost all their antifolate resistance; this result was consistent with the inability of ABCG2 to extrude long-chain antifolate polyglutamates. Ko143, a specific and potent ABCG2 inhibitor reversed methotrexate resistance in both G482-and R482-ABCG2 cells. Consistently, whereas the pool of free methotrexate in parental human embryonic kidney cells was prominent after 4 hours of transport with 1 Mmol/L [ 3 H]methotrexate, in R482-and G482-ABCG2 cells, it was minimal. Furthermore, G482-ABCG2 cells contained marked decreases in the di-and triglutamate species of [ 3 H]methotrexate at 4 hours of incubation with methotrexate and in the tetra-and pentaglutamates at 24 hours. These changes were not associated with any significant decrease in folylypoly-;-glutamate synthetase activity. These results provide the first evidence that the G482-ABCG2 mutation confers high-level resistance to various hydrophilic antifolates. (Cancer Res 2005; 65(18): 8414-22)

Structural Determinants of Imidazoacridinones Facilitating Antitumor Activity Are Crucial for Substrate Recognition by ABCG2

“…Previous reports have established that the Arg482Gly and Arg482Thr ABCG2 mutations resulted in altered substrate specificity and augmented cellular drug resistance (Robey et al, 2003;Shafran et al, 2005;Bram et al, 2006). To determine whether these mutations alter IA substrate specificity and thereby facilitate the efflux of non-ABCG2 substrates of group B IAs, a flow cytometric IA accumulation assay was employed using HEK293 cells stably transfected with wild-type Arg482-as well as mutant Gly482-or T482-ABCG2 (Robey et al, 2003) (Fig.…”

ATP-binding cassette, subfamily G (ABCG family)

“…ABCG2 confers resistance to anticancer drugs, such as mitoxantrone, doxorubicin, and daunorubicin, by decreasing their cellular accumulation, and thus, ABCG2 has been referred to as breast cancer resistant protein (BCRP) [17]. Subsequent analyses have shown that overexpression of ABCG2 results in the acquisition of resistance to anticancer drugs, such as camptothecin derivatives (topotecan [4], SN-38 [49], 9-aminocamptothecin [49]), antifolates (methotrexate and its polyglutamate form, GW1843 and tomudex) [68,79,78], flavopiridol [64], and bisantrene [46]. Attention should be paid to the acquired mutations of ABCG2 in some tumor cell lines (R482G and R482T), which have been shown to alter the spectrum of resistance to anticancer drugs.…”

Gene expression profiling of leukemia T-cells resistant to methotrexate and 7-hydroxymethotrexate reveals alterations that preserve intracellular levels of folate and nucleotide biosynthesis

“…A c c e p t e d M a n u s c r i p t 4 The metabolite 7-OHMTX ( Fig. 1) results from oxidation of MTX primarily by hepatic aldehyde oxidase.…”

“…Decreased cellular uptake via the reduced folate carrier (RFC/SLC19A1) [3] or increased active efflux mediated by ABC transporters such as the multidrug resistance-associated proteins 1-5 (MRP 1-5), the breast cancer resistance protein (BCRP/ABCG2) [4,5] and P-glycoprotein (MDR or ABCB1) [6] can lead to resistance to hydrophilic and lipophilic antifolates. The ability of cells to accumulate long chain MTX polyglutamates (MTX-PGs) is highly dependent on the activities of folylpolyglutamate synthetase (FPGS) and folylpolyglutamate hydrolase (FPGH).…”