Re‐evaluation and functional classification of non‐synonymous single nucleotide polymorphisms of the human ATP‐binding cassette transporter ABCG2

Abstract

H uman ABCG2 (1-3) is a member of the ABC transporter gene family. The ABCG2 gene is located on chromosome 4q22, spans over 66 kb, and consists of 16 exons ranging from 60 to 532 bp. (4) The ABCG2 protein is a so-called 'half ABC transporter', existing as a homodimer linked through a cysteinyl disulfide bond at Cys603. (5-7) ABCG2 protein is expressed endogenously in placental trophoblast cells, the epithelium of the small intestine and liver canalicular membrane, as well as in ducts and lobules of the breast. Apical localization in the epithelium of the small intestine and colon indicates a possible role for human ABCG2 in regulating the uptake of orally ( p.o.) administered drugs as well as xenobiotics. (8) Overexpression of ABCG2 reportedly confers cancer cell resistance to anticancer drugs, such as topotecan, irinotecan (CPT-11) and mitoxantrone. (9)(10)(11)(12) In the case of drug resistance to irinotecan, SN-38-resistant PC-6/SN2-5H human lung carcinoma cells were shown to overexpress ABCG2 with reduced intracellular accumulation of SN-38, an active metabolite of CPT-11, and the SN-38-glucuronide conjugate. (12) Plasma membrane vesicles prepared from those cancer cells or ABCG2-transfected cells transported both SN-38 and SN-38-glucuronide ATP-dependently. (13,14) It has also been reported that ABCG2-transfected cells are resistant to photosensitizers, such as hematoporphyrin IX, pheophorbide a, and chlorine e6, suggesting a possible role for ABCG2 in cellular resistance to photodynamic therapy. (15) In this regard, we have most recently demonstrated that ABCG2 transports hematoporphyrin in an ATP-dependent manner. (16) SNP of ABCG2 have been suggested as a significant factor in a patient's response to medication and the risk of diseases. (17)(18)(19)(20)(21)(22) Sequencing of the ABCG2 gene from human samples has revealed over 80 different, naturally occurring sequence variations. (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) However, information is still limited regarding the functional impact of genetic polymorphisms of ABCG2. In addition, it was noticed that some discrepancies existed among reports in terms of the transport function and drug resistance profile of SNP variants of ABCG2. (25,28,30,31) The reason for such discrepancies is not known, but it may be due to differences in their experimental procedures.In the present study, we aimed to re-evaluate the impact of the genetic polymorphisms of ABCG2 on drug resistance by functionally validating its SNP in vitro. To analyze quantitatively the effect of non-synonymous SNP of ABCG2 on the protein expression level and the drug resistance profile, we used the Flp-In method to integrate one single copy of ABCG2 variant cDNA into FRTtagged genomic DNA. By using this method, we could exclude the random integration of ABCG2 cDNA into the chromosomal DNA of host cells. Furthermore, we have examined the pharmacological potency of our new camptothecin analogs (32,33) to determine whether they could circumvent ABCG2-associated drug resistance in human tumor cel...

Single-nucleotide polymorphisms of ABCG2 increase the efficacy of tyrosine kinase inhibitors in the K562 chronic myeloid leukemia cell line

“…The impact of these SNPs on TKI efficacy might have been due to the lower transcriptional activity seen in K562/ABCG2 34 and 1582 cells rather than the specific SNPs. In a previous study, ABCG2 34G>A induced higher resistance to the camptothecin analog SN-38 compared to wild type ABCG2 expressed in human cell lines [33]. We could not identify any increased resistance to TKIs in K562/ABCG2 34 cells.…”

Genetic and Dietary Regulation of Glyburide Efflux by the Human Placental Breast Cancer Resistance Protein Transporter

“…1). Considering these findings, altered BCRP function in our experiments may be due to reduced BCRP protein trafficking to the cell surface, in line with other studies reporting decreased BCRP protein expression and function in C421A-BCRP-overexpressing cells (Imai et al, 2002;Kondo et al, 2004;Tamura et al, 2007, Furukawa et al, 2009Woodward et al, 2013). In human tissue, the heterozygous variant genotype (421C/A) did not change BCRP protein expression in the intestine or placenta, but there was a significant decrease in BCRP protein expression in placentas of individuals homozygous for the SNP (421A/A) (Zamber et al, 2003;Kobayashi et al, 2005;Urquhart et al, 2008).…”

Deletion ofAbcg2Has Differential Effects on Excretion and Pharmacokinetics of Probe Substrates in Rats

“…This SNP was not detected in African Americans, but 25.9% of European Americans and 50 to 60% of Asian Americans had at least one variant allele (Zamber et al, 2003). ABCG2 421CϾA SNP results in a lysine to glutamine acid change at codon 141 (Q141K), which leads to decreased protein expression level or reduced drug resistance to anticancer agents in transfected cells (Imai et al, 2002;Mizuarai et al, 2004;Morisaki et al, 2005;Tamura et al, 2006Tamura et al, , 2007Furukawa et al, 2009). The Q141K variant was associated with increased risk for gefitinib-induced diarrhea (Cusatis et al, 2006).…”