Purpose-Single nucleotide polymorphisms (SNPs) of the ATP-binding cassette (ABC) transporter ABCG2 gene have been suggested to be a significant factor in patients' responses to medication and/or the risk of diseases. We aimed to evaluate the impact of the major non-synonymous SNP Q141K on lysosomal and proteasomal degradations. Methods-ABCG2WT and the Q141K variant were expressed in Flp-In-293 cells by using the Flp recombinase system. Their expression levels and cellular localization was measured by immunoblotting and immunofluorescence microscopy, respectively.Results-The protein level of the Q141K variant expressed in Flp-In-293 cells was about half that of ABCG2 WT, while their mRNA levels were equal. The protein expression level of the Q141K variant increased about two-fold when Flp-In-293 cells were treated with MG132. In contrast, the protein level of ABCG2 WT was little affected by the same treatment. After treatment with bafilomycin A 1 , the protein levels of ABCG2 WT and Q141K increased 5-and 2-fold in Flp-In-293 cells, respectively. Conclusions-The results strongly suggest that the major non-synonymous SNP Q141K affects the stability of the ABCG2 protein in the endoplasmic reticulum and enhances its susceptibility to ubiquitin-mediated proteasomal degradation.

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...

The ATP-binding cassette (ABC) transporter ABCG2 has been implicated to play a significant role in the response of patients to medication and/or the risk of diseases. To clarify the possible physiological or pathological relevance of ABCG2 polymorphisms, we have functionally validated single nucleotide polymorphisms (SNP) of ABCG2. In the present study, based on the currently available data on SNPs and acquired mutations, we have created a total of 18 variant forms of ABCG2 (V12M,

Human ABCG2 belongs to the ATP-binding cassette (ABC) transporter family and plays an important role in various biological reactions, such as xenobiotic elimination and homeostasis of protoporphyrin. We previously reported that ABCG2 exists in the plasma membrane as a homodimer bound via a disulfide bond at Cys-603. In the present study, we examined the importance of an intramolecular disulfide bond for stability of the ABCG2 protein.Substitution of either Cys-592 or Cys-608 located in the extracellular loop to glycine resulted in a significant decrease in protein levels of ABCG2 when expressed in Flp-In-293 cells. Interestingly, the protein levels of those ABCG2 variants were remarkably enhanced by treatment with the proteasome inhibitor MG132. Concomitantly, increases in ubiquitinated forms of those variant proteins were detected by immunoprecipitation. In contrast, neither the protein level nor the ubiquitinated state of the ABCG2 wild-type (WT) was affected by MG132 treatment. Ubiquitin-mediated protein degradation is suggested to be involved in degradation of misfolded ABCG2 proteins lacking the intramolecular disulfide bond. On the other hand, the protein level of ABCG2 WT increased more than 4-fold when cells were treated with bafilomycin A 1 , which inhibits lysosomal degradation, whereas the C592G or C608G variant was little affected by the same treatment. These results strongly suggest that two distinct pathways exist for protein degradation of ABCG2 WT and mutants lacking the intramolecular disulfide bond. Namely, the WT ABCG2 is degraded in lysosomes, and the misfolded ABCG2 lacking intramolecular disulfide bond undergoes ubiquitin-mediated protein degradation in proteasomes.