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...

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.

No abstract

Since porphyrins are regarded as endogenous substrates for the ATP-binding cassette (ABC) transporter ABCG2, it is hypothesized that functional impairment owing to genetic polymorphisms or inhibition of ABCG2 by drugs may result in a disruption of cellular porphyrin homeostasis. In the present study, we expressed ABCG2 genetic variants, i.e., V12M, Q141K, S441N, and F489L, as well as the wild type (WT) in Flp-In-293 cells to examine the hypothesis. Cells expressing S441N and F489L variants exhibited high levels of both cellularly accumulated pheophorbide a and photosensitivity, when those cells were incubated with pheophorbide a and irradiated with visible light. To further elucidate the significance of ABCG2 in cellular porphyrin homeostasis, we observed cellular accumulation and compartmentation of porphyrin and pheophorbide a by means of a new fluorescence microscopy technology, and found that accumulation of porphyrin and pheophorbide a in the cytoplasm compartment was maintained at low levels in Flp-In-293 cells expressing ABCG2 WT, V12M, or Q141K. When ABCG2 was inhibited by imatinib or novobiocin, however, those cells became sensitive to light. Based on these results, it is strongly suggested that certain genetic polymorphisms and/or inhibition of ABCG2 by drugs can enhance the potential risk of photosensitivity.

Clinical relevance is implicated between the genetic polymorphisms of the ABC (ATP-binding cassette) transporter ABCG2 (ABC subfamily G, member 2) and the individual differences in drug response. We expressed a total of seven non-synonymous SNP (single nucleotide polymorphism) variants in Flp-In-293 cells by using the Flp (flippase) recombinase system. Of these, ABCG2 F208S and S441N variants were found to be expressed at markedly low levels, whereas their mRNA levels were equal to those of the other SNP variants and ABCG2 WT (wild-type). Interestingly, protein expression levels of the ABCG2 F208S and S441N variants increased 6- to 12-fold when Flp-In-293 cells were treated with MG132, a proteasome inhibitor. Immunoprecipitation followed by immunoblot analysis showed that the ABCG2 F208S and S441N variant proteins were endogenously ubiquitinated in Flp-In-293 cells, and treatment with MG132 significantly enhanced the level of these ubiquitinated variants. Immunofluorescence microscopy demonstrated that MG132 greatly affected the ABCG2 F208S and S441N variants in terms of both protein levels and intracellular distribution. Immunoblot analysis revealed that those variants were N-glycosylated; however, their oligosaccharides were immature compared with those present on ABCG2 WT. The ABCG2 F208S and S441N variant proteins do not appear to be processed in the Golgi apparatus, but undergo ubiquitin-mediated protein degradation in proteasomes, whereas ABCG2 WT is sorted to the plasma membrane and then degraded via the lysosomal pathway. The present study provides the first evidence that certain genetic polymorphisms can affect the protein stability of ABCG2. Control of proteasomal degradation of ABCG2 would provide a novel approach in cancer chemotherapy to circumvent multidrug resistance of human cancers.

A computationally efficacious free-energy functional for studies of inhomogeneous liquid water J. Chem. Phys. 137, 044107 (2012) Fourth virial coefficients of asymmetric nonadditive hard-disk mixtures J. Chem. Phys. 136, 184505 (2012) Equation of state and jamming density for equivalent bi-and polydisperse, smooth, hard sphere systems J. Chem. Phys. 136, 124508 (2012) On the theoretical determination of the Prigogine-Defay ratio in glass transition J. Chem. Phys. 136, 124502 (2012) Communication: Thermodynamics of condensed matter with strong pressure-energy correlations Hugoniot data of diamond was obtained using laser-driven shock waves in the terapascal range of 0.5-2 TPa. Strong shock waves were generated by direct irradiation of a 2.5 ns laser pulse on an Al driver plate. The shock wave velocities in diamond and Al were determined from optical measurements. Particle velocities and pressures were obtained using an impedance matching method and known Al Hugoniot. The obtained Hugoniot data of diamond does not show a marked difference from the extrapolations of the Pavlovskii Hugoniot data in the TPa range within experimental errors.

In the post-genome-sequencing era, emerging genomic technologies are shifting the paradigm for drug discovery and development. Nevertheless, drug discovery and development still remain high-risk and high-stakes ventures with long and costly timelines. Indeed, the attrition of drug candidates in preclinical and development stages is a major problem in drug design. For at least 30% of the candidates, this attrition is due to poor pharmacokinetics and toxicity. Thus, pharmaceutical companies have begun to seriously re-evaluate their current strategies of drug discovery and development. In that light, we propose that a transport mechanism-based design might help to create new, pharmacokinetically advantageous drugs, and as such should be considered an important component of drug design strategy. Performing enzyme- and/or cell-based drug transporter, interaction tests may greatly facilitate drug development and allow the prediction of drug-drug interactions. We recently developed methods for high-speed functional screening and quantitative structure-activity relationship analysis to study the substrate specificity of ABC transporters and to evaluate the effect of genetic polymorphisms on their function. These methods would provide a practical tool to screen synthetic and natural compounds, and these data can be applied to the molecular design of new drugs. In this review article, we present an overview on the genetic polymorphisms of human ABC transporter ABCG2 and new camptothecin analogues that can circumvent AGCG2-associated multidrug resistance of cancer.