Re-evaluation and functional classification of non-synonymous single nucleotide polymorphisms of the human ATP-binding cassette transporter ABCG2
Re-evaluation and functional classification of non-synonymous single nucleotide polymorphisms of the human ATP-binding cassette transporter ABCG2
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DOI：
10.1111/j.1349-7006.2006.00371.x
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发表时间：
2007-02-01
期刊：
影响因子：
5.7
通讯作者：
Ishikawa, Toshihisa
中科院分区：
文献类型：
--
作者：
Tamura, Ai;Wakabayashi, Kanako;Ishikawa, Toshihisa
ATP结合盒（ABC）转运蛋白BCRP/MXR 1/ABCP（ABCG 2）的遗传多态性对药物反应的影响已被牵连;然而，迄今为止报道的数据涉及一些不一致。为了重新评估ABCG 2的单核苷酸多态性（SNP）在体外的作用，我们通过定点突变创建了总共七个变体cDNA（V12 M、Q141 K、F208 S、S248 P、F431 L、S441 N和F489 L），并使用Flp重组酶系统在Flp-In-293细胞中稳定表达它们中的每一个。多色荧光原位杂交图谱分析显示，ABCG 2 cDNA的一个单拷贝被纳入染色体12 p的端粒区域。证明这些整合的ABCG 2变体的mRNA在Flp-In-293细胞中均匀表达。然而，蛋白质表达水平在这些变体之间变化。特别地，F208 S和S441 N变体的表达显著低，表明这些变体蛋白的不稳定性。表达两种主要SNP变体（V12 M和Q141 K）的Flp-In-293细胞对药物SN-38的耐药性谱表明，Q141 K的IC 50值（产生50%细胞生长减少的药物浓度）约为野生型的50%。次要SNP变体（F208 S、S248 P、F431 L、S441 N和F489 L）对SN-38、米托蒽醌、多柔比星、柔红霉素或依托泊苷的耐药性的贡献显著低于野生型。基于我们的功能验证，上述非同义多态性以及获得性突变体（R482 G和R482 T）的ABCG 2分为四组。此外，我们的研究小组合成的新喜树碱类似物在规避ABCG 2介导的耐药性方面具有有效作用，而不受主要非同义多态性的影响。
Impacts of genetic polymorphisms of the ATP-binding cassette (ABC) transporter BCRP/MXR1/ABCP (ABCG2) on drug response have been implicated; however, the hitherto reported data involve some inconsistencies. To re-evaluate the effect of single nucleotide polymorphisms (SNP) of ABCG2 in vitro, we created a total of seven variant cDNAs (V12M, Q141K, F208S, S248P, F431L, S441N and F489L) by site-directed mutagenesis and stably expressed each of them in Flp-In-293 cells using the Flp recombinase system. Multicolor fluorescence in situ hybridization mapping analysis revealed that one single copy of ABCG2 cDNA was incorporated into the telomeric region of chromosome 12p. It was proven that mRNAs of those integrated ABCG2 variants were expressed evenly in Flp-In-293 cells. However, the protein expression levels varied among those variants. In particular, expression of the F208S and S441N variants was markedly low, suggesting the instability of these variant proteins. Drug resistance profiles of Flp-In-293 cells expressing two major SNP variants (V12M and Q141K) toward the drug SN-38 demonstrated that the IC50 value (drug concentrations producing a 50% reduction of cell growth) for Q141K was approximately 50% of that for wild type. The contributions of the minor SNP variants (F208S, S248P, F431L, S441N and F489L) to drug resistance toward SN-38, mitoxantrone, doxorubicin, daunorubicin or etoposide were significantly lower than wild type. Based on our functional validation, the above-mentioned non-synonymous polymorphisms as well as acquired mutants (R482G and R482T) of ABCG2 were classified into four groups. Furthermore, new camptothecin analogs synthesized by our research group had potent effects in circumventing ABCG2-mediated drug resistance without any influence from major non-synonymous polymorphisms.