Materials and Methods

Cells and Culture Conditions.

The 72 established cell lines used in the present study, which originated from different Japanese individuals, were purchased from the Health Science Research Resource Bank (Osaka, Japan) or the Japanese Collection of Research Bioresources (National Institute of Health Sciences, Tokyo, Japan). All of the cells originated from various tissues and were cultured according to the suppliers' instructions.

Sequence analyses of cMOAT/MRP2/ABCC2 Gene.

We sequenced approximately 800 base pairs of the 5′-upstream region from the translation initiation codon and all of the 32 exons of the cMOAT/MRP2/ABCC2 gene after performing a single polymerase chain amplification reaction using multiplex primers, which are essentially described by Toh et al. (1999). DNA that was extracted from the cells was used as a template. The amplification and sequencing were done as previously described (Nakamura et al., 2001).

Results and Discussion

We sequenced the 800 base pairs of the 5′-upstream region and all the exonic regions of the cMOAT/MRP2/ABCC2 gene. As for the nucleotide position, the adenine of the translation initiation codon, ATG, was set as the nucleotide position 1. Frequently appearing nucleotide variations (c-24t, g1249a, and c3972t) were consistently observed between the present study and the article by Ito et al. (2001). Three additional polymorphic sites were found in the 5′-upstream region, and 19 variations that were not reported by Ito et al. (2001) (Table 1) were detected in the exons. Of those, 12 nucleotide changes were nonsynonymous, and the frequency for c1457t (T486I) was approximately 0.03 (Table 1). In exon 8, a deletion of seven adenines was observed resulting in a frameshift. A strong association was observed between the c-24t and c3972t substitutions. The statistical analyses gave a χ² value of 106.6, with degrees of freedom of 4 and a p value less than 0.0001 using the program Prism 3.0 (GraphPad Software, San Diego, CA).

The amino acid substitutions S789F (c2366t) and S1367C (c4100g) were reportedly located in nucleotide binding domains 1 and 2, respectively, those of V417I (g1249a) and T486I (c1457t) in membrane-spanning domain 2, and that of Q1019H (g3057t) in membrane spanning domain 3 (Toh et al., 1999). Basic amino acids that have a key functional role in drug transport have been identified for human MRP2 (Ryu et al., 2000). None of the amino acid alterations in the present study (Table 1) were found to be located within the Walker A, B, and C motifs or to be the basic amino acids in the transmembrane domains (TMs). It was strongly suggested that functional changes by the V417I, S789F, D883N, Q1019H, N1244K, S1367C, and C1515Y alterations seemed to be worth assessing because the TM1 to TM5 domains (200 N-terminal amino acids) were dispensable for transportation of substrates (Ryu et al., 2000). In the SNP database of National Center for Biotechnology Information, c-24t, c56t (P19L), and g4290t were registered with accession numbers of rs717620, rs927344, and rs1137968, respectively, indicating the usefulness of searching for possible novel SNPs from established cell lines.

In the present study, we have found a number of novel SNPs, including nonsynonymous ones in the cMOAT/MRP2/ABCC2 gene of 72 cell lines of Japanese subjects. The c-24t associated with c3972t may be of importance in the regulation of the cMOAT/MRP2/ABCC2 gene.