Abstract
Twelve single nucleotide polymorphisms (SNPs) in the human CES2 gene, which encodes a carboxylesterase, hCE-2 [human carboxylesterase 2 (EC 3.1.1.1)], have been reported in the Japanese [S. R. Kim, T. Nakamura, Y. Saito, K. Sai, T. Nakajima, H. Saito, K. Shirao, H. Minami, A. Ohtsu, T. Yoshida, et al. (2003) Drug Metab Pharmacokinet 18:327–332). In this report, we have examined functional alterations of three SNPs, a nonsynonymous SNP (100C>T, R34W), an SNP at the splice acceptor site in intron 8 (IVS8-2A>G), and one newly discovered nonsynonymous SNP (424G>A, V142M). For the two nonsynonymous SNPs, the corresponding variant cDNAs were expressed in COS-1 cells. Both the R34W and V142M variants showed little esterase activities toward the anticancer agent irinotecan and two typical carboxylesterase substrates, p-nitrophenol acetate and 4-methylumbelliferyl acetate, although increased levels of cDNA-mediated protein expression were observed by Western blotting as compared with the wild type. To investigate a possible splicing aberration in IVS8-2A>G, an in vitro splicing assay was utilized and transcripts derived from CES2 gene fragments of the wild type and IVS8-2A>G were compared. Sequence analysis of the cloned transcripts revealed that IVS8-2A>G yielded mostly aberrantly spliced transcripts, including a deleted exon or a 32-bp deletion proximal to the 5′ end of exon 9, which resulted in truncated hCE-2 proteins. These results suggested that 100C>T (R34W), 424G>A (V142M), and IVS8-2A>G are functionally deficient SNPs.
Footnotes
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T.K. and S.-R.K. contributed equally to this study. This study was supported by the Program for the Promotion of Fundamental Studies in Health Sciences (MPJ-1, -5, and -6) of the Pharmaceuticals and Medical Devices Agency (PMDA) of Japan.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.105.005587.
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ABBREVIATIONS: hCE-2, human carboxylesterase 2 (EC 3.1.1.1); SNP, single nucleotide polymorphism; RT, reverse transcriptase; PCR, polymerase chain reaction; AUC, area under the plasma concentration curve; CES2, the human carboxylesterase 2 gene; SN-38, 7-ethyl-10-hydroxycamptothecin; SN-38G, glucuronide conjugate of SN-38; HPLC, high-performance liquid chromatography; bp, base pair(s).
- Received May 20, 2005.
- Accepted July 13, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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