title: Expression and characterization of a human carboxylesterase 2 splice variant.
abstract: CPT-11 [7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin or Irinotecan] is a carbamate prodrug that is activated in vivo by carboxylesterase (CES)-2 to SN-38 (7-ethyl-10-hydroxycamptothecin), a potent topoisomerase I inhibitor. There is high interindividual variation when CPT-11 is used in the treatment of colorectal cancer. Several splice variants of CES2 are reported in the expressed sequence tag database. Real-time polymerase chain reaction was used to determine the abundance of the CES2 and splice variant of human carboxylesterase 2 (CES2Delta(458-473)) transcripts in 10 paired samples of human tumor and normal colon tissue. The results showed that the CES2Delta(458-473) transcript accounts for an average of 6% of total CES2 transcripts in colon tissue, and there is large interindividual variation in CES2 expression in both tumor and normal colon samples. The carboxylesterase activity of the colon samples was determined by 4-methylumbelliferyl acetate hydrolysis assays and nondenaturing polyacrylamide gel electrophoresis followed by activity staining. Significant, positive correlations were found between CES2 expression levels and both measures of carboxylesterase activity. We cloned and expressed the CES2Delta(458-473) protein in Sf9 insect cells. The purification profiles and preliminary characterization of the CES2Delta(458-473) protein indicated that the expressed protein is folded and glycosylated like CES2. However, in vitro assays show that the CES2Delta(458-473) protein lacks 4-methylumbelliferyl acetate and irinotecan hydrolase activities. In conclusion, we found that the CES2Delta(458-473) protein is an inactive splice variant of CES2 and that its transcript is spliced at a relatively constant rate in tumor and normal colon tissue.
authors.affiliation: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA.
authors.forename: Marissa A
authors.initials: MA
authors.lastname: Schiel
authors.forename: Scheri-lyn
authors.initials: SL
authors.lastname: Green
authors.forename: Wilhelmina I
authors.initials: WI
authors.lastname: Davis
authors.forename: Paresh C
authors.initials: PC
authors.lastname: Sanghani
authors.forename: William F
authors.initials: WF
authors.lastname: Bosron
authors.forename: Sonal P
authors.initials: SP
authors.lastname: Sanghani
mesh_terms: D017398:Alternative Splicing; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology* / Q000627:therapeutic use; D001483:Base Sequence; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000627:therapeutic use; D043182:Carboxylesterase / Q000096:biosynthesis* / Q000235:genetics; D003001:Cloning, Molecular; D003106:Colon / Q000201:enzymology*; D003110:Colonic Neoplasms / Q000188:drug therapy / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D000077146:Irinotecan; D007527:Isoenzymes; D008969:Molecular Sequence Data; D011355:Prodrugs / Q000494:pharmacology* / Q000627:therapeutic use; D020133:Reverse Transcriptase Polymerase Chain Reaction; D059004:Topoisomerase I Inhibitors
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D007527:Isoenzymes; D011355:Prodrugs; D059004:Topoisomerase I Inhibitors; D000077146:Irinotecan; C517204:CES2 protein, human; D043182:Carboxylesterase; D002166:Camptothecin