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METABOLISM, TRANSPORT, AND PHARMACOGENOMICS
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana
Received June 8, 2007; accepted July 16, 2007.
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Abstract |
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458–473) transcripts in 10 paired samples of human tumor and normal colon tissue. The results showed that the CES2458–473 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 CES2458–473 protein in Sf9 insect cells. The purification profiles and preliminary characterization of the CES2458–473 protein indicated that the expressed protein is folded and glycosylated like CES2. However, in vitro assays show that the CES2458–473 protein lacks 4-methylumbelliferyl acetate and irinotecan hydrolase activities. In conclusion, we found that the CES2458–473 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.
; Delord et al., 2005; Reardon et al., 2005; Stathopoulos et al., 2005). Metabolism of CPT-11 is complex with the involvement of several enzymatic reactions (Rivory, 2000; Slatter et al., 2000). CYP3A4-dependent oxidation of CPT-11 results in the formation of APC and NPC (Haaz et al., 1998; Sai et al., 2001); carboxylesterase-dependent hydrolysis of CPT-11 and NPC results in the formation of the active metabolite SN-38 (Humerickhouse et al., 2000; Khanna et al., 2000; Sanghani et al., 2004) and UGT1A1 glucuronidates SN-38 to form the inactive SN-38-glucuronide (Hanioka et al., 2001). The key step in the metabolism of CPT-11 is its activation by carboxylesterases. There are three known human carboxylesterases, CES1, CES2, and CES3. CES2 has 100- and 2000-fold greater catalytic efficiency for irinotecan hydrolysis than CES1 and CES3, respectively (Sanghani et al., 2004). Therefore, CES2, which is predominantly expressed in the liver, small intestines, and colon (Satoh et al., 2002), is the most important carboxylesterase for the activation of CPT-11. Clinical data, reported by Xu et al. (2002), indicate that CPT-11 is not effective therapy for lymphomas and gallbladder tumors that do not express CES2. Therefore, understanding the expression of CES2 and its variants is vital for the effective use of CPT-11.
A major problem associated with CPT-11 therapy is high interindividual variation both in patient response and in plasma SN-38 levels (Canal et al., 1996; Lokiec et al., 1996; Gupta et al., 1997; Rivory et al., 1997; Couteau et al., 2000). One possible explanation for this is variable expression of CES2. Our study in 24 colon tumors found a 23-fold variation in CES2 expression (Sanghani et al., 2003). Hosokawa et al. (1995) have reported high interindividual variation in the specific activities of 12 human liver carboxylesterase samples, ranging from 5 to 45-fold with respect to 10 different substrates. Likewise, a 15- and 3-fold variation in the expression of CES2 has been reported in cytosolic and microsomal fractions of 13 liver samples (Xu et al., 2002). Large interpatient variability in plasma levels of SN-38 could be indicative of genetic polymorphism. However, pharmacogenomics studies for CES2 have not come to a consensus on the association of SNPs with CES2 expression or activity (Wu et al., 2003; Charasson et al., 2004; Marsh et al., 2004; Kubo et al., 2005).
Our recent analysis of the full-length expressed sequence tags (ESTs) for CES2 revealed that alternate splicing could give rise to multiple CES2 proteins. The genomic locus of the CES2 gene contains 12 exons (Fig. 1). The complexity of the CES2 gene arises from the presence of two in-frame ATGs (ATG-1 and ATG-2) in exon 1 (Wu et al., 2003) and alternative splicing in exons 1 and 10. Combination of in-frame ATGs and alternative splicing can give rise to six possible CES2 variant proteins. The two proteins that are the focus of this report are shown in Fig. 1. The normal CES2 protein is generated from translation starting at ATG-2 (boxed in Fig. 1) and has an N-terminal endoplasmic reticulum signal peptide and a C-terminal endoplasmic reticulum retention signal. Alternative splicing in exon 10 (marked by a star in Fig. 1) removes the last 48 nucleotides of that exon from the final transcript. The resulting CES2458–473 protein lacks the 16 amino acids directly following the active site histidine. Currently, there is no information regarding the activity, expression, or function of the CES2458–473 variant. In this study, we report the cloning, expression, and irinotecan hydrolase activity of CES2458–473. We also report the expression of CES2 and CES2458–473 in colon adenocarcinomas with paired normal colon tissue.
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Materials and Methods |
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RNA Isolation and Reverse Transcription. Tissues, 
5 to 30 mg, were disrupted in RLT buffer (QIAGEN, Valencia, CA) using a disposable mortar and pestle (Kontes, Vineland, NJ). Disruption was followed by homogenization via Qiashreddar columns (QIAGEN). Total RNA was isolated using the RNeasy Plus Mini Kit (QIAGEN). RNA was quantified using a ND-1000 Spectrophotometer (Nanodrop, Wilmington, DE). RNA quality was assessed by running 500 ng of total RNA on a 1% agarose gel. The GeneAmp Gold RNA PCR kit (Applied Biosystems, Foster City, CA) was used for reverse transcription with 1 µg of total RNA added to a 50-µl reaction. The reaction components were 2.5 mM MgCl2, 250 µM of each deoxynucleotide triphosphate, 1.25 µM oligodeoxythymidylic acid primer, 0.5 U/µl RNase inhibitor, and 0.75 U/µl MultiScribe reverse transcriptase. Reaction conditions were described previously (Sanghani et al., 2003).
Real-Time PCR. We modified our real-time PCR protocol from that previously published (Sanghani et al., 2003) to distinguish between the two CES2 variants. Variant-specific forward primers were designed for CES2,5'-CCATGGTGATGAGCTTCCTTTTGT-3', and CES2458–473,5'-AGGCAGACCATGTTAAATTCACTGA-3'. The reverse primer 5'-AGGTATTGCTCCTCCTGGTCGAA-3' was common to both transcripts. The expected lengths of the PCR products were 186 and 145 bp for CES2 and CES2458–473, respectively. The PCR conditions were 3 mM Mg2+, 0.5 µM of each primer, and 0.2 mM of deoxynucleotide triphosphates using the SYBR Green kit (Applied Biosystems). The cDNA equivalent to 20 ng of RNA was added to each 25-µl PCR reaction. PCR cycling conditions were 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 30 s, 65°C for 30 s, and 72°C for 1 min on an Eppendorf Mastercycler EP instrument (Eppendorf, Westbury, NY). The uniformity of the PCR products was determined using the melt curve function. A standard curve for each gene was generated using clones that were constructed in our laboratory. The copy numbers of each variant present in the tissues were determined by comparison with the appropriate standard curve. All cDNA samples were analyzed in triplicate for each transcript.
Tissue Lysate Preparation. Approximately 10 to 60 mg of colon tissue was homogenized in 100 to 200 µl of 25 mM KH2PO4, pH 7.5, plus 0.05% Triton X-100, leupeptin (10 µg/ml), pepstatin (1 µg/ml), and DNase (0.5 µg/ml) using motorized pestles with microfuge tubes (Kontes). The lysates were centrifuged at 40,000g for 15 to 20 min at 4°C. The supernatant was collected for use in activity assays. Protein content was quantified via the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA) with bovine serum albumin used for a standard curve.
Cloning of the CES2458–473 Variant. A partial EST clone for CES2458–473 was obtained from American Type Culture Collection (Image Clone identification no. 6195662; American Type Culture Collection, Manassas, VA). The EST clone was digested with XbaI and SmaI restriction enzymes, and the 0.65-kb fragment was gel purified (QIAGEN) for ligation. A CES2 clone in the pVL1392 baculovirus transfer vector was subjected to complete digestion with XbaI and partial digestion with SmaI. The digested vector was treated with 1 µl of calf intestine phosphatase (NEB, Ipswich, MA) for 1 h at 37°C and then purified using a QIAGEN PCR purification kit. The 0.65-kb CES2458–473 insert was ligated into the vector using T4 DNA ligase (Fastlink; Epicenter, Madison, WI). The clones were analyzed by restriction digest, and two clones with the correct digestion patterns were isolated. Sequencing confirmed the presence of the 48-bp deletion.
Purification of the CES2458–473 Protein Expressed in Insect Cells. The CES2458–473 protein was expressed using the baculovirus system (BD Biosciences PharMingen, San Diego, CA) as reported previously (Sanghani et al., 2004; Sun et al., 2004). The CES2458–473-pVL1392 vector was cotransfected with linearized Baculogold DNA (BD Biosciences PharMingen) into 2.5 x 106 Sf9 cells following the manufacturer's protocol. The CES2458–473 recombinant virus was collected on day 8 post-transfection. The recombinant virus was amplified and used for protein expression. An 800-ml culture of Sf9 insect cells was infected with the CES2458–473 recombinant virus. The cells were collected 70 h postinfection by centrifuging at 500g for 5 min. The cell pellet was sonicated in 30 ml of 20 mM Tris buffer, pH 7.4, containing 1 mM benzamidine, 1 mM dithiothreitol, 1 µM leupeptin, and 0.1% Triton X-100. The lysate was ultracentrifuged at 100,000g for 45 min. The recombinant CES2458–473 variant was purified by a two-step purification protocol (Sanghani et al., 2004) using concanavalin A affinity chromatography and preparative nondenaturing PAGE (Sun et al., 2004). CES activity and protein concentrations were monitored in the eluted fractions. The protein elution profile for the CES2458–473 variant was compared with that for CES2. A protein peak was detected at the same position as CES2; however, it did not have any activity for the substrate 4-methylumbelliferyl acetate. Fractions from this peak were concentrated and loaded onto an 8.5-cm 6% nondenaturing preparative polyacrylamide gel as described earlier (Sun et al., 2004). Once again, a protein peak was identified that eluted similarly to CES2. The eluted fractions representing the protein peak were pooled and concentrated.
Characterization of the CES2458–473 Protein. Two micrograms each of purified CES2 and CES2458–473 proteins were separated by SDS-PAGE followed by Western blotting analysis. Antibodies were raised in rabbit to the deglycosylated human CES2 protein. The antibody was purified from 8 ml of serum using the DEAE Affi-Gel Blue (Bio-Rad) according to the manufacturer's protocol. The purified anti-CES2 antibody was diluted 5000x, and the secondary antibody was horseradish peroxidase-conjugated. SDS-PAGE and Western blotting identified a protein band of approximately 60 kDa. Matrix-assisted laser desorption ionization/time of flight and liquid chromatography/electrospray ionization quadruple time-of-flight analysis were used to further confirm the identity of the 60-kDa band.
Circular dichroism spectra for the proteins were determined using a J-720 spectropolarimeter (Jasco, Easton, MD). Both the CES2 and CES2458–473 proteins were buffer-exchanged and equilibrated in 5 mM potassium phosphate, pH 7.5, containing 10% ethylene glycol. A final concentration of 0.22 mg/ml of each protein was used for analysis. Each spectrum was an average of three scans using a 0.1-cm quartz cell. Spectra were recorded in the 250 to 190-nm range at a speed of 100 nm/min and with a resolution of 0.2 nm. The mean residue molar ellipticity [
]MRW was calculated using eq. 1:
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is in millidegree, n is the number of amino acid residues in the protein, Cp is the molar concentration of the protein (in moles per liter), and l is the cuvette path length in centimeters. For secondary structure analysis, the ellipticity was expressed as 
MRW in molar per centimeter units using eq. 2. The spectra were then analyzed using the CDPro software developed by Sreerama and Woody (1993, 2004). An extended protein basis set of 43 soluble and 13 membrane proteins was used for analysis. Of the three methods for calculating the secondary structure composition of the protein in the CDPro software, CONTIN/LL produced the most reliable results (Provencher and Glockner, 1981).
Carboxylesterase Activity Assays. The hydrolysis of 4-methylumbelliferyl acetate was monitored by a spectrophotometric assay described earlier (Brzezinski et al., 1997; Sanghani et al., 2003). CES2 band density was determined by analyzing the samples on a nondenaturing PAGE and staining for carboxylesterase activity using 4-MUA as described earlier (Dean et al., 1995; Sanghani et al., 2003).
CPT-11 Hydrolysis Assay. The CPT-11 hydrolase activities of the purified CES2 and CES2458–473 proteins were evaluated as reported earlier (Sanghani et al., 2004). In 250-µl reactions, 55 µM CPT-11 was incubated with varying concentrations of CES2 (0–30 µg) and CES2458–473 (0–50 µg) in HEPES buffer, pH 7, at 37°C for 30 min and 2 h, respectively. The reaction was stopped by the addition of 670 µl of 0.1 M HCl. Fifteen microliters of 0.14 mM camptothecin was added as internal standard. Samples were extracted using solid-phase columns (Oasis HLB column; Waters, Milford, MA), processed, and analyzed as described earlier (Sanghani et al., 2004). The standard curve samples, 0 to 5 µM SN-38, were processed in a similar fashion. The ratios of area under the curve for SN-38 and camptothecin were plotted against concentration (GraphPad Prism 4.0, San Diego, CA) and the unknown concentrations were calculated from the standard curve.
Statistical Analysis. Skewness, kurtosis, and overall normality of copy number, 4-MUA hydrolase activity, and band density data were assessed using the methods of D'Agostino et al. (1990). The copy numbers for CES2 were independently subjected to linear regression analysis against both 4-MUA hydrolase activity and band density using JMP 4.0 (SAS Institute Inc., Cary, NC). The data are considered significant if p < 0.05.
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Results |
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; Khanna et al., 2000; Sanghani et al., 2004). A variant DNA sequence for CES2 was reported in the EST database. This variant transcript has a 48-bp deletion (Fig. 2A) that gives rise to a protein with a 16-amino acid deletion immediately following the active site His457 residue (Fig. 2B). Use of the SPIDEY software (http://www.ncbi.nlm.nih.gov/IEB/Research/Ostell/Spidey/index.html) to align the CES2 (GI:37622884) and CES2458–473 (GI:37622886) mRNA transcripts with the CES2 genomic clone (GI:56788327) demonstrates that the exon 10 sequence contains good 5' splice sites for both of the transcripts. Hence, the two transcripts are created by the use of alternate 5' donor splice sites in exon 10. This evidence is further supported by the identification of 14 EST sequences in the human EST database as determined by blasting the database with a 25-nucleotide sequence unique to CES2458–473 (shown in bold in Fig. 2A).
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458–473 Expression in Human Colon Samples. Real-time PCR was used to quantify the expression levels of the CES2 and CES2458–473 transcripts in 10 paired tumor and normal colon samples. Forward primers (shown in bold in Fig. 2A) were designed to be variant-specific for CES2 and CES2458–473. Analysis of RNA quality by agarose gel electrophoresis showed little to no degradation. Standard curves were created for each variant using DNA clones constructed in our lab. Amplification of each standard curve was linear over a 100,000-fold range (r2 = 0.99) with respect to copy numbers. The amplification efficiency for each of the variants was 
0.9. All cDNA samples were analyzed in triplicate with the S.D., generally <12% for both CES2 and CES2458–473.
The repeatability of all the steps involved in real-time PCR was studied in control experiments. In one experiment, RNA was isolated from five different pieces of the same tumor sample. It was determined that <1.6-fold variation in the CES2 copy numbers could be attributed to the entire method from tissue disruption through real-time PCR. In another experiment, one sample RNA was reverse transcribed in quintuplicate. It was determined that <1.3-fold variation in copy number could be attributed to reverse transcription.
CES2 and CES2458–473 were found in all of the tumor and normal colon samples. There was a 45-fold variation in expression of CES2 transcript in normal tissue and a 27-fold variation in tumor tissue. Four patients expressed more CES2 in tumor as compared with normal tissue (Fig. 3A). No significant linear correlation (r2 = 0.025) was found in CES2 transcript expression between the paired tumor and normal tissue samples. CES2458–473 varied by 34-fold in normal tissue and 25-fold in the tumor tissues. The percentage of CES2458–473 transcript in colon tissue accounts for approximately 6% (S.D. < 1.3%) of the total CES2 transcripts (Fig. 3B).
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Carboxylesterase Activity in Colon Tissue Lysates. The carboxylesterase activities of the colon tissue lysates were quantified using the 4-methylumbelliferyl acetate hydrolysis assay and band densitometry analysis as described under Materials and Methods. All samples showed nonspecific carboxylesterase activity with 4-MUA as substrate (Table 1). The range for normal samples was 2.5 to 9.8 µmol mg–1 h–1 with a 4-fold variation, whereas the range for the tumor samples was 2.0 to 7.4 µmol mg–1 h–1 with a 3.7-fold variation. Nondenaturing PAGE was used to separate the carboxylesterases in the samples, and purified CES2 protein was loaded on each gel as a control. The band density attributable to CES2 was determined for each sample. All samples demonstrated activity for 4-MUA in the nondenaturing PAGE assay (Table 1). There was a 23-fold variation in normalized band density among the normal colon samples and an 8-fold variation among the tumor samples.
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Statistical Analysis. There was evidence of kurtosis (data not shown) for both the CES2 copy numbers (p = 0.03) and band densities (p = 0.04) of the tumor samples. Kurtosis is often an indication of bimodality (Darlington, 2007). All other measurements in tumor and all measurements in normal tissue appeared to be normally distributed. CES2 copy numbers, determined by real-time PCR, were subjected to linear regression analysis (JMP 4.0) with 4-MUA hydrolase activity and normalized band density (Fig. 4). Correlations of CES2 expression with 4-MUA hydrolase activity and band density are considered significant if p 
0.05 (Takahashi et al., 2000). CES2 copy numbers, in tumor samples, positively and significantly (p 0.0001) correlated with both 4-MUA hydrolase activity (r2 = 0.857) and band density (r2 = 0.982). For the normal samples, CES2 copy numbers significantly correlated with 4-MUA hydrolase activity (p < 0.0436), but correlation with band density (p < 0.0546) was just outside the range of significance. Data from the normal and tumor samples were analyzed collectively, and significant correlation existed between CES2 copy numbers and both 4-MUA (p < 0.0001) and band density (p < 0.001).
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458–473 Protein Variant. The CES2458–473 variant was expressed in Sf9 insect cells using a recombinant baculovirus containing the CES2458–473 cDNA. The recombinant CES2458–473 virus was confirmed by PCR. A two-step purification protocol comprised of concanavalin A chromatography and preparative nondenaturing gel electrophoresis was used to purify CES2458–473 to near homogeneity. Due to its lack of 4-MUA hydrolase activity, during each purification step, the protein elution profile of CES2458–473 was compared with CES2 to identify the protein peak of interest. The protein elution profile for CES2458–473 was similar to CES2 for both purification steps.
Characterization of the CES2458–473 Protein. The purified CES2 and CES2458–473 proteins were analyzed by SDS-PAGE (Fig. 5A), Western blot analysis (Fig. 5B), nondenaturing polyacrylamide gel electrophoresis (Fig. 5, C and D), circular dichroism (Fig. 5E), and mass spectroscopy to assess their identity, purity, and physical properties. The calculated molecular mass of CES2458–473 protein from the SDS-PAGE gel (Fig. 5A) is 59 kDa, and the expected molecular mass is 57.3 kDa before glycosylation. In the Western blot analysis, both CES2 and CES2458–473 were identified with an anti-CES2 antibody (Fig. 5B). Activity staining of the nondenaturing polyacrylamide gel showed that the CES2458–473 protein lacks carboxylesterase activity for 4-MUA (Fig. 5C). Coomassie Blue stain of the same gel shows the protein in the CES2458–473 lane (lane 2, Fig. 5D) migrating to the same place as CES2 (lane 1, Fig. 5D). The identity of the recombinant CES2458–473 protein was confirmed by mass spectroscopy, where a tryptic peptide with one miscleavage, ADHVKFTEEEEQLSR, specific for CES2458–473 (underlined in Fig. 2B) was identified. Folding of CES2458–473 protein was evaluated by circular dichroism. The mean residue molar ellipticity was plotted as a function of wavelength (Fig. 5E). The secondary structure composition analysis was done with CDpro software. Results from the CONTIN/LL method showed that the compositions of CES2 and CES2458–473 were 15% 
-helical, 34% 
-strand, 22% turn, and 29% unordered and 20% -helix, 29% -strand, 22% turn, and 29% unordered, respectively.
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458–473 protein (0–50 µg) was incubated with 55 µM CPT-11 for 2 h to determine its CPT-11 hydrolase activity. Under these conditions, no significant SN-38 (< 5 nM) peak was detected in any of the CES2458–473 samples after 2 h. In comparison, 1.98 µM SN-38 was detected when CES2 (30 µg) was incubated with the same concentration of CPT-11 for just 30 min (Fig. 6).
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Discussion |
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; Sanghani et al., 2004). Irinotecan has shown to be ineffective therapy for lymphomas and gallbladder tumors that lack CES2 expression, suggesting that local expression of CES2 in tumors may influence treatment outcome (Xu et al., 2002). CES2 SNPs have been identified, but there is no consensus as to the effect of the SNPs on expression and activity (Wu et al., 2003; Charasson et al., 2004; Marsh et al., 2004; Kubo et al., 2005). Analysis of CES2 transcripts in the EST database reveals that combinations of alternate ATG start sites and two splicing events could lead to six potential proteins. A 25-nucleotide sequence (shown in bold in Fig. 2A) unique to the CES2458–473 transcript was identified in 14 ESTs. The transcript is missing the final 48 nucleotides from exon 10, and the resulting protein CES2458–473 lacks the 16 amino acid residues directly following the active site histidine. The proximity of the deleted amino acids to the active site suggested that CES2458–473 may differ in activity from CES2.
The expression levels of the CES2458–473 and CES2 transcripts were analyzed in tumor and normal colon tissue pairs using variant specific primers and real-time PCR. Primers were able to distinguish between alternate splicing events in exon 10 but could not detect any splicing events occurring in exon 1. Thus, copy numbers for CES2458–473 and CES2 represented the total transcript levels differentiating only between normal and alternate splicing in exon 10, with no regard for alternative splicing in exon 1. Both the CES2458–473 and CES2 variants were present in all 20 tissue samples, and CES2458–473 accounted for approximately 6% of the total CES2 transcript, suggesting that it was being spliced at a constant rate. There was large interindividual variation in CES2 expression among the tumor tissue samples and the normal colon samples, 27- and 45-fold, respectively. There was no significant difference in CES2 expression between tumor and normal colon samples, which supports the findings of our previous report (Sanghani et al., 2003). In six paired samples, CES2 expression was greater in the normal tissues, whereas the other four pairs have higher expression in tumor. This variability suggests that interindividual variation in expression of CES2 could account for differences in patient response to irinotecan therapy. CES2 expression levels, especially in tumor tissue, may dictate whether or not irinotecan therapy is successful, whereas CES2 expression levels in normal tissue may contribute to the severity of deleterious side effects.
CES2 expression levels were subjected to linear regression analysis with two different measures of carboxylesterase activity, 4-MUA hydrolysis and band density. Unlike our previous study (Sanghani et al., 2003), variant-specific primers were used to quantify the transcript for CES2 separately from the transcript for CES2458–473. CES2 expression in the colon tumor samples had significant positive correlation with 4-MUA hydrolase activity and band density. These findings are in concordance with the data previously published by Sanghani et al. (2003), although the correlation coefficients are increased. These increased correlations may be attributable to the use of variant specific primers as well as improved methods. CES2 expression in normal tissue was also found to have significant, positive correlation with 4-MUA hydrolase activity and positive correlation with band density (p 0.055). Correlation values between expression and activity for the tumor samples were greater than the corresponding correlation values in normal samples. It is possible that the tumor samples are more homogenous in cell type than the normal samples, which may include muscular tissue in addition to mucosal tissue.
If interindividual variation in response to CPT-11 treatment may be explained by variations in CES2 expression, it is important to examine the properties of the CES2458–473 variant. CES2458–473 was cloned and expressed to characterize its activity and physical properties. When purifying the CES2458–473 variant protein, the protein elution profile of CES2 was used as a guide. The CES2458–473 protein had an elution profile identical to CES2 for purification on concanavalin A and preparative nondenaturing PAGE. Binding of the CES2458–473 protein to concanavalin A resin indicates that it is glycosylated. CES2 and CES2458–473 proteins migrated similarly on nondenaturing PAGE (Fig. 5D). When incubated with 4-MUA, a substrate for which CES2 has high activity with a reported Kcat/Km value of 60,000 mM–1 min–1 (Pindel et al., 1997), CES2458–473 demonstrated no activity.
The lack of esterase activity demonstrated by the CES2458–473 protein raised questions regarding its folding. The circular dichroism spectrum for CES2458–473 (Fig. 5E) shows the presence of a secondary structure similar to that of CES2. The small increase in -helical content and small decrease in -strand content of CES2458–473 in comparison with CES2 could be expected from the 16-amino acid deletion. The identities of the recombinant proteins were further confirmed by mass spectroscopy. Although the CES2458–473 protein lacked 4-MUA hydrolase activity, it was feasible that it retained activity for other substrates. The CPT-11 hydrolase activity of the CES2458–473 protein was evaluated. Even at the highest concentration of CES2458–473 protein, no significant amount of SN-38 (<5 nM) was detected. Based on these results, we concluded that the CES2458–473 protein lacked irinotecan hydrolase activity.
Alternative splicing is emerging as a major mechanism for proteome diversity and differential splicing occurs in disease specific manner, especially in cancer (Garcia-Blanco et al., 2004; Venables, 2004, 2006). The role of CES2 splice variants in ester drug metabolism has not been evaluated. We show that the CES2458–473 variant is expressed at a low and constant level of 6% of total CES2. The low expression and lack of activity of CES2458–473 suggest that it is not a determinant for CPT-11 pharmacokinetics and therapeutic outcome. However, if any of the remaining variants are found to have significant expression levels or activities that differ from CES2, it may further the understanding of the interindividual variation in ester drug metabolism.
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Acknowledgements |
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Footnotes |
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1 CES2 is the normal form that has been extensively studied. CES2458–473 is a splice variant of CES2 that has a 48-bp deletion in exon 10 and results in formation of a protein with a 16-amino acid deletion from 458 to 473. For the sake of simplicity, the transcript and the protein are called by the same name in the article; however, the transcript is shown in italics.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: CPT-11, 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin or Irinotecan; APC, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxycamptothecin; NPC, 7-ethyl-10-[4-(1-piperidino)-1-amino]-carbonyloxycamptothecin; SN-38, 7-ethyl-10-hydroxycamptothecin; CES, carboxylesterase; EST, expressed sequence tag; CES2 (GI:37622884), human carboxylesterase 2; CES2458–473 (GI:37622886), splice variant of human carboxylesterase 2; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; 4-MUA, 4-methylumbelliferyl acetate; ATG, translation initiation codon.
Address correspondence to: Sonal P. Sanghani, 1345 West 16th Street, Indianapolis, IN 46202. E-mail: ssanghan{at}iupui.edu
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) removes 48 nucleotides from the message. Translation initiation at ATG-2 and alternative splicing in exon 10 yields two proteins, normal CES2 and CES2
) and a C-terminal ER retention signal (). Positions of the catalytic residues serine (S), glutamic acid (E), and histidine (H) and glycosylation sites (GLY) are marked.
