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Clinical Cancer Research Vol. 12, 1794-1803, March 2006
© 2006 American Association for Cancer Research

Cancer Therapy: Preclinical

Gemcitabine Pharmacogenomics: Cytidine Deaminase and Deoxycytidylate Deaminase Gene Resequencing and Functional Genomics

Judith A. Gilbert1, Oreste E. Salavaggione1, Yuan Ji1, Linda L. Pelleymounter1, Bruce W. Eckloff2, Eric D. Wieben2, Matthew M. Ames1 and Richard M. Weinshilboum1

Authors' Affiliations: Departments of 1 Molecular Pharmacology and Experimental Therapeutics and 2 Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota

Requests for reprints: Richard M. Weinshilboum, Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic College of Medicine, 200 First Street Southwest, Rochester, MN 55905. Phone: 507-284-2246; Fax: 507-284-9111; E-mail: Weinshilboum.Richard{at}mayo.edu.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Gemcitabine is a nucleoside analogue with activity against solid tumors. Gemcitabine metabolic inactivation is catalyzed by cytidine deaminase (CDA) or, after phosphorylation, by deoxycytidylate deaminase (DCTD). We set out to study the pharmacogenomics of CDA and DCTD.

Experimental Design: The genes encoding CDA and DCTD were resequenced using DNA from 60 African American and 60 Caucasian American subjects. Expression constructs were created for nonsynonymous coding single nucleotide polymorphisms (cSNP) and reporter gene constructs were created for 5'-flanking region polymorphisms. Functional genomic studies were then conducted after the transfection of mammalian cells.

Results: CDA resequencing revealed 17 polymorphisms, including one common nonsynonymous cSNP, 79 A>C (Lys27Gln). Recombinant Gln27 CDA had 66 ± 5.1% (mean ± SE) of the wild-type (WT) activity for gemcitabine but without a significant decrease in level of immunoreactive protein. The apparent Km (397 ± 40 µmol/L) for the Gln27 allozyme was significantly higher than that for the WT (289 ± 20 µmol/L; P < 0.025). CDA 5'-flanking region reporter gene studies showed significant differences among 5'-flanking region haplotypes in their ability to drive transcription. There were 29 SNPs in DCTD, including one nonsynonymous cSNP, 172 A>G (Asn58Asp), in Caucasian American DNA. Recombinant Asp58 DCTD had 11 ± 1.4% of WT activity for gemcitabine monophosphate with a significantly elevated level of immunoreactive protein. No DCTD polymorphisms were observed in the initial 500 bp of the 5'-flanking region.

Conclusions: These results suggest that pharmacogenomic variation in the deamination of gemcitabine and its monophosphate might contribute to variation in therapeutic response to this antineoplastic agent.

Gemcitabine (2',2'-difluorodeoxycytidine) is a deoxycytidine analogue with antineoplastic activity against solid tumors (1). The mechanism of action of gemcitabine parallels that of 1-ß-D-arabinofuranosylcytosine and is thought to require the transport of gemcitabine into cells, followed by metabolic activation to form a triphosphate that is both incorporated into DNA and competitively inhibits DNA synthesis (1). The reactions involved in metabolic activation are depicted graphically in Fig. 1 , which also shows gemcitabine inactivation by deamination catalyzed by cytidine deaminase (CDA) as well as the deamination of gemcitabine monophosphate, which is catalyzed by deoxycytidylate deaminase (DCTD). In the present study, we set out to study gemcitabine pharmacogenomics by applying a "genotype-to-phenotype" strategy to CDA and DCTD. That process began by resequencing the genes encoding these two proteins using DNA samples from subjects of differing ethnicity, followed by the creation of expression constructs for all nonsynonymous coding single nucleotide polymorphisms (cSNP) observed and reporter gene constructs for 5'-flanking region polymorphisms. Functional genomic studies were then conducted by transfecting mammalian cells with those constructs.


Figure 1
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  		Fig. 1. Gemcitabine biotransformation. DCK, deoxycytidine kinase; UMP-CMPK, deoxycytidylate kinase; dFdC, gemcitabine; dFdCMP, gemcitabine monophosphate; dFdCDP, gemcitabine diphosphate; dFdCTP, gemcitabine triphosphate; dFdU, 2',2'-difluorodeoxyuridine; and dFdUMP, 2',2'-difluorodeoxyuridine monophosphate.

 
We observed 17 CDA polymorphisms, including one common nonsynonymous cSNP that was present in DNA from both African American and Caucasian American subjects and resulted in a Lys27Gln change in the encoded amino acid sequence. This polymorphism had previously been seen (2–5) and was reported to result in either no change in activity with 1-ß-D-arabinofuranosylcytosine (ara-C) as substrate (5) or up to a 3-fold decrease in activity (4). In our hands, transfection of mammalian cells with a Gln27 variant allozyme expression construct resulted in decreased activity relative to wild-type (WT) enzyme with gemcitabine as substrate but without a significant change in the quantity of immunoreactive protein. Furthermore, the variant allozyme had a significantly higher apparent Km value for gemcitabine than did the WT allozyme. In addition, reporter gene experiments included in the present studies indicated that common polymorphisms present in the 5'-flanking region of CDA might influence transcription. Resequencing of DCTD showed 29 polymorphisms, including one nonsynonymous cSNP. Functional studies of the Asp58 DCTD variant allozyme showed little basal activity but had elevated levels of immunoreactive protein when compared with WT DCTD. Taken together, these observations suggest that inherited variation in the deamination of gemcitabine and its monophosphate might contribute to individual variation in the therapeutic efficacy and/or toxicity of this increasingly important antineoplastic drug.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
DNA samples. DNA from 60 African American and 60 Caucasian American subjects (sample sets HD100AA and HD100CAU) was obtained from the Coriell Cell Repository (Camden, NJ). These samples had been deposited by the National Institute of General Medical Sciences and all subjects had provided written consent for the use of their DNA for research purposes. Further information with regard to these anonymized samples can be obtained from the Coriell Cell Repository. The present studies were reviewed and approved by the Mayo Clinic Institutional Review Board.

CDA and DCTD resequencing. All CDA and DCTD exons and splice junctions, as well as a portion of the 5'-flanking regions for each gene, were amplified by use of the PCR. Primers were designed to hybridize within introns, in the 5'-flanking regions or in the 3'-untranslated regions (UTR), of the terminal exon at locations selected to avoid repetitive sequence. All primer sequences are listed in Supplementary data. Amplification reactions were done with AmpliTaq Gold DNA polymerase (Perkin-Elmer, Foster City, CA) with a "hot start." Because of the high GC content of DCTD exon 1, PCR reactions for that exon used as template a long PCR product that included ~500 bp of 5'-flanking region, plus exon 1 and intron 1. All PCR amplifications were done in a Perkin-Elmer model 9700 thermal cycler. Amplicons were sequenced in the Mayo Molecular Core Facility on both strands with an ABI 3700 DNA sequencer using BigDye (Perkin-Elmer) dye primer-sequencing chemistry (6). To exclude the possibility of PCR-induced artifacts, independent amplifications, followed by sequencing, were done for any SNP observed in only a single DNA sample or for any sample with an ambiguous chromatogram. The sequencing chromatograms were analyzed using the PolyPhred 3.0 (7), Consed 8.0 (8), and Mutation Surveyor 2.41 (9) programs. The University of Wisconsin GCG software package, version 10, was also used to analyze nucleotide sequence. NT_004610 and NM_001785 were the GenBank accession numbers for the CDA reference sequences used in these experiments, and NT_022792 and NM_001921 were the reference sequences for DCTD.

CDA and DCTD expression constructs and transient expression. Site-directed mutagenesis done with the QuikChange kit (Stratagene, La Jolla, CA) was used to create variant expression constructs or, in the case of CDA, the WT construct because the Gln27 variant was amplified initially. Sequences of the primers used for site-directed mutagenesis are also listed in Supplementary data. The sequences of inserts were confirmed by sequencing after cloning into the eukaryotic expression vector pCR3.1 (Invitrogen, Carlsbad, CA). Expression constructs for the WT and variant CDA allozyme cDNAs, as well as "empty" pCR3.1 that lacked an insert, were transfected into COS-1 cells in serum-free DMEM (BioWhittaker, Walkersville, MD) using the TransFast reagent (Promega, Madison, WI) at a charge ratio of 1:1. The same approach was used with the DCTD constructs except for the fact that those constructs were transfected into Chinese hamster ovary K1 cells because these cells had negligible DCTD and deoxycytidylate kinase background levels of activity. Specifically, 7 µg of construct DNA were cotransfected with 7 µg of pSV-ß-galactosidase DNA (Promega) as a control to make it possible to correct for transfection efficiency. The cells were harvested, homogenized, and cytosol preparations were made as described elsewhere (10).

CDA reporter gene constructs. Luciferase reporter gene constructs were created to study the possible effects of 5'-flanking region polymorphisms on the regulation of CDA transcription. Specifically, the PCR was used to amplify DNA segments from samples with known 5'-flanking region haplotypes, and those amplicons were used to create the constructs. Forward and reverse primers included MluI and XhoI restriction sites at their 5'-ends, respectively. Amplification products were digested overnight with these restriction enzymes, followed by purification with the QIAquick PCR Purification Kit (Qiagen, Valencia, CA). The digested products were then cloned into pGL3-basic (Promega), 5' of the firefly luciferase open reading frame. Each insert was sequenced, and these constructs were used to transfect HEK293T and PC-3 prostate carcinoma cells, both of which showed CDA protein expression by Western blot analysis (data not shown). Specifically, 1 µg of purified plasmid DNA was transfected into the cultured cells with 200 ng of pRL-TK (Promega) DNA that encoded Renilla luciferase. The Renilla luciferase activity expressed by pRL-TK was used as a control for transfection efficiency. The cells were also transfected with pGL3-basic that lacked insert. Transfections were done using the FuGENE reagent (Roche, Indianapolis, IN). The cells were incubated for 48 hours, followed by harvest in passive lysis buffer (Promega). These cell lysates were used to assay luciferase activity with a Dual-Luciferase Reporter Assay (Promega), and results were expressed as the ratio of firefly luciferase light units to Renilla luciferase light units.

Enzyme assays and substrate kinetics. CDA catalyzes the formation of 2',2'-difluorodeoxyuridine from gemcitabine (Fig. 1). CDA activity was measured with gemcitabine (Eli Lilly, Indianapolis, IN) as substrate using a modification of the assay of Miwa et al. (11) with the high-performance liquid chromatography analysis of Freeman et al. (12). Basal levels of enzyme activity for WT and variant allozymes were corrected for transfection efficiency on the basis of ß-galactosidase activity measured with the ß-Galactosidase Enzyme Assay System (Promega).

DCTD catalyzes the formation of 2',2'-difluorodeoxyuridine monophosphate from gemcitabine monophosphate (Fig. 1). A modification of the assay described by Heinemann et al. (13) was used to measure DCTD activity with the extraction procedure and high-performance liquid chromatography separation described by van Haperen et al. (14). This assay measured the "disappearance" of gemcitabine monophosphate (Eli Lilly) and was optimized with recombinant human WT DCTD that had been expressed in COS-1 cells.

Western blot analysis. Levels of immunoreactive recombinant protein, corrected for transfection efficiency, were analyzed by quantitative Western blot analysis. A rabbit polyclonal antibody to the COOH-terminal 14 amino acids of CDA (15) was provided by Dr. Richard L. Momparler (Universite de Montreal, Hopital Sainte-Justine, Montreal, Quebec, Canada). Rabbit polyclonal antibody to purified DCTD was provided by Dr. Frank Maley (Wadsworth Center, New York State Department of Health, Albany, NY; refs. 16, 17). Specifically, samples for Western blot analysis were loaded on a 15% acrylamide gel (Criterion, Bio-Rad, Hercules, CA) on the basis of the cotransfected ß-galactosidase activity to correct for possible variation in transfection efficiency. Immunoreactive proteins were detected with the Enhanced Chemiluminescence System (Amersham) and Hyperfilm-Enhanced Chemiluminescence System (Amersham), and the results were quantitated using the NIH Image program (http://rsb.info.nih.gov/nih-image/).

Data analysis. Linkage analysis was done by testing all possible pairwise combinations of polymorphisms and calculating D' values, a method for reporting linkage data that is independent of allele frequency (18, 19). These data were then depicted graphically. Haplotypes were inferred computationally by using a program based on the Expectation-Maximization algorithm (20–22). Apparent Km values were calculated by nonlinear least squares regression analysis done with the GraphPad Prism program (GraphPad Software, San Diego, CA).


   Results
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 Materials and Methods
 Results
Discussion
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CDA and DCTD gene resequencing. Before resequencing, 10 kb of human CDA genomic DNA sequence located 5' of the ATG translation initiation codon were compared with the mouse genome sequence. The CDA cDNA open reading frame was also used to search the human EST database. Comparison with the mouse genome resulted in the identification of an area 377 bp in length in the human genome that was located 90 bp 5' of CDA exon 1 that was 70.6% identical to the mouse genome sequence, compatible with the conclusion that this 5'-flanking region sequence probably included the core promoter for the gene. Our search of the human EST database indicated that CDA mRNA was expressed in human bone marrow, prostate, pancreas, liver, and spleen. In addition, a small number of EST sequences, obtained mainly from leukocytes, included an out-of-frame, rarely represented, alternatively spliced exon (referred to here as "exon 1b") located between coding exons 1 and 2 (see Fig. 2A ).


Figure 2
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  		Fig. 2. CDA and DCTD polymorphisms. A, CDA gene structure; arrows, locations of polymorphisms. Black rectangles, open reading frame; white rectangles, portions of exons that encode UTR sequence. Cross-hatched box, a rare, out-of-frame alternatively spliced exon. A change in encoded amino acid resulting from the presence of a nonsynonymous cSNP is also indicated. I/D, an insertion/deletion event. B, the DCTD gene structure; arrows, locations of polymorphisms.

 
CDA was resequenced in 120 DNA samples. When this sequence was compared with the consensus CDA sequence, 17 polymorphisms, including 16 SNPs and 1 insertion-deletion, were observed in the 240 alleles resequenced (Fig. 2A; Table 1 ). Fourteen polymorphisms were present in DNA samples from African American and 11 in DNA from Caucasian American subjects. Six of the SNPs in DNA from the African American subjects were not observed in the Caucasian American subjects, and three of those present in the Caucasian American samples were absent from the African American DNA samples. Thirteen of the 14 polymorphisms in DNA from African American subjects and 8 of the 11 in DNA from Caucasian American subjects had frequencies of >1%, and, as a result, would be considered "common" in these populations. One nonsynonymous cSNP, 79 A>C (Lys27Gln), was observed, with allele frequencies of 10.8% and 29.8% in African American and Caucasian American subjects, respectively (Fig. 2A; Table 1). The 208 G>A (Ala70Thr) polymorphism previously reported in Japanese and African American subjects (5, 23) was not observed in any of our samples. We also compared the polymorphisms that we observed with those deposited in publicly accessible databases. A total of 11 of the 17 polymorphisms that were identified in this study had been deposited in dbSNP build 124 (http://www.ncbi.nlm.nih.gov/SNP), including the nonsynonymous cSNP that resulted in the Lys27Gln change in encoded amino acid. Four of the six polymorphisms not represented in dbSNP were observed only in the African American DNA samples.


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  		Table 1. Human CDA polymorphisms

 
A search of the human EST database before resequencing DCTD resulted in the identification of numerous transcripts that included sequence located ~1.0 and 1.9 kb 5'-upstream from the ATG start codon. Furthermore, these noncoding exons were bounded by appropriately placed "GT-AG" canonical splice sequences (24). As a result, we also resequenced these upstream noncoding exons, as well as ~500 bp of sequence located immediately upstream of the initial noncoding exon, plus the five DCTD coding exons and all splice junctions. DCTD was also resequenced in 120 DNA samples. When those data were compared with consensus DCTD sequence, 29 polymorphisms were observed (Fig. 2B; Table 2 ). Twenty-four of these polymorphisms were present in DNA samples from African American, with 15 in DNA from Caucasian American subjects. Fourteen of the SNPs in the African American subjects were not observed in the Caucasian American subjects, and five of those present in the Caucasian American samples were absent from African American subjects. Nineteen of the 24 polymorphisms in DNA from African American subjects and 13 of the 15 in DNA from Caucasian American subjects had frequencies >1% and, as a result, would be considered common in these populations. One nonsynonymous cSNP, 172 A>G (Asn58Asp), was observed in a single Caucasian American subject (Fig. 2B; Table 2). No SNPs were observed in the 500 bp immediately upstream of DCTD. We also compared the DCTD SNPs with those in dbSNP build 124. A total of 11 of the 29 polymorphisms that we observed had been deposited in dbSNP. Of the 18 polymorphisms that were not in the database, 12 were found only in the African American and 4 were observed only in Caucasian American DNA samples.


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  		Table 2. Human DCTD polymorphisms

 
Linkage disequilibrium and haplotype analysis. Linkage disequilibrium analysis was done for all of the polymorphisms that we observed in CDA and DCTD. Specifically, D' values were calculated for pairwise combinations of all polymorphisms in each gene (18, 19). The results for both African American and Caucasian American subjects are depicted graphically in Fig. 3 . Because haplotype is very useful for molecular epidemiology and pharmacogenomic studies (25), we also did haplotype analyses for both genes (Tables 3 and 4 ). Eight unambiguous CDA haplotypes (i.e., those deduced entirely on the basis of genotype) were observed in the 120 DNA samples resequenced, including four that were unique to African American and three to Caucasian American subjects (Table 3). These unequivocal haplotypes accounted for 69% and 68% of all samples in the two ethnic groups, respectively. The Expectation-Maximization algorithm (20–22) was used to infer a total of 25 and 16 additional CDA haplotypes for DNA samples from African American and Caucasian American subjects, respectively. However, only inferred haplotypes with frequencies ≥1% have been listed in Table 3. Information with regard to inferred haplotypes with frequencies <1% can be obtained from the authors on request. Haplotype designations were made initially on the basis of the encoded amino acid sequence, with the WT amino acid sequence being designated *1. We then assigned letter designations based on descending allele frequencies, starting with haplotypes present in African American subjects. Specifically, *1A was more common than *1B, etc. (Table 3). *2 haplotypes for CDA encoded the common Gln27 variant allozyme. A similar analysis was done for DCTD, and nine unambiguous haplotypes were observed in the 120 DNA samples studied, including six in African American and five in Caucasian American subjects (Table 4). Unequivocal DCTD haplotypes accounted for 59% and 61% of all samples from the two ethnic groups, respectively. There was also a total of 36 and 22 inferred haplotypes for African American and Caucasian American subjects, respectively, and those with frequencies of ≥1% are listed in Table 4. DCTD haplotype designations were made using a process similar to that used for CDA, with *2 indicating the haplotype that encoded the Asp58 variant allozyme.


Figure 3
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  		Fig. 3. Human CDA and DCTD linkage disequilibrium. The extent of population-specific linkage disequilibrium, depicted as pairwise |D'| values, is displayed graphically for both genes.

 

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  		Table 3. Human CDA haplotype analysis

 

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  		Table 4. Human DCTD haplotype analysis

 
Functional genomic studies. To characterize possible functional consequences of alterations in encoded amino acid sequence resulting from the nonsynonymous SNPs in CDA and DCTD, mammalian expression constructs were created for the WT and variant allozymes for both genes. Those constructs were used to transfect mammalian cells, and basal levels of enzyme activity in cytosolic preparations were measured under optimal conditions. In each case, two rounds of transfection were done, each in triplicate, and levels of enzyme activity or protein were corrected for the cotransfected ß-galactosidase to correct for possible variation in transfection efficiency. Average levels of basal enzyme activity for the two CDA allozymes studied are depicted graphically in Fig. 4A . The Gln27 variant had 66 ± 5.1% (mean ± SE) of the WT activity (P < 0.05). When substrate kinetic studies were done, the apparent Km value of the Gln27 allozyme, 397 ± 40 µmol/L, was significantly higher than that for WT CDA, 289 ± 20 µmol/L; P < 0.025 (Fig. 5 ). When recombinant allozyme protein concentrations, corrected for transfection efficiency, were measured by quantitative Western blot analysis, immunoreactive protein levels did not differ significantly between WT and Gln27 CDA allozymes (Fig. 4B). Expression constructs were also created for the DCTD WT and Asp58 variant allozymes. Recombinant Asp58 DCTD had very little enzyme activity but levels of immunoreactive protein for this allozyme were significantly elevated when compared with those for the WT enzyme (Fig. 6 ). Unfortunately, because of the very low level of activity for the Asp58 variant allozyme, substrate kinetic studies were not possible.


Figure 4
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  		Fig. 4. Recombinant human CDA allozyme activity and immunoreactive protein. A, columns, mean enzyme activity for recombinant allozymes and an empty vector control, corrected for transfection efficiency (n = 6); bars, SE. *, P < 0.05, compared with WT. B, mean immunoreactive protein corrected for transfection efficiency (n = 6). Inset, representative Western blot used to obtain the data shown in (B).

 

Figure 5
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  		Fig. 5. Recombinant CDA substrate kinetics. Gemcitabine substrate kinetics for the WT (A) and Gln27 (B) allozymes (n = 6). Points, mean; bars, SE.

 

Figure 6
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  		Fig. 6. Recombinant human DCTD allozyme activity and immunoreactive protein. A, columns, mean enzyme activity for recombinant allozymes and an empty vector control, corrected for transfection efficiency (n = 6); bars, SE. *, P < 0.005, compared with WT. B, mean immunoreactive protein corrected for transfection efficiency (n = 6). *, P < 0.005, compared with WT. Inset, representative Western blot used to obtain the data shown in (B).

 
The final series of functional genomic studies involved an attempt to determine whether polymorphisms located in the 5'-flanking region of CDA might influence transcription. Because we had not detected polymorphisms in the 5'-flanking region of DCTD, reporter gene studies were limited to CDA. Specifically, reporter gene constructs were created for common CDA 5'-flanking region haplotypes (Table 5 ). A total of 10 different constructs were transfected into HEK293T and PC-3 cells. Reporter gene activity showed similar patterns in both cell lines (Fig. 7 ). There were significant differences among 5'-flanking region haplotypes in their ability to drive transcription.


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  		Table 5. Human CDA 5'-FR haplotypes used in the reporter gene experiments

 

Figure 7
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  		Fig. 7. CDA 5'-flanking region luciferase reporter gene studies. Luciferase activity levels for reporter gene constructs containing CDA 5'-flanking region haplotypes transfected into HEK293T (A) or PC-3 (B) cells. Construct names refer to the 5'-flanking region haplotype designations listed in Table 5. 5'-Flanking region insert lengths are also indicated. Columns, mean (n = 6); bars, SE.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Gemcitabine can be inactivated by deamination catalyzed by CDA and DCTD. It also undergoes phosphorylation-dependent metabolic activation, which converts the drug to an active triphosphate nucleotide (Fig. 1). In the present study, we set out to determine the nature and extent of sequence variation in CDA and DCTD and to do initial functional genomic experiments as a step toward pharmacogenetic studies of this important antineoplastic drug. We began by resequencing exons, splice junctions, and a portion of the 5'-flanking region of these two genes using DNA samples from both African American and Caucasian American subjects. For CDA, 17 polymorphisms, including 1 nonsynonymous cSNP, were observed in the 120 DNA samples studied (Table 1; Fig. 2A) whereas 29 DCTD polymorphisms, including 1 novel nonsynonymous cSNP, were observed (Table 2; Fig. 2B). For both genes, more SNPs were present in DNA samples from African American than in those from Caucasian American subjects. This observation is compatible with previous reports of greater DNA sequence diversity in African populations than in other ethnic groups (26). Finally, a series of haplotypes were observed for both genes in each population (Tables 3 and 4).

The next step in our studies involved functional genomic experiments. Expression constructs were created for the nonsynonymous cSNPs that we observed in both CDA and DCTD, and those constructs were used to transiently transfect mammalian cells. The common CDA 79 A>C (Lys27Gln) polymorphism resulted in a moderate decrease in the level of CDA activity that was associated with a modest alteration in apparent Km value. The functional consequences of this polymorphism had been studied previously (4, 5). Kirch et al. (4) reported that deamination of ara-C by bacterial recombinant WT CDA was 1.3- to 3.3-fold that of Gln27. Yue et al. (5) reported an allele frequency of 20.1% for the C79 allele in a study of 221 Japanese subjects but did not observe a significant difference in enzyme activity between the variant and WT allozymes after expression in yeast with either cytidine or ara-C as substrates. Our apparent Km value of 289 µmol/L for recombinant WT CDA with gemcitabine as a substrate can be compared with values of 95.7 µmol/L for purified human placental CDA (27) and 264 µmol/L for bacterially expressed recombinant human CDA (11), both measured with the same substrate that we used. In our studies, the DCTD Asp58 allozyme exhibited very little enzyme activity but did have an elevated level of immunoreactive protein when compared with the WT enzyme (Fig. 6). These observations raise the possibility that this alteration in amino acid sequence might disrupt the ability of the protein to catalyze the enzyme reaction. However, because of the very low level of activity for this variant allozyme, substrate kinetic studies were not possible. Although this variant is rare, its striking effect on DCTD activity indicates that it might be associated with a clinically relevant phenotype, so its clinical implications should be pursued in future clinical pharmacogenetic studies. The mechanism(s) responsible for the alterations in levels of immunoreactive protein for CDA Gln27 and DCTD Asp58 should be explored in the course of future studies. We also did reporter gene experiments to study the possible effect of CDA 5'-flanking region variant haplotypes on transcription. Patterns of reporter gene expression were similar in the two cell lines studied but there were significant differences among 5'-flanking region haplotypes in their ability to drive transcription (Fig. 7). Demontis et al. (28) have reported that the CDA 5'-flanking region contains numerous potential transcription factor binding sites, so the present experiments represent merely an initial step in defining mechanisms responsible for the variations in transcription that we observed.

In summary, we have characterized the nature and extent of common sequence variation in the human CDA and DCTD genes in two ethnic groups. We also did functional studies of this sequence variation. The in vivo functional and clinical implications of these observations remain to be determined, as do the possible functional consequences of the other polymorphisms that we observed. However, the present results raise the possibility that ethnic-specific genetic variation in CDA and DCTD might contribute to individual differences in therapeutic response to treatment with gemcitabine.


   Acknowledgments
 
We thank Luanne Wussow for her assistance with the preparation of this manuscript.


   Footnotes
 
Grant support: NIH grants R01 GM28157 (O.E. Salavaggione, Y. Ji, and R.M. Weinshilboum), R01 GM35720 (O.E. Salavaggione, Y. Ji, and R.M. Weinshilboum), and U01 GM61388, The Pharmacogenetics Research Network (O.E. Salavaggione, Y. Ji, B.W. Eckloff, L.L. Pelleymounter, E.D. Wieben, and R.M. Weinshilboum), and a grant from Eli Lilly (J.A. Gilbert, M.M. Ames, E.D. Wieben, and R.M. Weinshilboum).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

Received 9/ 9/05; revised 11/11/05; accepted 1/ 4/06.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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