ABSTRACT
The drug fluorouracil (5-FU) is a widely used antimetabolite chemotherapy in the treatment of colorectal cancer. The gene uridine monophosphate synthetase (UMPS) is thought to be primarily responsible for conversion of 5-FU to active anticancer metabolites in tumor cells. Mutation or aberrant expression of UMPS may contribute to 5-FU resistance during treatment. We undertook a characterization of UMPS mRNA isoform expression and sequence variation in 5-FU-resistant cell lines and drug-naive or -exposed primary and metastatic tumors. We observed reciprocal differential expression of two UMPS isoforms in a colorectal cancer cell line with acquired 5-FU resistance relative to the 5-FU-sensitive cell line from which it was derived. A novel isoform arising as a consequence of exon skipping was increased in abundance in resistant cells. The underlying mechanism responsible for this shift in isoform expression was determined to be a heterozygous splice site mutation acquired in the resistant cell line. We developed sequencing and expression assays to specifically detect alternative UMPS isoforms and used these to determine that UMPS was recurrently disrupted by mutations and aberrant splicing in additional 5-FU-resistant colorectal cancer cell lines and colorectal tumors. The observed mutations, aberrant splicing and downregulation of UMPS represent novel mechanisms for acquired 5-FU resistance in colorectal cancer.
Subject(s)
Colorectal Neoplasms/genetics , Fluorouracil/administration & dosage , Multienzyme Complexes/genetics , Orotate Phosphoribosyltransferase/genetics , Orotidine-5'-Phosphate Decarboxylase/genetics , RNA Isoforms/genetics , RNA, Messenger/genetics , Alternative Splicing/genetics , Cell Line, Tumor , Colorectal Neoplasms/drug therapy , Down-Regulation , Drug Resistance, Neoplasm/genetics , Fluorouracil/adverse effects , Gene Expression Regulation, Neoplastic/drug effects , Humans , Multienzyme Complexes/metabolism , Mutation , Orotate Phosphoribosyltransferase/metabolism , Orotidine-5'-Phosphate Decarboxylase/metabolismABSTRACT
Cytochrome P450 2A6 (CYP2A6) is the main nicotine (NIC)-metabolizing enzyme in humans. We investigated the relationships between CYP2A6 genotype, baseline plasma trans- 3'-hydroxycotinine/cotinine (3HC/COT) (a phenotypic marker of CYP2A6 activity), and smoking behavior in African-American light smokers. Cigarette consumption, age of initiation, and dependence scores did not differ among 3HC/COT quartiles or CYP2A6 genotype groups. Slow metabolizers (SMs; both genetic and phenotypic) had significantly higher plasma NIC levels, suggesting that cigarette consumption was not reduced to adjust for slower rates of NIC metabolism. Individuals in the slowest 3HC/COT quartile had higher quitting rates with both placebo and NIC gum treatments (odds ratio 1.85, 95% confidence interval (CI) 1.08-3.16, P = 0.03). Similarly, the slowest CYP2A6 genotype group had higher quitting rates, although this trend did not reach significance (odds ratio 1.61, 95% CI 0.95-2.72, P = 0.08). The determination of the 3HC/COT ratio, and possibly CYP2A6 genotype, may be useful in the future for personalizing the choice of smoking cessation treatment in African-American light smokers.
Subject(s)
Aryl Hydrocarbon Hydroxylases/genetics , Aryl Hydrocarbon Hydroxylases/metabolism , Black or African American , Nicotine/metabolism , Smoking Cessation , Adult , Aged , Body Mass Index , Cotinine/analogs & derivatives , Cotinine/blood , Cytochrome P-450 CYP2A6 , Double-Blind Method , Female , Genotype , Humans , Male , Middle Aged , Polymorphism, GeneticABSTRACT
Cytochrome P450 2A6 (CYP2A6) is the human enzyme responsible for the majority of nicotine's metabolism. CYP2A6 genetic variants contribute to the interindividual and interethnic variation in nicotine metabolism. We examined the association between the CYP2A6*1B variant and nicotine's in vivo metabolism. Intravenous infusions of deuterium-labeled nicotine were administered to 292 volunteers, 163 of whom were White and did not have common CYP2A6 variants, other than CYP2A6*1B. We discovered three novel CYP2A6*1B variants in the 3'-flanking region of the gene that can confound genotyping assays. We found significant differences between CYP2A6*1A/*1A, CYP2A6*1A/*1B, and CYP2A6*1B/*1B groups in total nicotine clearance (17.2+/-5.2, 19.0+/-6.4, and 20.4+/-5.9, P<0.02), non-renal nicotine clearance (16.4+/-5.0, 18.5+/-6.2, and 19.8+/-5.7, P<0.01), and the plasma trans-3'-hydroxycotinine/cotinine ratio (0.26+/-0.1, 0.26+/-0.1, and 0.34+/-0.1, P<0.001). There were also differences in total nicotine (29.4+/-12.9, 25.8+/-0.12.9, and 22.4+/-12.4, P<0.01), cotinine (29.2+/-8.1, 32.2+/-9.1, and 33.0+/-6.6, P<0.01) and trans-3'-hydroxycotinine (32.4+/-9.1, 34.2+/-12.3, and 41.3+/-11.3, P<0.001) excreted in the urine. We report evidence that CYP2A6*1B genotype is associated with faster nicotine clearance in vivo, which will be important to future CYP2A6 genotype association studies.
