Aims Dihydropyrimidine dehydrogenase (DPD) catalyses the reduction of pyrimidines, including the anticancer agent 5-fluorouracil (5FU). Impaired 5FU degradation, through low DPD activity, has led to severe, life-threatening or fatal toxicity after administration of 5FU. Complete DPD deficiency is associated with the inherited metabolic disease thymine uraciluria. Several mutations in the gene encoding DPD have recently been identified, but the phenotype-genotype concordance of these alterations in the general population has not been reported. Methods Mononuclear cells were isolated from whole blood and DPD activity was determined after ex vivo incubation with 14 C-5FU followed by h.p.l.c. analysis of 5FU metabolites. Analysis of mutations in the DPD gene at an exon splice site, codons 534, 543, and 732, and a deletion at base 1897 (DC1897) were performed in 30 subjects with the lowest and 30 subjects with the highest enzyme activity using PCR-RFLP. Results DPD activity was measured in 226 Caucasian subjects and was highly variable (range 19.1-401.4 pmol min −1 mg −1 protein). Mutations were frequently observed at codons 543 (allele frequency 28%), 732 (allele frequency 5.8%), and 534 (allele frequency 0.8%), but were not associated with low DPD activity. There were no splice site or DC1897 mutations found in this population. Conclusions The five mutations analysed in this study are insufficient for identification of patients at risk for 5FU toxicity or thymine uraciluria. Both the splice site mutation and DC1897 are relatively rare in the general Caucasian population. Therefore, identification of further molecular alterations is required to facilitate the use of DPD analysis in genetic diagnosis and cancer therapeutics.
Summary Individuals with a deficiency in the enzyme dihydropyrimidine dehydrogenase (DPD) may experience severe life-threatening toxicity when treated with 5-fluorouracil (5-FU). As routine measurement of enzyme activity is not practical in many clinical centres, we have investigated the use of DNA mutation analysis to identify cancer patients with low enzyme levels. We have identified two new mutations at codons 534 and 543 in the DPD cDNA of a patient with low enzyme activity and screened the DNA from 75 colorectal cancer patients for these mutations and the previously reported splice site mutation (Vreken et al, 1996;Wei et al, 1996). In all cases, DPD enzyme activity was also measured. The splice site mutation was detected in a patient (1 out of 72) with low enzyme activity whereas mutations at codons 534 (2 out of 75) and 543 (11 out of 23) were not associated with low enzyme activity. These studies highlight the need to combine DPD genotype and phenotype analysis to identify mutations that result in reduced enzyme activity.Keywords: dihydropyrimidine dehydrogenase; 5-fluorouracil; polymorphism; colorectal cancer 5-Fluorouracil (5-FU) is widely used in the treatment of advanced solid tumours, including colorectal, breast and head/neck tumours. 5-FU is also frequently used in adjuvant chemotherapy for colorectal and breast cancers, in which its mild toxicity profile of mucositis and diarrhoea is well tolerated by patients who are at risk for tumour recurrence but have no current evidence of disease. 5-FU is a pyrimidine analogue and greater than 80% of a dose is degraded in a three-step pathway, initially catalysed by the enzyme dihydropyrimidine dehydrogenase (DPD; E.C. 1.3.1.2., Heggie et al, 1987). Deficiency in DPD enzyme activity is associated with a considerable delay in clearance of 5-FU from the plasma (Diasio et al, 1988;Fleming et al, 1992), leading to severe, life-threatening diarrhoea, neutropenia and in some cases neurotoxicity, incurring prolonged hospitalization . High concentrations of plasma and urine 5-FU, uracil and thymine may be detected along with low mononuclear cell DPD activity in these patients. The toxicity is thought to result from higher levels of 5-FU entering the anabolic pathway, resulting in an increased production of cytotoxic nucleotides. Although thymidine rescue has been attempted in one case (Takimoto et al, 1996), the majority of cases have been managed with supportive care after the cessation of 5-FU-based therapy. DPD activity is found in most human tissues, with the highest levels in the liver and lymphocytes. Population studies of peripheral blood mononuclear cell (PBMNC) DPD have shown that enzyme activity is variable with a seven-to 10-fold range observed (Lu et al, 1993;Etienne et al, 1994;McMurrough and McLeod, 1996). These studies suggest that although total deficiency is rare in adults, as many as 3% of the population may have low enzyme levels and thus be at One mutation in the DPYD gene has been reported to date in patients exhibiting severe toxicity after 5...
In vitro exposure of a human lymphoblastoid cell line (WIL-2) to the antifolate metoprine (DDMP), when followed by the addition of exogenous deoxyuridine, led to intracellular accumulation of deoxyuridine triphosphate (dUTP) and incorporation of deoxyuridine monophosphate (dUMP) into DNA.When newly synthesized DNA was extracted from DDMP-treated cells that had been labeled with deoxyuridine for up to 3 min, most of the DNA synthesized was no larger than 4 Prokaryotic and eukaryotic cells contain a potent deoxyuridine triphosphatase (1-4) and a uracil N-glycosylase-dependent repair system (5-9) that normally prevent accumulation of dUMPcontaining DNA. We have found that, when intact WIL-2 human lymphoblastoid cells are inhibited by antifolates methotrexate (MTX) or metoprine [2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine, DDMP], the apparent levels of intracellular dUMP can be proportionally increased by exposure to increasing exogenous concentrations of deoxyuridine (10). In this study we examined the fate of exogenously added deoxyuridine in DDMP-treated cells, in order to determine whether dUTP becomes detectable and leads to dUMP misincorporation. We also looked for perturbations of DNA synthesis that might occur as a result of this lesion. Specifically, it was important to determine whether antifolates and deoxyuridine might affect the normal progression of DNA synthesis and not simply decrease overall incorporation into DNA products of all sizes. For rapid pulse experiments, cells were injected directly into 60% methanol at -20°C. Extraction of nucleosides and nucleotides was carried out for at Itast 24 hr before further processing of the samples. MATERIALS AND METHODS Chemicals andNucleotide Pool Analysis by Cellulose Thin-Layer Chromatography. The methanol nucleotide pool extracts described above were lyophilized to dryness and resuspended in 30 ,ul of 10% isopropyl alcohol/10% acetic acid/80% water (vol/vol).DNA nucleotides (0.01 M) were used as standards for chromatography. Cellulose plastic plates (Kodak), impregnated with fluorescent dye indicator, were divided into 10 numbered channels. Portions of each sample were spotted at the origin, positioned at 2 cm from the bottom of the plate for each channel. Portions of standard solutions were spotted together with or beside each sample, and the position of migration was visualized by irradiating the plates with shortwave ultraviolet light. The solvent system utilized for the nucleotide separations was isobutyric acid/NH40H/H20(63:1:33 vol/vol). Thin-layer chromatography plates were developed for approximately 4 hr (or until the solvent had moved 17 cm from the bottom of the plate) Abbreviations: MTX, methotrexate; DDMP, 2,4-diamino-5-(3',4'-dichlorophenyl)-&methylpyrimidine (metoprine); PEI, polyethyleneimine. 917The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
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