Aberrant Methylation of 
 DPYD
 Promoter, 
 DPYD
 Expression, and Cellular Sensitivity to 5-Fluorouracil in Cancer Cells

Noguchi, Takuya; Tanimoto, Keiji; Shimokuni, Tatsushi; Ukon, Kei; Tsujimoto, H.; Fukushima, Masakazu; Noguchi, Tsuyoshi; Kawahara, Katsunobu; Hiyama, Keiko; Nishiyama, Masahiko

doi:10.1158/1078-0432.ccr-04-0337

Cited by 60 publications

(37 citation statements)

References 15 publications

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“…Studies in cancer cell lines (oral epidermoid carcinoma line KB, colon adenocarcinoma COLO20, oral squamous cell carcinoma lines HSC3, HSC4, and Ca9-22 and hepatoma HepG2 cell lines) showed the absence of genetic alterations in DPYD promoter region with full activity (8). Our results show the absence of DNA sequence variants in the DPYD promoter region in all studied individuals.…”

Section: Discussionsupporting

confidence: 57%

“…Recent advances in our understanding of molecular mechanisms involved in the activation and degradation of 5-FU have led to an increased awareness of the potential importance of epigenetic factors in deciding the sensitivity of patients to anticancer drugs. We hypothesize that in the absence of inactivating mutations, methylation of CpG islands located in the 5V regulatory region of the DPYD gene promoter, may inhibit the binding of transcriptional factors or stabilize the chromatin structure, thereby directly inhibiting transcription (8).…”

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confidence: 99%

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Methylation of the DPYD Promoter: An Alternative Mechanism for Dihydropyrimidine Dehydrogenase Deficiency in Cancer Patients

Ezzeldin

Lee

Mattison

et al. 2005

Clinical Cancer Research

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Purpose: Dihydropyrimidine dehydrogenase (DPD) deficiency, a known pharmacogenetic syndrome associated with 5-fluorouracil (5-FU) toxicity, has been detected in 3% to 5% of the population. Genotypic studies have identified >32 sequence variants in the DPYD gene; however, in a number of cases, sequence variants could not explain the molecular basis of DPD deficiency. Recent studies in cell lines indicate that hypermethylation of the DPYD promoter might downegulate DPD expression. The current study investigates the role of methylation in cancer patients with an unexplained molecular basis of DPD deficiency. Experimental Design: DPD deficiency was identified phenotypically by both enzyme assay and uracil breath test, and genotypically by denaturing high-performance liquid chromatography. The methylation status was evaluated in PCR products (209 bp) of bisulfite-modified DPYD promoter, using a novel denaturing high-performance liquid chromatography method that distinguishes between methylated and unmethylated alleles. Clinical samples included five volunteers with normal DPD enzyme activity, five DPD-deficient volunteers, and five DPD-deficient cancer patients with a history of 5-FU toxicity. Results: No evidence of methylation was detected in samples from volunteers with normal DPD. Methylation was detected in five of five DPD-deficient volunteers and in three of five of the DPD-deficient cancer patient samples. Of note, one of the two samples from patients with DPD-deficient cancer with no evidence of methylation had the mutation DPYD*2A, whereas the other had DPYD*13. Discussion: Methylation of the DPYD promoter region is associated with down-regulation of DPD activity in clinical samples and should be considered as a potentially important regulatory mechanism of DPD activity and basis for 5-FU toxicity in cancer patients.Dihydropyrimidine dehydrogenase (DPD) enzyme deficiency, a known pharmacogenetic syndrome detected in 3% to 5% of the population (1), has been associated with toxicity to 5-fluorouracil (5-FU) cancer chemotherapy and death in some cases with profound deficiency of the enzyme (2). DPD is the first enzyme in a three-step catabolic pathway responsible for the degradation of f85% of administered 5-FU. Genotypic studies have identified >32 sequence variants in the DPYD gene (1, 3). Expression analysis of these variants showed that many were polymorphisms with no obvious functional significance (4), with the exception of a few mutations. One example commonly associated with DPD deficiency and severe toxicity to 5-FU is an intronic sequence variation (IVS14 + 1 G > A, DPYD*2A), which results in a truncated protein that lacks 55 amino acids due to the skipping of exon 14 (5). A second example is a less common mutation (1679T > G, I560S, DPYD*13), which is associated with decreased DPD activity and 5-FU toxicity (6) due to a nonconservative amino acid change from isoleucine to serine at codon 560 (I560), which is 100% conserved among human, mouse, rat, bovine, and pig species, suggesting its imp...

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Section: Discussionsupporting

confidence: 57%

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confidence: 99%

Methylation of the DPYD Promoter: An Alternative Mechanism for Dihydropyrimidine Dehydrogenase Deficiency in Cancer Patients

Ezzeldin

Lee

Mattison

et al. 2005

Clinical Cancer Research

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“…Despite its easiness, many contradictory results regarding the predictive power of expression analyses were obtained [38,39]. The influence of DPYD promoter methylation on DPYD gene expression was also considered, however contrary to initial reports latest studies demonstrated limited or no methylation of DPYD promoter in high-toxicity patients [27,[40][41][42]. The most common concept for prediction of serious toxicity in 5-FU-treated patients represents pharmacogenomic analyses.…”

Section: Discussionmentioning

confidence: 99%

Influence of dihydropyrimidine dehydrogenase gene (DPYD) coding sequence variants on the development of fluoropyrimidine-related toxicity in patients with high-grade toxicity and patients with excellent tolerance of fluoropyrimidine-based chemotherapy

Kleibl

Fidlerová²,

Kleiblová³

et al. 2009

neo

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Alterations in dihydropyrimidine dehydrogenase gene (DPYD) coding for the key enzyme (DPD) of fluoropyrimidines (FPs) catabolism contribute to the development of serious FPs-related toxicity. We performed mutation analysis of DPYD based on cDNA sequencing in 76 predominantly colorectal cancer patients treated by FPs with early development of high (grade 3-4) hematological and/or gastrointestinal toxicity. Six previously described [85T>C (C29R), 496A>G (M166V), 775A>G (K259E), 1601G>A (S534N), 1627A>G (I543V), IVS14+1G>A, 2194G>A (V732I)] and two novel [187A>G (K63E) and 1050 G>A (R357H)] non-synonymous DPYD variants were found in 56/76 (73.7%) high-toxicity patients. Subsequently, these alterations were analyzed in 48 patients with excellent long-term tolerance of FPs and in 243 controls and were detected in 37/48 (77.1%) and 166/243 (68.3%) cases, respectively. Analysis of these alterations as risk factors for development of toxicity in pooled FPs-treated population demonstrated that C29R negatively correlated with overall gastrointestinal toxicity (OR = 0.48; 95%CI 0.23-1.0) and M166V in women protected against overall hematological toxicity and neutropenia (both OR = 0.26; 95%CI 0.07-0.89), whereas IVS14+1G>A (found in five high-toxicity patients only) increased risk of mucositis in overall population (OR = 7.0; 95%CI 1.1-44.53), and thrombocytopenia in women (OR = 10.8; 95%CI 1.24-93.98). Moreover, we identified a strong association of V732I with leucopenia (OR = 8.17; 95%CI 2.44 -27.31) and neutropenia (OR=2.78; 95% CI 1.03-7.51). Our data enabled characterization of "high risk" haplotypes (carriers of IVS14+1G>A or V732 lacking M166V) representing small (22% female and 11% male patients), population in high risk of serious hematological toxicity development, and in patients with "lower risk" that unlikely develop serious hematological toxicity [carriers of M166V without IVS14+1G>A and V732I in females (32% women), and non-carriers of C29R, M166V, IVS14+1G>A, and V732I in males (46% men)]. Our results indicate that genotyping of several DPYD variants may lead to stratification of patients with respect to the risk of serious hematological toxicity development during FPs treatment. Key words: dihydropyrimidine dehydrogenase gene [DPYD], fluoropyrimidines, 5-fluorouracil toxicity, mutation analysis, haplotypes, association study* Corresponding author † These authors contributed equally to this work.Fluoropyrimidines -5-fluorouracil (5-FU) and its derivates (e.g. capecitabine) -belong to the most frequently used anticancer drugs in treatment of solid cancers. Mechanism of action of 5-FU involves its anabolic conversions to 5-fluoropyrimidine nucleotides that exert profound inhibitory effect on thymidylate synthetase activity and interfere with RNA and DNA metabolism [1]. The development of severe toxicity is the critical complication of 5-FU-based therapy. It occurs in nearly one third of cases with progression to life-threatening complications in approximately 0.5% patients [2].About 80% of 5-FU is quickly ina...

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“…However, there is a general consensus that there can be some discrepancies among mRNA, protein expressions and the enzyme activity, because there can be several regulatory mechanisms including posttranscriptional and posttranslational regulation in the expression of a gene and also there can be a conditional regulation concerning the enzyme activity. Regulatory mechanism of DPD activity may be associated with negative-feedback system, regulation by transcriptional factor of DPD gene, such as AP-1, 48 methylation of the promoter region 49 or unknown DPD inhibitor. Further investigation should be required for precise explication of regulatory mechanism of DPD activity.…”

Section: Discussionmentioning

confidence: 99%

Synergistic effects of docetaxel and S‐1 by modulating the expression of metabolic enzymes of 5‐fluorouracil in human gastric cancer cell lines

Wada

Yoshida

Suzuki

et al. 2006

Intl Journal of Cancer

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We have recently demonstrated in a Phase I/II study that combination chemotherapy with docetaxel (TXT) and S-1 is active against metastatic gastric carcinomas. To elucidate the mechanisms underlying the synergistic effects of these drugs, both the growth inhibitory effects and the expression profiles of enzymes involved in fluorouracil (5-FU) metabolism were examined in vitro and in vivo. TXT alone and in combination with 5-FU inhibited the growth of each of the 5 gastric cancer cell lines that we examined , in a time-and dosedependent manner. Moreover, striking synergistic effects were observed in TMK-1 cells in vitro with IC 50 values of between 4.73 and 0.61 nM 5-FU. Furthermore, in TMK-1 xenografts, 5-FU/ TXT cotreatments exhibited synergistic antitumor effects. The combination of S-1 and TXT, however, exhibited greater growthinhibitory effects than the 5-FU/TXT cotreatments. The mechanisms underlying these synergistic effects of S-1 and TXT were examined by expression and activity analyses of the 5-FU metabolic enzymes. The expression of thymidylate synthase (TS), and dihydropyrimidine dehydrogenase (DPD) were decreased 50 and 73% of control levels, respectively, and that of orotate phosphorybosyl transferase (OPRT) was increased by 3.9-fold at the protein level. These findings suggested that biochemical modulation of the 2 drugs had occurred, which was further confirmed by the results of the activity assays. These data strongly indicate that a combination chemotherapy of TXT and S-1 is effective against gastric carcinomas and is therefore a good candidate as a standard chemotherapeutic strategy in treating these tumors. ' 2006 Wiley-Liss, Inc.Key words: gastric cancer; TMK-1; docetaxel; 5-fluorouracil; S-1; TS, thymidylate synthase; DPD, dihydropyrimidine dehydrogenase; OPRT, orotate phosphorybosyl transferase; combination effectIn spite of recent advances in the development of both diagnostic and therapeutic tools, gastric cancer is still a major cause of death throughout the world. Moreover, because many patients are still diagnosed at only the late stages of this disease, recurrent tumors are often detected even after curative surgery. In such patients, chemotherapy is one of the main treatment strategies that is employed, as it has been shown to improve prognoses. 1-3 However, a standard regimen of the treatment of metastatic gastric cancer has not been yet established in Japan.5-Fluorouracil (5-FU) is widely used an anticancer agent and is currently considered to be a key drug in clinical chemotherapeutic treatments for gastrointestinal cancers, such as esophageal, 4 gastric 5 and colorectal. 6 In addition, 5-FU has been also used to treat both breast 7 and cervical 8 cancers. 5-FU is catabolized to 2-fluorob-alanin by the first and rate-limiting enzyme of its metabolic pathway, dihydropyrimidine dehydrogenase (DPD). The functional effects of 5-FU are thought to occur through its active metabolite, 5-fluorodeoxyuridine diphosphate (FdUMP) 9,10 which, together with the coenzyme 5,10-methylenethtrahydr...

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Aberrant Methylation of DPYD Promoter, DPYD Expression, and Cellular Sensitivity to 5-Fluorouracil in Cancer Cells

Cited by 60 publications

References 15 publications

Methylation of the DPYD Promoter: An Alternative Mechanism for Dihydropyrimidine Dehydrogenase Deficiency in Cancer Patients

Methylation of the DPYD Promoter: An Alternative Mechanism for Dihydropyrimidine Dehydrogenase Deficiency in Cancer Patients

Influence of dihydropyrimidine dehydrogenase gene (DPYD) coding sequence variants on the development of fluoropyrimidine-related toxicity in patients with high-grade toxicity and patients with excellent tolerance of fluoropyrimidine-based chemotherapy

Synergistic effects of docetaxel and S‐1 by modulating the expression of metabolic enzymes of 5‐fluorouracil in human gastric cancer cell lines

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