orotate phosphoribosyltransferase

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1 orotate phosphoribosyltransferase (II) Expression of orotate phosphoribosyltransferase in colorectal cancer Takehiro Takahashi (Department of digestive and general surgery, Saitama Medical School, Moroyama, Iruma- gun, Saitama , Japan) Backgroud: Activation of 5 -fluorouracil (5 -FU) into nucleotides requires phosphorylation by orotate phosphoribosyltransferase (OPRT). In this study, we investigated the correlation between enzymatic activity and gene expression of OPRT in colorectal cancer tissues, and the association between OPRT gene expression and anti-tumor effect. Materials and Methods: Enzymatic activity and gene expression of OPRT were measured by radioassay method and a real - time reverse transcriptional -polymerase chain reaction method, respectively, in 20 primary colorectal cancer tissues. In 37 patients treated with tegafur -uracil and leucovorin for metastatic colorectal cancer, OPRT gene expressions were analyzed by the same method. Results: There was a positive correlation between enzymatic activity and gene expression of OPRT (r 0.788, P ). Responding tumors had statistically higher OPRT gene expression than nonresponding tumours (P ), and patients with a high OPRT mrna expression suvived longer than those with a low OPRT mrna expression (P ). Conclusion: The expression of OPRT gene might be useful as predictive parameter for the efficacy of fluoropyrimidine- based chemotherapy. Keywords: orotate phosphoribosyltransferase, colorectal cancer, 5-fluorouracil 5-fluorouracil 5-FU 1) 5-FU dihydropyrimidine dehydrogenase DPD 2 -fluoro - -alanine 2) 5-FU 5-fluoro-deoxyuridine monophosphate FdUMP 3, 4) 5, 10-methylene tetrahydrofolatedna de novo thymidylate synthase TS ternary complexts 5) DNA de novo DNA 6) 5-FU TS DPD TS DPD TS DPD 75 7) 5-FUTS DPD 8)

2 5-FU FdUMP orotate phosphoribosyltransferase OPR T 9, 10) uridine phosphorylase UP 11, 12) thymidine phosphorylase TP 13) 1 14) OPRT 5-phosphoribosyl-1-pyrophosphate PRPP 5-fluorouridine mono-phosphate FUMP 5-FU 9, 10) in vitro 5-FU OPRT 15, 16) OPRT in vivo OPRT OPRT mrna OPRT mrna 1 OPRTOPRT mrna I II 1OPRT OPRT mrna 5 mm OPRT OPRT mrna 1 OPRT 500 mg OPRTnmol/min/mg-protein radioassay phosphoribosylpyrophosphate 3H -5-FU H -5-FU FUMP 1 17, 18) 2 OPRT mrna 14) 20 mg RNA RNeasy MiniKit QIAGEN Inc. Chatsworth, CA, USA RNA 19) RNA cdna OPRT mrna glyceralaldehyde- 3 -phosphate dehydrogenase GAPDH mrna ABI PRISM 7700 Sequence Detection System 1. Metabolism of 5 -FU (5 -fluorouracil) anabolism. (Pathway 1) OPRT; orotate phosphoribosyltransferase, PRPP; 5 - phosphoribosyl-1-pyrophosphate, (pathway 2) UP; uridine phosphorylase, Rib-1-P; ribose-1-phosphate, FUR; 5-fluorouridine, (pathway 3) TP; thymidine phosphor ylase, FUdR; 2 -deoxy -5 -fluorouridine, drib -1 -P; deoxyribose -1 -phosphate catabolism. DPD; dihydropyrimidine dehydrogenase, F- -Ala; 2-fluoro- alanine, FUMP; 5-fluoroudine monophosphate, FUDP; 5-fluorouridine, diphosphate, FUTP; 5-fluoro-uridine-5 -triphosphate, FdUMP; 5-fluoro-deoxyuridine-monophosphate, TS; thymidylate synthase, CH 2 THF; 5,10-methylene tetrahydrofolate.

3 orotate phosphoribosyltransferase Taqman Perkin -Elmer Applied Biosystems, Foster City, CA, USA 20-22) 1 primer/probeoprt mrna OPRT mrna GAPDH mrna OPRT/GAPDH 2 OPRTOPRT mrna OPRTOPRT mrna Pearson p 0.05 III OPRTOPRT mrna2 OPRTOPRT mrna p OPRT mrna 20 mg 80 OPRT mrna OPRT mrna GAPDH mrna OPRT/GAPDH 3. Mann-Whitney U test two sided Fisher s exact test Kaplan-Meier log-lank test p 0.05 III. 2 OPRT mrna I UFT LV 37 7) first-line 1 PS score 2 23) II 1 UFT 400 mg/m 2 /daylv 15 mg/ body , 24) 2 8 CT UICC 25) 2 CR 10PR 16 NC 9 PD /37 1. OPRT mrna 37 OPRT mrna UFT LV OPRT mrna OPRT mrna P OPRT mrna OPRT mrna 1.0 cut-off OPRT mrna 1.0OPRT 1.0 OPRTOPRT 63 12/19 OPRT P OPRT mrna OPRT OPRT OPRT P FU FdUMP OPRT UP TP uridine kinase UK ribonucleotide reductase thymidine kinase TK 1. The primers and probe sequences for GAPDH (glyceraldehyde-3-phosphate dehydrogenase), and OPRT (orotate phosph oribosyltransferase)

4 1 1 : 5- phosphoribosyl -1 -pyrophosphate PRPP OPRT FUMP 9, 10) 2 : Rib-1-P UPFUR UK FUMP 11, 12) 3 : drib-1-p TP FUdRTK FdUMP 13) 3 2. OPRT mrna expression plotted against enzymatic activity of OPRT. OPRT mrna shows a positive correlation against enzymatic activity of OPRT (correlation coefficient 0.788, p ). 5-FU 5-FU OPRT15-FU 9, 10) Inaba 5-FU OPRT 26) Fujii54 in vitro 5-FU OPRT 16) OPRT 100 mg 10 mg mrna OPRTOPRT OPRT OPRT Uetake Ichikawa RT-PCR DPD mrna DPD 27) RT-PCR 3. OPR T gene expression in terms of response. Responding tumors statistically had higher OPRT gene expression than nonresponding tumors (P ). 4. Survival curves in terms of OPRT gene expression. The median survival time was 12.5 months in patients with high OPRT mrna expression (range: months), but 8.5 months (range: months) in patients with low expression (P ). Patients with a high OPRT mrna expression suvived longer than those with a low OPRT mrna expression.

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