Quantitative measurement of thymidylate synthase and dihydropyrimidine dehydrogenase mRNA level in gastric cancer by real-time RT-PCR.
We used real-time reverse-transcription polymerase chain reaction (RT-PCR) to assay expression of the mRNA of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) in gastric cancer tissue with the objective of establishing a system to measure TS and DPD in ultra-low-volume samples. Nude mouse xenografts of 5 human gastric cancer cell lines and 85 clinical samples were used as the specimens in this study. Sensitivity to 5-fluorouracil (5-FU) was determined on the basis of the relative tumor proliferation rate in mice and the results of ATP assay using serum-free cultures of the clinical samples. mRNA expression was measured in tumor tissue by real-time RT-PCR using the ABI PRISM 7700 system. The values for expression of the mRNA for TS and DPD were corrected according to the level of glyceraldehyde-3-phosphate dehydrogenase mRNA expression. The xenografts yielded correlations between TS and DPD mRNA expression and the activity of the enzymes (TS: rs=0.700, DPD: rs=0.900), and an inverse correlation was noted between the mRNA levels and sensitivity to 5-FU (TS: rs=-0.900, DPD: rs=-0.800). The clinical samples showed an inverse correlation between 5-FU sensitivity and mRNA expression (TS: rs=-0.518, DPD: rs=-0.564). Sensitivity to 5-FU was noted only in cases in which TS mRNA expression and DPD mRNA expression were both low. Real-time RT-PCR can provide a highly sensitive assessment of TS and DPD mRNA expression in gastric cancer, and it was useful for predicting 5-FU sensitivity.
Fujiwara
Hisataka
H
Department of Surgery, Iwate Medical University, Morioka 020-8505, Japan.
Terashima
Masanori
M
Irinoda
Takashi
T
Takagane
Akinori
A
Abe
Kaoru
K
Kashiwaba
Masahiro
M
Oyama
Kennichi
K
Takahashi
Masanori
M
Maesawa
Chihaya
C
Saito
Kazuyoshi
K
Takechi
Teiji
T
Fukushima
Masakazu
M
D000818:Animals; D042943:Dihydrouracil Dehydrogenase (NADP); D005260:Female; D005472:Fluorouracil / Q000494:pharmacology; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009368:Neoplasm Transplantation; D010088:Oxidoreductases / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013274:Stomach Neoplasms / Q000188:drug therapy / Q000201:enzymology*; D013940:Thymidylate Synthase / Q000235:genetics*; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D010088:Oxidoreductases; D042943:Dihydrouracil Dehydrogenase (NADP); D013940:Thymidylate Synthase; D005472:Fluorouracil