Omura K, Morishita M, Kawakami K, Kanehira E, Ishida Y, Watanabe Y
Department of Surgery 1, Kanazawa University School of Medicine Faculty of Medicine, Japan.
Hepatogastroenterology. 1999 Mar-Apr;46(26):985-90.
BACKGROUND/AIMS: Thymidylate synthase (TS) is a target enzyme for 5-fluorouracil (5-FU). It is important to know the TS expression level in tumor tissue for chemotherapy. We evaluated the TS expression level using competitive polymerase chain reaction (cPCR), and estimated the reliability and reproducibility of this method.
TS expression was assessed by both Fluorodeoxyuridine-monophosphate (FdUMP) binding assay and cPCR in 9 human adenocarcinoma cell lines and 50 human adenocarcinoma tissues obtained by surgical resection. TS expression was also assessed by cPCR in nine biopsy specimens obtained before surgery. We synthesized TS and beta-actin competitors. Following cPCR, PCR products were quantified on ethidium bromide-stained gels using a digital image analyzer. TS mRNA/beta-actin mRNA ratio was used to determine relative TS expression level.
The number of FdUMP binding sites on TS and TS mRNA/beta-actin mRNA ratio were significantly correlated in both cell lines and surgically resected specimens (p < 0.01). A linear regression line formed from the data points was obtained for TS mRNA/beta-actin mRNA ratios in surgical specimens versus TS mRNA/beta-actin mRNA ratios in biopsy specimens (p < 0.01).
Assessment of TS expression by cPCR using our competitors was accurate and reproducible.
背景/目的：胸苷酸合成酶（TS）是5-氟尿嘧啶（5-FU）的靶酶。了解肿瘤组织中TS的表达水平对化疗很重要。我们使用竞争性聚合酶链反应（cPCR）评估TS的表达水平，并评估该方法的可靠性和可重复性。
通过氟脱氧尿苷单磷酸（FdUMP）结合试验和cPCR对9个人类腺癌细胞系和50例手术切除获得的人类腺癌组织进行TS表达评估。还通过cPCR对9例术前活检标本进行TS表达评估。我们合成了TS和β-肌动蛋白竞争物。cPCR后，使用数字图像分析仪对溴化乙锭染色凝胶上的PCR产物进行定量。TS mRNA/β-肌动蛋白mRNA比值用于确定相对TS表达水平。
在细胞系和手术切除标本中，TS上FdUMP结合位点的数量与TS mRNA/β-肌动蛋白mRNA比值均显著相关（p < 0.01）。手术标本中TS mRNA/β-肌动蛋白mRNA比值与活检标本中TS mRNA/β-肌动蛋白mRNA比值的数据点形成了一条线性回归线（p < 0.01）。
使用我们的竞争物通过cPCR评估TS表达是准确且可重复的。
