title: Associations between gene polymorphisms of thymidylate synthase with its protein expression and chemosensitivity to 5-fluorouracil in pancreatic carcinoma cells.
abstract: BACKGROUND
  Thymidylate synthase (TS) is a key regulatory enzyme for de novo DNA synthesis. TS activity is also an important determinant of the response to chemotherapy with fluoropyrimidine prodrugs, and its expression may be affected by gene polymorphisms. In this study, we investigated the associations between polymorphisms of the TS gene and its protein expression, and the implications on the efficacy of 5-fluorouracil (5-FU) in pancreatic cancer cells.
  
  
  METHODS
  Genotypes based on the 28-bp TS tandem repeat for pancreatic cell lines were determined by electrophoretic analysis of PCR products. A single nucleotide polymorphism (SNP) at nucleotide 12 of the second 28-bp repeat of the 3R allele was determined by nucleotide sequencing. The chemosensitivity of pancreatic carcinoma cells to 5-FU in vitro was evaluated using Cell Counting Kit-8 (CCK-8). TS protein expression was analyzed by Western blotting.
  
  
  RESULTS
  Seven pancreatic carcinoma cell lines had different genotypes in terms of the 28-bp TS tandem repeat, as follows: homozygous 2R/2R (T3M4 and BxPC-3 cells), heterozygous 2R/3R (AsPC-1, Capan-1, and SU86.86), and homozygous 3R/3R (PANC-1 and COLO357). The optical density ratio of genotypes 3R/3R, 2R/2R and 2R/3R was 1.393 ± 0.374, 0.568 ± 0.032 and 0.561 ± 0.056, respectively. Cells with the 2R/3R or 3R/3R genotypes were further analyzed for the G to C SNP at nucleotide 12 of the second 28-bp repeat of the 3R allele, yielding heterozygous 2R/3Rc (AsPC-1, Capan-1, and SU86.86), homozygous 3Rg/3Rg (COLO357) and homozygous 3Rc/3Rc (PANC-1). The optical density ratio of homozygous 3Rg/3Rg cells and homozygous 3Rc/3Rc cells was 1.723 ± 0.062 and 1.063 ± 0.134, respectively, and this difference was statistically significant (P < 0.05). Cells with the 2R/2R and 2R/3R genotypes of TS were hypersensitive to 5-FU in vitro as compared with those with the 3R/3R cells.
  
  
  CONCLUSIONS
  Polymorphisms in the TS gene influenced its protein expression and affected sensitivity of 5-FU in seven pancreatic cancer cell lines. Cells with the 3R/3R genotype had higher TS protein expression than the 2R/2R or 2R/3R genotypes. Cells of the 3R/3R genotype with high TS protein expression were shown lower 5-FU sensitivity than cells with the 2R/2R or 2R/3R genotypes. These data warrant large-scale clinical studies to assess the role of polymorphisms in the TS gene on its protein expression and chemosensitivity to 5-FU in pancreatic cancer.
authors.affiliation: Department of General Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Zhang
authors.forename: Yu-Pei
authors.initials: YP
authors.lastname: Zhao
authors.forename: Quan
authors.initials: Q
authors.lastname: Liao
authors.forename: Ya
authors.initials: Y
authors.lastname: Hu
authors.forename: Qiang
authors.initials: Q
authors.lastname: Xu
authors.forename: Li
authors.initials: L
authors.lastname: Zhou
authors.forename: Hong
authors.initials: H
authors.lastname: Shu
mesh_terms: D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D005472:Fluorouracil / Q000494:pharmacology*; D006801:Humans; D018598:Minisatellite Repeats / Q000235:genetics; D010190:Pancreatic Neoplasms / Q000235:genetics; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D013940:Thymidylate Synthase / Q000235:genetics*
chemical_list: D013940:Thymidylate Synthase; D005472:Fluorouracil