title: Characterization of p53 wild-type and null isogenic colorectal cancer cell lines resistant to 5-fluorouracil, oxaliplatin, and irinotecan.
abstract: To elucidate mechanisms of resistance to chemotherapies currently used in the first-line treatment of advanced colorectal cancer, we have developed a panel of HCT116 p53 wild-type (p53(+/+)) and null (p53(-/-)) isogenic colorectal cancer cell lines resistant to the antimetabolite 5-fluorouracil (5-FU), topoisomerase I inhibitor irinotecan (CPT-11), and DNA-damaging agent oxaliplatin. These cell lines were generated by repeated exposure to stepwise increasing concentrations of each drug over a period of several months. We have demonstrated a significant decrease in sensitivity to 5-FU, CPT-11, and oxaliplatin in each respective resistant cell line relative to the parental line as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis, with increases in IC(50 (72 h)) concentrations ranging from 3- to 65-fold. Using flow cytometry, we have also demonstrated compromised apoptosis and cell cycle arrest in 5-FU-, oxaliplatin-, and CPT-11-resistant cell lines compared with the parental lines after exposure to each drug. In addition, we found that resistance to 5-FU and oxaliplatin was higher in parental p53(-/-) cells compared with parental p53(+/+) cells, with an approximately 5-fold increase in IC(50 (72 h)) for each drug. In contrast, the IC(50 (72 h)) doses for CPT-11 were identical in the p53 wild-type and null cell lines. Furthermore, apoptosis after treatment with 5-FU and oxaliplatin, but not CPT-11, was significantly reduced in parental p53(-/-) cells compared with parental p53(+/+) cells. These data suggest that p53 may be an important determinant of sensitivity to 5-FU and oxaliplatin but not CPT-11. Using semiquantitative reverse transcription-PCR, we have demonstrated down-regulation of thymidine phosphorylase mRNA in both p53(+/+) and p53(-/-) 5-FU-resistant cells, suggesting that decreased production of 5-FU active metabolites may be an important resistance mechanism in these lines. In oxaliplatin-resistant cells, we noted increased mRNA levels of the nucleotide excision repair gene ERCC1 and ATP-binding cassette transporter breast cancer resistance protein. In CPT-11-resistant cells, we found reduced mRNA levels of carboxylesterase, the enzyme responsible for converting CPT-11 to its active metabolite SN-38, and topoisomerase I, the SN-38 target enzyme. In addition, we noted overexpression of breast cancer resistance protein in the CPT-11-resistant lines. These cell lines are ideal tools with which to identify novel determinants of drug resistance in both the presence and absence of wild-type p53.
authors.affiliation: Department of Oncology, Queen's University Belfast, Belfast, Northern Ireland.
authors.forename: John
authors.initials: J
authors.lastname: Boyer
authors.forename: Estelle G
authors.initials: EG
authors.lastname: McLean
authors.forename: Somaiah
authors.initials: S
authors.lastname: Aroori
authors.forename: Peter
authors.initials: P
authors.lastname: Wilson
authors.forename: Andrea
authors.initials: A
authors.lastname: McCulla
authors.forename: P Declan
authors.initials: PD
authors.lastname: Carey
authors.forename: Daniel B
authors.initials: DB
authors.lastname: Longley
authors.forename: Patrick G
authors.initials: PG
authors.lastname: Johnston
mesh_terms: D000970:Antineoplastic Agents / Q000633:toxicity; D017209:Apoptosis / Q000187:drug effects; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000633:toxicity*; D015179:Colorectal Neoplasms / Q000235:genetics*; D019008:Drug Resistance, Neoplasm; D005472:Fluorouracil / Q000633:toxicity*; D016158:Genes, p53 / Q000235:genetics*; D006801:Humans; D000077146:Irinotecan; D009944:Organoplatinum Compounds / Q000633:toxicity*; D000077150:Oxaliplatin; D016159:Tumor Suppressor Protein p53 / Q000172:deficiency* / Q000235:genetics*
chemical_list: D000970:Antineoplastic Agents; D009944:Organoplatinum Compounds; D016159:Tumor Suppressor Protein p53; D000077150:Oxaliplatin; D000077146:Irinotecan; D005472:Fluorouracil; D002166:Camptothecin