Characterization of p53 Wild-Type and Null Isogenic Colorectal Cancer Cell Lines Resistant to 5-Fluorouracil, Oxaliplatin, and Irinotecan

• Published in: Clinical cancer research Vol. 10; no. 6; pp. 2158 - 2167
• Main Authors: BOYER, John, MCLEAN, Estelle G, AROORI, Somaiah, WILSON, Peter, MCCULLA, Andrea, CAREY, P. Declan, LONGLEY, Daniel B, JOHNSTON, Patrick G
• Format: Journal Article
• Language: English
• Published: Philadelphia, PA American Association for Cancer Research 15.03.2004
• Subjects: Antineoplastic agents; Antineoplastic Agents - toxicity; Apoptosis - drug effects; Biological and medical sciences; Camptothecin - analogs & derivatives; Camptothecin - toxicity; Colorectal Neoplasms - genetics; Drug Resistance, Neoplasm; Fluorouracil - toxicity; Genes, p53 - genetics; Humans; Medical sciences; Organoplatinum Compounds - toxicity; Pharmacology. Drug treatments; Tumor Suppressor Protein p53 - deficiency; Tumor Suppressor Protein p53 - genetics; Tumors; Enzyme; Transferases; Fluoropyrimidine derivatives; Colorectal cancer; DNA topoisomerase; Enzyme inhibitor; Thymidylate synthase; Wild type; Irinotecan; Isomerases; TP53 Gene; Oxaliplatin; Methyltransferases; Antimetabolic; Established cell line; Digestive diseases; Intestinal disease; Pyrimidine derivatives; Isogenic line; Fluorouracil; Tumor suppressor gene; Platinum II Complexes
• ISSN: 1078-0432; 1557-3265
• DOI: 10.1158/1078-0432.CCR-03-0362

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 ∼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.