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

John Boyer, Estelle G. McLean, Somaiah Aroori, Peter Wilson, Andrea McCulla, P. Declan Carey, Daniel B. Longley, Patrick G. Johnston
2004 Clinical Cancer Research  
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
more » ... riod 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 ϳ5-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. RESULTS Cytotoxicity Analyses. By repeated exposure to stepwise increasing concentrations of drug over a period of 10
doi:10.1158/1078-0432.ccr-03-0362 pmid:15041737 fatcat:sqd5w4cyrbflzacjzjxaqhjr2m