Characterization of p53 wild-type and null isogenic colorectal cancer cell lines resistant to 5-fluorouracil, oxaliplatin, and irinotecan
Characterization of p53 wild-type and null isogenic colorectal cancer cell lines resistant to 5-fluorouracil, oxaliplatin, and irinotecan
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DOI：
10.1158/1078-0432.ccr-03-0362
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发表时间：
2004-03-15
影响因子：
11.5
通讯作者：
Johnston, PG
中科院分区：
文献类型：
--
作者：
Boyer, J;McLean, EG;Johnston, PG
为了阐明目前用于晚期结直肠癌一线治疗的化疗耐药机制，我们开发了一组HCT 116 p53野生型（p53（+/+））和无效（p53（-/-））等基因结直肠癌细胞系，对抗代谢药5-氟尿嘧啶（5-FU）、拓扑异构酶I抑制剂伊立替康（CPT-11）和DNA损伤剂奥沙利铂耐药。这些细胞系是通过在几个月的时间内重复暴露于逐步增加浓度的每种药物而产生的。通过3（4，5-二甲基噻唑-2-基）-2，5-二苯基溴化四氮唑分析，我们已经证明，相对于亲本细胞系，各耐药细胞系对5-FU、CPT-11和奥沙利铂的敏感性显著降低，IC 50（72 h）浓度增加3- 65倍。使用流式细胞术，我们还证明了与暴露于每种药物后的亲本细胞系相比，5-FU-，奥沙利铂-和CPT-11耐药细胞系的细胞凋亡和细胞周期阻滞受损。此外，我们发现亲代p53（-/-）细胞对5-FU和奥沙利铂的耐药性高于亲代p53（+/+）细胞，两种药物的IC 50（72 h）增加了约5倍。相反，CPT-11的IC 50（（72 h））剂量在p53野生型和无效细胞系中相同。此外，与亲代p53（+/+）细胞相比，亲代p53（-/-）细胞经5-FU和奥沙利铂（而非CPT-11）处理后的细胞凋亡显著减少。这些数据表明，p53可能是5-FU和奥沙利铂敏感性的重要决定因素，但不是CPT-11。利用半定量逆转录-PCR，我们已经证明了在p53（+/+）和p53（-/-）5-FU耐药细胞中胸苷磷酸化酶mRNA的下调，这表明5-FU活性代谢产物的产生减少可能是这些细胞系中重要的耐药机制。在奥沙利铂耐药细胞中，我们注意到核苷酸切除修复基因ERCC 1和ATP结合盒转运体乳腺癌耐药蛋白的mRNA水平增加。在CPT-11耐药细胞中，我们发现羧酸酯酶（负责将CPT-11转化为其活性代谢物SN-38的酶）和拓扑异构酶1（SN-38靶酶）的mRNA水平降低。此外，我们注意到乳腺癌耐药蛋白在CPT-11耐药株系中的过表达。这些细胞系是在野生型p53存在和不存在的情况下鉴定新的耐药性决定因素的理想工具。
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 IC50 (72 h) concentrations ranging from 3- to 65-fold. Using now 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 similar to5-fold increase in IC50 ((72 h)) for each drug. In contrast, the IC50 ((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 1, 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.