Paclitaxel sensitivity correlates with p53 status and DNA fragmentation, but not G2/M accumulation

Purpose: The antitumor agent paclitaxel (Taxol(R)) has been shown to arrest cells in mitosis through microtubule stabilization and to induce apoptosis . The tumor suppressor gene p53, is implicated in the regulation of cell cy cle checkpoints and can mediate apoptotic cell death. Although initial stud ies demonstrated that various DNA-damaging agents can induce p53, more rece nt studies have also shown p53 induction following nonDNA-damaging agents, including paclitaxel. We investigated the influence of p53 abrogation on pa clitaxel-induced cell kill and correlated the extent of mitotic arrest and DNA fragmentation by paclitaxel with the drug's cytotoxic effect. Materials and Methods: The parental human colorectal carcinoma cell line (R KO) with wild-type p53 alleles, and two transfected RKO cell lines with ina ctivated p53 (RKO.p53.13 with transfected mutant p53,7 and RC 10.3 with HPV -16-derived E6 gene) were exposed to graded doses of paclitaxel (1-100 nM) for 24-h intervals. The functional status of p53 in cells was assessed by t hymidine and BrdU incorporation following exposure to ionizing radiation ( 4 Gy). Reproductive integrity following paclitaxel treatment was assessed b y clonogenic assay. Immunolabeling and microscopic evaluation were used to assess mitotic accumulation and micronucleation. Apoptosis was assayed usin g DNA fragmentation analyses. Results: A 4-fold increase in paclitaxel sensitivity was observed among RKO cells deficient in p53 function compared with wild-type RKO cells (IC 50: 4 nM I, 1 nM, 1nM for RKO, RKO,p53.13, RC 10.3, respectively). The increase d cytotoxic effect in RKO cells with inactive p53 correlated with an increa sed propensity towards micronucleation and DNA fragmentation following pacl itaxel treatment. However, no significant difference in peak mitotic accumu lation was observed among RKO cells with functional or abrogated p53. Conclusions: RKO cells lacking functional p53 demonstrate significantly enh anced sensitivity to paclitaxel compared with that of wild-type RKO cells. This response corresponded with increased micronucleation and DNA fragmenta tion in cells deficient in p53 function. Although previous published report s of enhanced paclitaxel sensitivity in p53-deficient cells correlated this finding with increased G2/M I arrest, we did not observe any significant c orrelation between paclitaxel-induced cell kill and the degree of mitotic a rrest. Our data suggest that apoptosis is the predominant mechanism of pacl itaxel cytotoxicity in RKO cells and is likely mediated by a p53-independen t process. (C) 1999 Elsevier Science Inc.