The p53
tumor suppressor is an important regulator of cellular response to chemotherapeutic agents by virtue of the
protein's ability, upon activation by phosphorylation, to transcriptionally activate a number of genes involved in cell proliferation, apoptosis, and metabolism. Transcriptome analysis following introduction of a constitutively active form of p53 (p53T18D/S20D) into colon
carcinoma cell lines identified transcriptional activation of the
carboxylesterase 2 (CES-2) gene, which is involved in
drug metabolism. We examined whether p53 activated by the
DNA-damaging
drug 5-fluorouracil (5-FU) also induces CES-2 expression. Our experiments showed that
5-FU induced CES-2 expression in two colon
carcinoma cell lines that express wild-type p53 (HCT116 p53(+/+) and RKO) but not in five lines that are p53-null (HCT116 p53(-/- )) or express mutated p53 (HT29, KM12C, KM12SM, and KM12L4A). Sequence analysis revealed a putative p53-binding
element in the first intron of CES-2 that differed from consensus by one
nucleotide. A reporter gene assay showed that the
luciferase construct with the p53-binding
element responded to
5-FU treatment, whereas the reporter construct without the binding
element did not.
Chromatin immunoprecipitation assay confirmed that p53 bound the CES-2 fragment containing the p53-binding
element after
5-FU treatment, whereas p21 binding to p53 was present with or without
chemotherapy. Knockdown of expression of CES-2 and p53 by small interference RNA in RKO and HCT116 p53(+/+) cells attenuated the anti-proliferation effects of
CPT11. These results taken together show that activated p53 directly regulates CES-2 expression via a p53-binding site, representing a novel mechanism through which the p53 pathway modulates
drug metabolism. In addition, the degree of homology in the p53-binding
element may determine the strength of p53 regulation.