p53-dependent Transcriptional Regulation of the APC Promoter in Colon Cancer Cells Treated with DNA Alkylating Agents

Aruna S. Jaiswal, Satya Narayan

Research output: Contribution to journalArticle

45 Scopus citations

Abstract

The APC (adenomatous polyposis coli) gene product is involved in cell cycle arrest and in apoptosis. The loss of APC function is associated with the development of colorectal carcinogenesis. In previous studies, we have shown that the APC gene is inducible and that the DNA damage-induced level of APC mRNA requires p53. In the present study, we examined the role of p53 in the transcriptional regulation of APC promoter and characterized two p53-binding sites on the cloned APC promoter (pAPCP). Results of electrophoretic mobility shift assay showed specific interactions of p53 protein with p53-binding site oligonucleotides. The DNA-protein complex formed in electrophoretic mobility shift assay was competed with unlabeled excess of p53-binding site oligonucleotide, unaffected with p53-binding site mutant or Sp1-binding site oligonucleotides, and supershifted with anti-p53 antibodies. In a transient transfection assay, the pAPCP promoter activity was lower in HCT-116(p53 +/+) cells versus HCT-116(p53-/-) cells. p53-dependent down-regulation was further confirmed after co-transfection of pAPCP plasmid with pCMV-p53 into HCT-116(p53-/-) and SAOS-2 (p53-negative) cells. However, the treatment of cells with DNA alkylating agents methylmethane sulfonate and N-methyl-N′-nitro-N-nitrosoguanidine, which cause phosphorylation of p53 at Ser15 and Ser392, induced pAPCP promoter activity in HCT-116(p53+/+) cells. Other than p53-binding sites, using deletion mutation constructs, we have shown that N-methyl-N′-nitro-N-nitrosoguanidine-induced transcriptional activation of the pAPCP promoter in HCT-116(p53+/+) cells depended upon the Sp1-binding site and the E-box B site. From these results, we conclude that unphosphorylated p53 can down-regulate and phosphorylated p53 can up-regulate the pAPCP promoter activity involving the p53, Sp1, or E-box B elements. These studies are important to understanding the role of p53 and APC in DNA damage-induced cell cycle arrest and/or apoptosis of cancer cells.

Original languageEnglish (US)
Pages (from-to)18193-18199
Number of pages7
JournalJournal of Biological Chemistry
Volume276
Issue number21
DOIs
StatePublished - Jan 25 2001
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'p53-dependent Transcriptional Regulation of the APC Promoter in Colon Cancer Cells Treated with DNA Alkylating Agents'. Together they form a unique fingerprint.

  • Cite this