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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 Ser(15) and Ser(392), 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.
Highlights
The development and progression of colon cancer is a multistep process in which growth control is progressively impaired
Other than p53-binding sites, using deletion mutation constructs, we have shown that N-methyl-N-nitro-N-nitrosoguanidine-induced transcriptional activation of the p53-binding sites on the cloned APC promoter (pAPCP) promoter in HCT116(p53؉/؉) cells depended upon the Sp1-binding site and the E-box B site
We and others have reported that p53 is phosphorylated on Ser15 and Ser392 in HCT-116 cells treated with DNA alkylating agents N-methyl-N-nitrosourea, N-methyl-NЈ-nitro-N-nitrosoguanidine (MNNG), and methylmethane sulfonate (MMS) [37]
Summary
The development and progression of colon cancer is a multistep process in which growth control is progressively impaired. Mutations of the APC1 (adenomatous polyposis coli), Ki-ras, Mutations in the APC gene are found in 60 – 80% of sporadic colorectal cancers and adenomas [1]. Mutations in APC are associated with malignant brain tumors (Turcot’s syndrome; Ref. 5), and the APC locus on chromosoyme 5q21 shows loss of heterozygosity in ϳ25% of breast cancers [6]. Loss of heterozygosity at the APC gene locus is prominent in the early stages of non-small cell lung cancers [8]. Wingless/Wnt signaling regulates the assembly of a complex consisting of Axin (and its homolog Axil and conductin), APC, -catenin, and glycogen synthase-3 kinase (GSK3). Tumor suppressor p53, which plays a critical role in the cellular response to DNA damage, oxidative stress, and hypoxia, is frequently mutated in colon cancer cells We and others have reported that p53 is phosphorylated on Ser and Ser392 in HCT-116 cells treated with DNA alkylating agents N-methyl-N-nitrosourea, N-methyl-NЈ-nitro-N-nitrosoguanidine (MNNG), and methylmethane sulfonate (MMS) [37].2
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