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Regulation of the human AP-endonuclease (APE1/Ref-1) expression by the tumor suppressor p53 in response to DNA damage.

Zaky A, Busso C, Izumi T, Chattopadhyay R, Bassiouny A, Mitra S, Bhakat KK - Nucleic Acids Res. (2008)

Bottom Line: Time-dependent decrease was observed in APE1 mRNA and protein levels in the human colorectal cancer line HCT116 p53(+/+), but not in the isogenic p53 mutant after treatment with camptothecin, a DNA topoisomerase I inhibitor.Furthermore, ectopic expression of WTp53 in the p53 cells significantly reduced both endogenous APE1 and APE1 promoter-dependent luciferase expression in a dose-dependent fashion.Taken together, our results demonstrate that WTp53 is a negative regulator of APE1 expression, so that repression of APE1 by p53 could provide an additional pathway for p53-dependent induction of apoptosis in response to DNA damage.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Sealy Center for Molecular Medicine, University of Texas Medical Branch, TX-77555, Galveston, USA.

ABSTRACT
The human AP-endonuclease (APE1/Ref-1), an essential multifunctional protein, plays a central role in the repair of oxidative base damage via the DNA base excision repair (BER) pathway. The mammalian AP-endonuclease (APE1) overexpression is often observed in tumor cells, and confers resistance to various anticancer drugs; its downregulation sensitizes tumor cells to those agents via induction of apoptosis. Here we show that wild type (WT) but not mutant p53 negatively regulates APE1 expression. Time-dependent decrease was observed in APE1 mRNA and protein levels in the human colorectal cancer line HCT116 p53(+/+), but not in the isogenic p53 mutant after treatment with camptothecin, a DNA topoisomerase I inhibitor. Furthermore, ectopic expression of WTp53 in the p53 cells significantly reduced both endogenous APE1 and APE1 promoter-dependent luciferase expression in a dose-dependent fashion. Chromatin immunoprecipitation assays revealed that endogenous p53 is bound to the APE1 promoter region that includes a Sp1 site. We show here that WTp53 interferes with Sp1 binding to the APE1 promoter, which provides a mechanism for the downregulation of APE1. Taken together, our results demonstrate that WTp53 is a negative regulator of APE1 expression, so that repression of APE1 by p53 could provide an additional pathway for p53-dependent induction of apoptosis in response to DNA damage.

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Effect of WTp53 on APE1 promoter reporter activity. (A) Basal promoter activity of APE1 promoter luciferase constructs containing −4500 or −1800 bp of APE1 promoter sequences linked to the luciferase gene. (B) p53(−/−) cells were transiently transfected with 0.5 μg of APE1 promoter–luciferase plasmid (−1800/+65) and WTp53 or control vector as before. Luciferase activity was measured 48 h after transfection as before; *P < 0.05.
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Figure 4: Effect of WTp53 on APE1 promoter reporter activity. (A) Basal promoter activity of APE1 promoter luciferase constructs containing −4500 or −1800 bp of APE1 promoter sequences linked to the luciferase gene. (B) p53(−/−) cells were transiently transfected with 0.5 μg of APE1 promoter–luciferase plasmid (−1800/+65) and WTp53 or control vector as before. Luciferase activity was measured 48 h after transfection as before; *P < 0.05.

Mentions: To identify the cis element(s) responsible for p53-mediated downregulation of the APE1 promoter, we carried out promoter deletion analysis. We have shown earlier that the APE1 promoter contains multiple negative regulatory and enhancer elements, including two nCaRE-B elements upstream (−4000 to −1800 bp) of the basal promoter (15). To test whether p53-induced repression of APE1 promoter activity is mediated through the cis elements located in this region, we cotransfected HCT116 p53(−/−) cells with WTp53 and the reporter plasmid containing −1800 to +65 bp (−1800/+65 APE1-Luc) of the APE1 promoter. Deletion of the promoter sequence from −4000 to −1800 bp significantly increased the basal promoter activity (∼9-fold, Figure 4A), confirming our earlier observation about the presence of negative regulatory elements within this sequence (15). Furthermore, ectopic expression of WTp53 caused a decrease in the promoter (−1800/+65 APE1-Luc) activity in a dose-dependent manner (Figure 4B).Figure 4.


Regulation of the human AP-endonuclease (APE1/Ref-1) expression by the tumor suppressor p53 in response to DNA damage.

Zaky A, Busso C, Izumi T, Chattopadhyay R, Bassiouny A, Mitra S, Bhakat KK - Nucleic Acids Res. (2008)

Effect of WTp53 on APE1 promoter reporter activity. (A) Basal promoter activity of APE1 promoter luciferase constructs containing −4500 or −1800 bp of APE1 promoter sequences linked to the luciferase gene. (B) p53(−/−) cells were transiently transfected with 0.5 μg of APE1 promoter–luciferase plasmid (−1800/+65) and WTp53 or control vector as before. Luciferase activity was measured 48 h after transfection as before; *P < 0.05.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2275136&req=5

Figure 4: Effect of WTp53 on APE1 promoter reporter activity. (A) Basal promoter activity of APE1 promoter luciferase constructs containing −4500 or −1800 bp of APE1 promoter sequences linked to the luciferase gene. (B) p53(−/−) cells were transiently transfected with 0.5 μg of APE1 promoter–luciferase plasmid (−1800/+65) and WTp53 or control vector as before. Luciferase activity was measured 48 h after transfection as before; *P < 0.05.
Mentions: To identify the cis element(s) responsible for p53-mediated downregulation of the APE1 promoter, we carried out promoter deletion analysis. We have shown earlier that the APE1 promoter contains multiple negative regulatory and enhancer elements, including two nCaRE-B elements upstream (−4000 to −1800 bp) of the basal promoter (15). To test whether p53-induced repression of APE1 promoter activity is mediated through the cis elements located in this region, we cotransfected HCT116 p53(−/−) cells with WTp53 and the reporter plasmid containing −1800 to +65 bp (−1800/+65 APE1-Luc) of the APE1 promoter. Deletion of the promoter sequence from −4000 to −1800 bp significantly increased the basal promoter activity (∼9-fold, Figure 4A), confirming our earlier observation about the presence of negative regulatory elements within this sequence (15). Furthermore, ectopic expression of WTp53 caused a decrease in the promoter (−1800/+65 APE1-Luc) activity in a dose-dependent manner (Figure 4B).Figure 4.

Bottom Line: Time-dependent decrease was observed in APE1 mRNA and protein levels in the human colorectal cancer line HCT116 p53(+/+), but not in the isogenic p53 mutant after treatment with camptothecin, a DNA topoisomerase I inhibitor.Furthermore, ectopic expression of WTp53 in the p53 cells significantly reduced both endogenous APE1 and APE1 promoter-dependent luciferase expression in a dose-dependent fashion.Taken together, our results demonstrate that WTp53 is a negative regulator of APE1 expression, so that repression of APE1 by p53 could provide an additional pathway for p53-dependent induction of apoptosis in response to DNA damage.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Sealy Center for Molecular Medicine, University of Texas Medical Branch, TX-77555, Galveston, USA.

ABSTRACT
The human AP-endonuclease (APE1/Ref-1), an essential multifunctional protein, plays a central role in the repair of oxidative base damage via the DNA base excision repair (BER) pathway. The mammalian AP-endonuclease (APE1) overexpression is often observed in tumor cells, and confers resistance to various anticancer drugs; its downregulation sensitizes tumor cells to those agents via induction of apoptosis. Here we show that wild type (WT) but not mutant p53 negatively regulates APE1 expression. Time-dependent decrease was observed in APE1 mRNA and protein levels in the human colorectal cancer line HCT116 p53(+/+), but not in the isogenic p53 mutant after treatment with camptothecin, a DNA topoisomerase I inhibitor. Furthermore, ectopic expression of WTp53 in the p53 cells significantly reduced both endogenous APE1 and APE1 promoter-dependent luciferase expression in a dose-dependent fashion. Chromatin immunoprecipitation assays revealed that endogenous p53 is bound to the APE1 promoter region that includes a Sp1 site. We show here that WTp53 interferes with Sp1 binding to the APE1 promoter, which provides a mechanism for the downregulation of APE1. Taken together, our results demonstrate that WTp53 is a negative regulator of APE1 expression, so that repression of APE1 by p53 could provide an additional pathway for p53-dependent induction of apoptosis in response to DNA damage.

Show MeSH
Related in: MedlinePlus