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The effect of p53-RNAi and p53 knockout on human 8-oxoguanine DNA glycosylase (hOgg1) activity

Department of Otolaryngology-Head and Neck Surgery, Head and Neck Cancer Research Division, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

²Correspondence: Department of Otolaryngology-Head and Neck Surgery, Head and Neck Cancer Research Division, 720 Rutland Ave., Johns Hopkins University School of Medicine, 818 Ross Research Building, Baltimore, MD 21205-2196, USA. E-mail: dsidrans{at}jhmi.edu

SPECIFIC AIMS

Base excision repair pathway repairs a wide range of DNA base modifications. Among them is 8-oxo-7, 8 dihydroguanine (8-oxoG), which pairs with adenine and cytosine during DNA replication, resulting in GC to TA transversion mutations that have been commonly observed in tumor suppressor genes in cancer. In humans, 8-oxoG incorporated into DNA is removed by hOgg1, a DNA glycosylase/AP lyase that specifically incises 8-oxoG opposite cytosine. The aim of this study was to determine the role of p53 in the regulation of the DNA repair gene hOGG1, and further determine the effect of p53 on hOgg1 protein activity.

PRINCIPAL FINDINGS

1. Suppression and/or knock out of p53 decreases hOGG1 mRNA and hOgg1 protein expression
MCF12A cells transfected with p53-RNAi oligo and the empty vector were selected with 350 µg/mL of G418. The selected colonies were screened for positive down-regulation of p53 in the MCF12A cell line by Western blot analysis. We performed RT-PCR to analyze hOGG1 expression in p53 knockdown cells (p53-RNAi) and in p53 knockout cells. Our results indicated that both nuclear (–hOGG1) and mitochondrial (ß–hOGG1) cDNA were down-regulated in the p53-RNAi transfected cells (Fig. 1 A). However, -hOGG1 was significantly down-regulated (>50%) by the presence of either p53-RNAi or hOGG1-RNAi. The effect of p53 on hOGG1 transcription was further confirmed by testing hOGG1 expression in isogenic cell lines, HCT116^p53+/+ and HCT116^p53–/–. RT-PCR analysis showed that hOGG1 mRNA expression was severely reduced in the HCT116^p53–/– cells when compared with the HCT116^p53+/+ cells (Fig. 1A ). Western blot analysis also indicated that hOgg1 expression was reduced in MCF12A-p53-RNAi cells. Western blot analysis was unable to detect hOgg1 protein in the HCT116^p53–/– cells (Fig. 1B ) despite the slight expression of cDNA. Figure 1B also shows that the p53 null cells were able to overexpress hOgg1 upon transient transfection of the hOGG1 expression plasmid. These results indicate that suppression of p53 resulted in decreased expression of hOGG1 both at the mRNA and protein levels.

View larger version (43K): [in this window] [in a new window]   Figure 1. Regulation of hOGG1 expression by p53.A) RT-PCR analysis of hOGG1 in cells transfected with vector, p53-RNAi, and hOGG1-RNAi and RT-PCR analysis of hOGG1 in HCT116p53+/+ and HCT116p53–/– cells. B) Western blot analysis of hOgg1 in HCT116p53+/+, HCT116p53–/–, and HCT116p53–/– transfected with hOGG1 expression vector.

2. Knockout of p53 abolished hOgg1 activity
To determine the effect of p53 on hOgg1 protein activity, we measured the 8-oxoG incision activity in the nuclear extracts of MCF12A-p53-RNAi, MCF12A-Vector, and HCT116^p53+/+ and HCT116^p53–/–. The assay is based on the cleavage of an 8-oxoG containing double-stranded oligonucleotide. Figure 2 A shows representative results of the incision assay. Fpg, an enzyme known to incise 8-oxoG opposite cytosine resulted in the expected 10-mer product (Fig. 2A , lane Fpg). The nuclear extracts incised 8-oxoG, producing the expected 10-mer product (Fig. 2A , lanes 10–150, indicating the microgram quantity of protein lysate used). No incision was observed when the control oligonucleotide lacking 8-oxoG was incubated with the nuclear extracts (Fig. 2A , lane C). No incision product was observed in the absence of the extract (Fig. 2A , lanes labeled 0), indicating the specificity of the incision for the lesion. Figure 2B represents a kinetic analysis of 8-oxoG incision using 50 µg of protein extracts. Knockout of p53 in HCT116 cells severely reduced 8-oxoG incision, resulting in 80% decrease (at all time points except 24 h) in activity compared with the HCT116^p53+/+ cells (Fig. 2B ). We further supplemented the HCT116^p53–/– cells with hOGG1, by transiently transfecting the HCT116 p53 null cells with hOGG1 expression plasmid, and determined the 8-oxoG incision activity. These results showed that hOGG1 plasmid transfection corrected the repair defect in the p53 null cells (compare and in Fig. 2B ) to a level higher than the wild-type HCT116^p53+/+ cells. The increased activity in the hOGG1 transfected cells was due to overexpression of the hOgg1 protein as observed in Fig. 1B .

View larger version (44K): [in this window] [in a new window]   Figure 2. 8-oxoG incision activity by nuclear extracts of p53 down-regulated cell lines. A) Dose-dependent incision of 8-oxoG. 5' end-labeled duplex oligonucleotide containing a single 8-oxoG was incubated at 37°C with indicated amount of MCF12A-p53-RNAi and MCF12A-Vector nuclear extracts for 6 h. Lane C indicates control oligonucleotide lacking 8-oxoG, and lanes labeled 0–150 indicate the amounts (in micrograms) of nuclear extract used per reaction. Lane Fpg indicates incubation of 8-oxoG oligonucleotide with formamidopyrimidine glycosylase, an enzyme specific for 8-oxoG cleavage. B) Graphical representation of the kinetic analysis of 8-oxoG incision assay using 50 µg of nuclear extracts from HCT116p53+/+ (), HCT116p53–/– (), and HCT116p53–/– cells transfected with hOGG1 expression plasmid ().

CONCLUSIONS AND SIGNIFICANCE

Our findings that p53 knockout resulted in a severe reduction in hOGG1 mRNA and hOgg1 protein levels suggest that p53 is a major regulator of hOGG1. Our results also showed that p53 knockout almost completely abolished hOgg1 activity when compared with the isogenic p53^+/+ cells. To our knowledge, this is the first report to show that p53 knockout results in an almost complete abolishment of the hOgg1 activity. The fact that very little hOGG1 cDNA was detected in HCT116^p53–/– but hOgg1 protein was undetectable in the same cell line suggests that p53 may have a stabilizing role of hOgg1 protein in addition to regulating its expression. Recent evidence in the literature showed a direct interaction of p53 and hOgg1 proteins and that p53 knockout in HCT116 cell line resulted in increased 8-oxoG lesions in the genomic DNA. However, there was no demonstration that hOGG1 itself is regulated by p53. Using reconstitution studies, Achanta et al. showed that addition of p53 protein increased the ability to incise 8-oxoG from a synthetic oligonucleotide, suggesting involvement of p53 in damage recognition and enhancement of glycosylase activity. The down-regulation of hOGG1 mRNA in p53 knockdown and knockout cells suggest a transcriptional regulation of hOGG1 by p53. Further analysis of the hOGG1 promoter revealed a putative p53 binding element in the region –945 to –726, which was shown to bind the p53 protein as evidenced by the EMSA results. Our findings provide a new and different mechanism of p53 in 8-oxoG repair from the current literature. Our findings and those in the current literature suggest that p53 acts at several levels that include interaction with APE and Pol ß, binding to hOgg1 and binding to the damaged DNA, enhancement of incision of damaged base, and possibly transcriptional regulation of hOGG1. Based on our findings that the p53 knockout cells were almost devoid of hOGG1 mRNA and hOgg1 protein expression and that the 8-oxoG incision activity was severely decreased, we believe that p53 is a major regulator of hOGG1 expression and hOgg1 activity, and that the other regulators of this gene most likely occur downstream of p53. Because of the potential role of hOGG1 in cancer and the established role of p53 in cancer, it is important to understand the relationships between these two genes in their contribution to carcinogenesis.

View larger version (24K): [in this window] [in a new window]   Figure 3. Schematic representation of the mechanism of hOGG1 gene activation and glycosylase activity enhancement by p53. p53 protein binds to the p53 response element on the hOGG1 promoter resulting in direct transactivation of the hOGG1 promoter. Binding of p53 to the hOGG1 promoter results in increased hOGG1 mRNA, hOgg1 protein levels, and increased gylcosylase activity. This direct binding is responsible for part of the promoter activity. p53 can also activate other p53-inducible genes whose gene products can bind directly to the hOGG1 promoter thereby resulting in increased hOGG1 transcription and hOgg1 protein activity. Knockout of p53 leads to severe reduction in hOGG1 mRNA and hOgg1 protein and to abolishment of Ogg1 activity.

FOOTNOTES

¹ These authors contributed equally to this work.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3423fje;

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