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Tuberin haploinsufficiency is associated with the loss of OGG1 in rat kidney tumors.

Habib SL, Simone S, Barnes JJ, Abboud HE - Mol. Cancer (2008)

Bottom Line: Loss of heterozygosity (LOH) at the TSC2 locus has been detected in TSC-associated renal cell carcinoma (RCC) and in RCC in the Eker rat.These results demonstrate that OGG1 localizes with tuberin preferentially in kidney cortex.In addition, the predominant expression of OGG1 in the cortex and its decreased expression and activity in the Eker rat may account for the predominant cortical localization of renal cell carcinoma.

View Article: PubMed Central - HTML - PubMed

Affiliation: O'Brien Kidney Research Center, Department of Medicine, University of Texas Health Science Center, San Antonio, TX 78229, USA. habib@uthscsa.edu

ABSTRACT

Background: Tuberous sclerosis complex (TSC) is caused by defects in one of two tumor suppressor genes, TSC-1 or TSC-2. TSC-2 gene encodes tuberin, a protein involved in the pathogenesis of kidney tumors. Loss of heterozygosity (LOH) at the TSC2 locus has been detected in TSC-associated renal cell carcinoma (RCC) and in RCC in the Eker rat. Tuberin downregulates the DNA repair enzyme 8-oxoguanine DNA-glycosylase (OGG1) with important functional consequences, compromising the ability of cells to repair damaged DNA resulting in the accumulation of the mutagenic oxidized DNA, 8-oxo-dG. Loss of function mutations of OGG1 also occurs in human kidney clear cell carcinoma and may contribute to tumorgenesis. We investigated the distribution of protein expression and the activity of OGG1 and 8-oxo-dG and correlated it with the expression of tuberin in kidneys of wild type and Eker rats and tumor from Eker rat.

Results: Tuberin expression, OGG1 protein expression and activity were higher in kidney cortex than in medulla or papilla in both wild type and Eker rats. On the other hand, 8-oxo-dG levels were highest in the medulla, which expressed the lowest levels of OGG1. The basal levels of 8-oxo-dG were also higher in both cortex and medulla of Eker rats compared to wild type rats. In kidney tumors from Eker rats, the loss of the second TSC2 allele is associated with loss of OGG1 expression. Immunostaining of kidney tissue shows localization of tuberin and OGG1 mainly in the cortex.

Conclusion: These results demonstrate that OGG1 localizes with tuberin preferentially in kidney cortex. Loss of tuberin is accompanied by the loss of OGG1 contributing to tumorgenesis. In addition, the predominant expression of OGG1 in the cortex and its decreased expression and activity in the Eker rat may account for the predominant cortical localization of renal cell carcinoma.

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Distribution of renal OGG1 activity and 8-oxodG in cortices (C) and medulla (M) of wild type and Eker rat. A. 21-mer containing an 8-oxoG lesion was labeled at its 5' end using [32P] ATP and incubated with cortex and medulla kidney homogenate of wild type and Eker rat. Oligonucleotide cleavage products were analyzed on DNA sequencing gels and subjected to autoradiography. Pure human OGG1 enzyme (E) and buffer alone (S) were analyzed as positive and negative controls, respectively. The top arrow indicates the 21-mer of 8-oxodG as a substrate and the bottom arrow is the DNA cleavage product (13-mer). B. Histograms represent means ± SE (n = 3). C. DNA was extracted and digested with nuclease P1. The detection of dG and 8-oxodG was performed by HPLC-EC analysis. Authentic standards of 8-oxodG and dG were analyzed simultaneously. Standard curves for dG and 8-oxodG were prepared and quantitated by linear regression analyses. Significant difference from wild type rat is indicated by * P < 0.05 and ** P < 0.01.
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Figure 2: Distribution of renal OGG1 activity and 8-oxodG in cortices (C) and medulla (M) of wild type and Eker rat. A. 21-mer containing an 8-oxoG lesion was labeled at its 5' end using [32P] ATP and incubated with cortex and medulla kidney homogenate of wild type and Eker rat. Oligonucleotide cleavage products were analyzed on DNA sequencing gels and subjected to autoradiography. Pure human OGG1 enzyme (E) and buffer alone (S) were analyzed as positive and negative controls, respectively. The top arrow indicates the 21-mer of 8-oxodG as a substrate and the bottom arrow is the DNA cleavage product (13-mer). B. Histograms represent means ± SE (n = 3). C. DNA was extracted and digested with nuclease P1. The detection of dG and 8-oxodG was performed by HPLC-EC analysis. Authentic standards of 8-oxodG and dG were analyzed simultaneously. Standard curves for dG and 8-oxodG were prepared and quantitated by linear regression analyses. Significant difference from wild type rat is indicated by * P < 0.05 and ** P < 0.01.

Mentions: To determine if the decreases in OGG1 protein expression correlates with the enzymatic activity, OGG1 activity was compared in kidneys from wild type or Eker rats. DNA glycosylase activity of the OGG1 enzyme was assayed as the cleavage of an 8-oxoG-containing oligomer, which releases oxidized guanine base from a 32P-labeled 21 oligonuleotide. OGG1 activity was higher in cortex compared to the medulla (Fig. 2A). OGG1 activity was significantly lower in kidney cortex and medulla of Eker rats compared to wild type rats (Fig. 2B). OGG1 is the major DNA base excision repair enzyme that recognizes and excises 8-oxodG, therefore we determined whether the change in OGG1 abundance influenced the accumulation of 8-oxodG, a substrate of OGG1 enzyme. In order to evaluate the basal level of oxidative DNA damage in the cortices and inner medullae of Eker and wild type rats, we examined the content of 8-oxo-dG in the nuclear DNA extracted from both section using HPLC-EC. DNA was isolated from kidney cortices and medulla of Eker and wild type rats and 8-oxodG levels were analyzed by HPLC. 8-OxodG levels were higher in kidney tissue from Eker compared to wild type rats (Fig. 2C). The levels of 8-oxodG in nuclear DNA were significantly higher (2 fold) in cortices of Eker compared to wild type rat. The levels of 8-oxodG were significantly higher in kidney medullae compared to kidney cortices. These data suggest that OGG1 protein and activity express the low levels in inner medullae may not be sufficient to repair the generated 8-oxodG compared to cortex region.


Tuberin haploinsufficiency is associated with the loss of OGG1 in rat kidney tumors.

Habib SL, Simone S, Barnes JJ, Abboud HE - Mol. Cancer (2008)

Distribution of renal OGG1 activity and 8-oxodG in cortices (C) and medulla (M) of wild type and Eker rat. A. 21-mer containing an 8-oxoG lesion was labeled at its 5' end using [32P] ATP and incubated with cortex and medulla kidney homogenate of wild type and Eker rat. Oligonucleotide cleavage products were analyzed on DNA sequencing gels and subjected to autoradiography. Pure human OGG1 enzyme (E) and buffer alone (S) were analyzed as positive and negative controls, respectively. The top arrow indicates the 21-mer of 8-oxodG as a substrate and the bottom arrow is the DNA cleavage product (13-mer). B. Histograms represent means ± SE (n = 3). C. DNA was extracted and digested with nuclease P1. The detection of dG and 8-oxodG was performed by HPLC-EC analysis. Authentic standards of 8-oxodG and dG were analyzed simultaneously. Standard curves for dG and 8-oxodG were prepared and quantitated by linear regression analyses. Significant difference from wild type rat is indicated by * P < 0.05 and ** P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Distribution of renal OGG1 activity and 8-oxodG in cortices (C) and medulla (M) of wild type and Eker rat. A. 21-mer containing an 8-oxoG lesion was labeled at its 5' end using [32P] ATP and incubated with cortex and medulla kidney homogenate of wild type and Eker rat. Oligonucleotide cleavage products were analyzed on DNA sequencing gels and subjected to autoradiography. Pure human OGG1 enzyme (E) and buffer alone (S) were analyzed as positive and negative controls, respectively. The top arrow indicates the 21-mer of 8-oxodG as a substrate and the bottom arrow is the DNA cleavage product (13-mer). B. Histograms represent means ± SE (n = 3). C. DNA was extracted and digested with nuclease P1. The detection of dG and 8-oxodG was performed by HPLC-EC analysis. Authentic standards of 8-oxodG and dG were analyzed simultaneously. Standard curves for dG and 8-oxodG were prepared and quantitated by linear regression analyses. Significant difference from wild type rat is indicated by * P < 0.05 and ** P < 0.01.
Mentions: To determine if the decreases in OGG1 protein expression correlates with the enzymatic activity, OGG1 activity was compared in kidneys from wild type or Eker rats. DNA glycosylase activity of the OGG1 enzyme was assayed as the cleavage of an 8-oxoG-containing oligomer, which releases oxidized guanine base from a 32P-labeled 21 oligonuleotide. OGG1 activity was higher in cortex compared to the medulla (Fig. 2A). OGG1 activity was significantly lower in kidney cortex and medulla of Eker rats compared to wild type rats (Fig. 2B). OGG1 is the major DNA base excision repair enzyme that recognizes and excises 8-oxodG, therefore we determined whether the change in OGG1 abundance influenced the accumulation of 8-oxodG, a substrate of OGG1 enzyme. In order to evaluate the basal level of oxidative DNA damage in the cortices and inner medullae of Eker and wild type rats, we examined the content of 8-oxo-dG in the nuclear DNA extracted from both section using HPLC-EC. DNA was isolated from kidney cortices and medulla of Eker and wild type rats and 8-oxodG levels were analyzed by HPLC. 8-OxodG levels were higher in kidney tissue from Eker compared to wild type rats (Fig. 2C). The levels of 8-oxodG in nuclear DNA were significantly higher (2 fold) in cortices of Eker compared to wild type rat. The levels of 8-oxodG were significantly higher in kidney medullae compared to kidney cortices. These data suggest that OGG1 protein and activity express the low levels in inner medullae may not be sufficient to repair the generated 8-oxodG compared to cortex region.

Bottom Line: Loss of heterozygosity (LOH) at the TSC2 locus has been detected in TSC-associated renal cell carcinoma (RCC) and in RCC in the Eker rat.These results demonstrate that OGG1 localizes with tuberin preferentially in kidney cortex.In addition, the predominant expression of OGG1 in the cortex and its decreased expression and activity in the Eker rat may account for the predominant cortical localization of renal cell carcinoma.

View Article: PubMed Central - HTML - PubMed

Affiliation: O'Brien Kidney Research Center, Department of Medicine, University of Texas Health Science Center, San Antonio, TX 78229, USA. habib@uthscsa.edu

ABSTRACT

Background: Tuberous sclerosis complex (TSC) is caused by defects in one of two tumor suppressor genes, TSC-1 or TSC-2. TSC-2 gene encodes tuberin, a protein involved in the pathogenesis of kidney tumors. Loss of heterozygosity (LOH) at the TSC2 locus has been detected in TSC-associated renal cell carcinoma (RCC) and in RCC in the Eker rat. Tuberin downregulates the DNA repair enzyme 8-oxoguanine DNA-glycosylase (OGG1) with important functional consequences, compromising the ability of cells to repair damaged DNA resulting in the accumulation of the mutagenic oxidized DNA, 8-oxo-dG. Loss of function mutations of OGG1 also occurs in human kidney clear cell carcinoma and may contribute to tumorgenesis. We investigated the distribution of protein expression and the activity of OGG1 and 8-oxo-dG and correlated it with the expression of tuberin in kidneys of wild type and Eker rats and tumor from Eker rat.

Results: Tuberin expression, OGG1 protein expression and activity were higher in kidney cortex than in medulla or papilla in both wild type and Eker rats. On the other hand, 8-oxo-dG levels were highest in the medulla, which expressed the lowest levels of OGG1. The basal levels of 8-oxo-dG were also higher in both cortex and medulla of Eker rats compared to wild type rats. In kidney tumors from Eker rats, the loss of the second TSC2 allele is associated with loss of OGG1 expression. Immunostaining of kidney tissue shows localization of tuberin and OGG1 mainly in the cortex.

Conclusion: These results demonstrate that OGG1 localizes with tuberin preferentially in kidney cortex. Loss of tuberin is accompanied by the loss of OGG1 contributing to tumorgenesis. In addition, the predominant expression of OGG1 in the cortex and its decreased expression and activity in the Eker rat may account for the predominant cortical localization of renal cell carcinoma.

Show MeSH
Related in: MedlinePlus