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

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.

Figure 1

Distribution of tuberin and OGG1 protein in kidney cortex (C), medulla (M) and papilla (P) of wild type and Eker rats. A. Immunoblot analysis of tuberin and OGG1 in different kidney regions. Different kidney regions were homogenized and protein extracts were loaded onto 7% SDS-polyacrylamide gels and transfered to PVDF membrane. The membrane was incubated with anti-tuberin or anti-OGG1 followed by different specific HRP-conjugated secondary antibodies. The proteins were visualized by ECL. GAPDH was used as loading control. B. Histograms represent means ± SE (n = 3). Significant difference from wild type rat is indicated by * P < 0.05 and ** P < 0.01.

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.

Figure 3

(A) Kidney from wild type and (B) Eker rats were harvested at 12 months. Kidney from wild type rat show normal size and appearing while large size and multiple tumors were appeared in kidney of Eker rat. H&E staining of kidney from (C) wild type and (D) Eker rats. Kidney section of wild type and Eker rats shows normal tubular architecture of kidney in wild type rat and fibrovascular stroma (indicated by one head arrow) with clear cytoplasm and excentric in large nuclei of clear cell carcinoma type (indicated by two head arrow) in Eker rat.

Figure 4

Tuberin and OGG1 staining are mainly localized in the kidney cortex. (A) H&E staining in kidney section of wild type rats (TSC2^+/+) show normal tubular architecture. The majority staining of tuberin (B) and OGG1 (C) are localized in the cortical region compared to medulla region.

Figure 5

Loss of tuberin is associated with loss of OGG1 in kidney tumor of Eker rat. (A) H&E staining in kidney section of Eker rats (TSC-2^+/-) show cortical tumors. The tuberin (B) and OGG1 (C) staining are not detected in tumor (T) compared to normal tissue (N) of cortical region.

Figure 6

A deficiency in tuberin is associated with loss of OGG1 expression in Eker rat kidney tumors. A. Immunoblot analysis of tuberin and OGG1 protein expression in normal kidney of wild type rats, normal and tumor kidney tissue from Eker rats. GAPDH was used as loading control. B. Histograms represent means ± SE (n = 3). Significant difference from wild type rat is indicated by ** P < 0.01.