Samy L. Habib

Mechanism of Oxidative DNA damage in diabetes: tuberin inactivation and downregulation of DNA repair enzyme OGG1

OBJECTIVE—To investigate potential mechanisms of oxidative DNA damage in a rat model of type 1 diabetes and in murine proximal tubular epithelial cells and primary culture of rat proximal tubular epithelial cells. RESEARCH DESIGN AND METHODS—Phosphorylation of Akt and tuberin, 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) levels, and 8-oxoG-DNA glycosylase (OGG1) expression were measured in kidney cortical tissue of control and type 1 diabetic animals and in proximal tubular cells incubated with normal or high glucose. RESULTS—In the renal cortex of diabetic rats, the increase in Akt phosphorylation is associated with enhanced phosphorylation of tuberin, decreased OGG1 protein expression, and 8-oxodG accumulation. Exposure of proximal tubular epithelial cells to high glucose causes a rapid increase in reactive oxygen species (ROS) generation that correlates with the increase in Akt and tuberin phosphorylation. High glucose also resulted in downregulation of OGG1 protein expression, paralleling its effect on Akt and tuberin. Inhibition of phosphatidylinositol 3-kinase/Akt significantly reduced high glucose–induced tuberin phosphorylation and restored OGG1 expression. Hydrogen peroxide stimulates Akt and tuberin phosphorylation and decreases OGG1 protein expression. The antioxidant N-acetylcysteine significantly inhibited ROS generation, Akt/protein kinase B, and tuberin phosphorylation and resulted in deceased 8-oxodG accumulation and upregulation of OGG1 protein expression. CONCLUSIONS—Hyperglycemia in type 1 diabetes and treatment of proximal tubular epithelial cells with high glucose leads to phosphorylation/inactivation of tuberin and downregulation of OGG1 via a redox-dependent activation of Akt in renal tubular epithelial cells. This signaling cascade provides a mechanism of oxidative stress–mediated DNA damage in diabetes.

Diabetes and Risk of Cancer

Diabetes and cancer represent two complex, diverse, chronic, and potentially fatal diseases. Cancer is the second leading cause of death, while diabetes is the seventh leading cause of death with the latter still likely underreported. There is a growing body of evidence published in recent years that suggest substantial increase in cancer incidence in diabetic patients. The worldwide prevalence of diabetes was estimated to rise from 171 million in 2000 to 366 million in 2030. About 26.9% of all people over 65 have diabetes and 60% have cancer. Overall, 8–18% of cancer patients have diabetes. In the context of epidemiology, the burden of both diseases, small association between diabetes and cancer will be clinically relevant and should translate into significant consequences for future health care solutions. This paper summarizes most of the epidemiological association studies between diabetes and cancer including studies relating to the general all-site increase of malignancies in diabetes and elevated organ-specific cancer rate in diabetes as comorbidity. Additionally, we have discussed the possible pathophysiological mechanisms that likely may be involved in promoting carcinogenesis in diabetes and the potential of different antidiabetic therapies to influence cancer incidence.

Diabetes and risk of renal cell carcinoma

Background and objectives: There is evidence that the incidence of solid tumors is markedly increased in patients with diabetes mellitus. In the current study, we investigate the association between diabetes and renal cancer. Patients and Methods: A single-center retrospective analysis of 473 patients who underwent nephrectomy for renal cell carcinoma (RCC) was performed. Diabetic RCC patients were screened for age, gender, ethnicity, HgA1C, glucose levels and renal function. Results: Of the 473 cases with RCC, we identified 120 patients (25.4%) with a history of diabetes. The incidence of diabetes in RCC patients was higher in female than male subjects and in Hispanic compared to White and Other ethnic backgrounds. At diagnosis, the majority of diabetic RCC patients were 50-59 years of age. In diabetic RCC cases, clear cell type histology (92.0%), nuclear grade 2 (56.1%) and tumor size range from 1-5 cm (65.7%) were the most common in each category. Conclusion: Our findings indicate that diabetic RCC patients have a predominance of localized, small clear cell RCC. In addition, females with a history of RCC have a higher frequency of diabetes compared to males. This is the first report of clinical and histopathological features of RCC associated with diabetes.

The Ser(326)Cys Polymorphism of 8-Oxoguanine Glycosylase 1 (OGG1) Is Associated with Type 2 Diabetes in Mexican Americans

Objective: Human 8-oxoguanine glycosylase 1 (OGG1) excises oxidatively damaged promutagenic base 8-oxoguanine, a lesion previously observed in a rat model of type 2 diabetes (T2DM). The objective of the present study is to determine whether genetic variation in OGG1 is associated with type 2 diabetes (T2DM) in a Mexican American cohort. Methods: Ten SNPs including two tagging SNPs (rs1052133, rs2072668) across the OGG1 gene region were selected from the Hapmap database and genotyped in the entire cohort (n = 670; 29% diabetes; 39 families) by TaqMan assay. Association analyses between the SNPs and T2DM were performed using the measured genotype approach as implemented in the program SOLAR. Results: Of the ten SNPs genotyped, only five were polymorphic. The minor allele frequencies of these 5 SNPs ranged from 1–38%. Of the SNPs examined for association, the Ser(326)Cys (rs1052133) exhibited significant association with T2DM (p = 0.016) after accounting for age and sex effects. Another intronic variant (rs2072668), which was in strong linkage disequilibrium (r2 = 0.96) with Ser(326)Cys also exhibited significant association with T2DM (p = 0.031). Conclusions: These results suggest for the first time that the variants in OGG1 could influence diabetes risk in these Mexican American families and support a role for alterations of OGG1 in the pathogenesis of T2DM.

TSC2 deficiency increases PTEN via HIF1α

Substantial evidence suggests roles of TSC2 and PTEN in the development of cancer predisposition syndromes. Loss of TSC2 results in benign tumors, neurological disorders, and angiomyolipomas. We found that PTEN mRNA and protein levels are elevated in Tsc2−/− mouse embryo fibroblasts with concomitant reduction in Akt phosphorylation. Reconstitution of TSC2 in Tsc2−/− mouse embryo fibroblasts decreases PTEN levels. Interestingly, increased HIF1α activity present in Tsc2 null cells is required for PTEN transcription and protein expression. We identified a canonical hypoxia-responsive element in the PTEN promoter, which regulates the transcription of this tumor suppressor protein in a TSC2-dependent manner. Finally, we demonstrate a positive correlation between expression of HIF1α and PTEN in renal angiomyolipomas from TSC patients. Our results reveal a unique function of HIF1α in up-regulation of PTEN and provide a new mechanism of reduced Akt phosphorylation in Tsc2 null cells. These data suggest that PTEN may safeguard against developing malignant tumors in patients with TSC deficiency.

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

BackgroundTuberous 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.ResultsTuberin 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.ConclusionThese 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.

Tuberin Inhibits Production of the Matrix Protein Fibronectin in Diabetes

Exposure of proximal tubular epithelial cells to high glucose contributes to the accumulation of tubulointerstitial and matrix proteins in diabetic nephropathy, but how this occurs is not well understood. We investigated the effect of the signaling molecule tuberin, which modulates the mammalian target of rapamycin pathway, on renal hypertrophy and fibronectin expression. We found that the kidney mass was significantly greater in partially tuberin-deficient (TSC2(+/-) ) diabetic rats than wild-type diabetic rats. Furthermore, TSC2(+/-) rats exhibited significant increases in the basal levels of phospho-tuberin and fibronectin expression in the kidney cortex. Increased levels of phosphorylated tuberin associated with an increase in fibronectin expression in both wild-type and TSC2(+/-) diabetic rats. Treatment with insulin abrogated the diabetes-induced increase in fibronectin expression. In vitro, high glucose enhanced fibronectin expression in TSC2(+/-) primary proximal tubular epithelial cells; both inhibition of Akt and inhibition of the mammalian target of rapamycin could prevent this effect of glucose. In addition, forced expression of tuberin in tuberin-null cells abolished the expression of fibronectin protein. Taken together, these data suggest that tuberin plays a central role in the development of renal hypertrophy and in modulating the production of the matrix protein fibronectin in diabetes.

Genetic polymorphisms in OGG1 and their association with angiomyolipoma, a benign kidney tumor in patients with tuberous sclerosis.

The enzyme 8-oxoguanine glycosylase 1 (OGG1) repairs 8-oxo-2- deoxyguanosine residue (8-oxodG) an oxidatively damaged promutagenic base. Genetic variations in OGG1 gene have been shown to modulate DNA repair capacity and are related risk of tumor development. However, epidemiologic findings have been inconsistent. The purpose of this study is to determine whether genetic variants in OGG1 play a role in susceptibility to angiomyolipoma, a benign kidney tumor associated with tuberous sclerosis complex (TSC) patients. To identify genetic variations, all 7 exons of the OGG1 gene were amplified by PCR and sequenced in 22 TSC patients with angiomyolipoma (cases) and 18 controls. By direct sequencing, we identified four missense mutations in OGG1: Arg(45)Gln, Ala(85)Ser, Arg(229)Gln, and Ser(326)Cys. Genotypic association with angiomyolipoma was performed using the measured genotype approach as implemented in the variance component analytical tools. Association analysis showed the presence of significant association (p=0.01) only between the Ser(326)Cys polymorphism of OGG1 and angiomyolipoma. We also assessed the presence of oxidative DNA damage in kidney section from normal healthy subjects, normal kidney tissue from TSC patients and kidney angiomyolipoma tissue from TSC patients by immunostaining for 8-oxodG. 8-OxodG staining was highly abundant in kidney angiomyolipoma tissue from TSC patients compared to weak staining in uninvolved tissue from the same TSC patients or normal kidney from healthy subjects. Taken together, our findings suggest that Ser(326)Cys variant of OGG1 may confer risk for development of angiomyolipomas by increasing oxidative DNA damage.

Regulation of PI 3-K, PTEN, p53, and mTOR in Malignant and Benign Tumors Deficient in Tuberin.

The tuberous sclerosis complex (TSC) is caused by mutation in either of 2 tumor suppressor genes, TSC-1 (encodes hamartin) and TSC-2 (encodes tuberin). In humans, deficiency in TSC1/2 is associated with benign tumors in many organs, including renal angiomyolipoma (AML) but rarely renal cell carcinoma (RCC). In contrast, deficiency of TSC function in the Eker rat is associated with RCC. Here, we have investigated the activity of PI 3-K and the expression of PTEN, p53, tuberin, p-mTOR, and p-p70S6K in both Eker rat RCC and human renal AML. Compared to normal tissue, increased PI 3-K activity was detected in RCC of Eker rats but not in human AML tissue. In contrast, PTEN was highly expressed in AML but significantly reduced in the renal tumors of Eker rats. Phosphorylation on Ser(2448) of mTOR and Thr(389) of p70S6K were significantly increased in both RCC and AML compared to matching control tissue. Total tuberin was significantly decreased in AML while completely lost in RCC of Eker rats. Our data also show that while p53 protein expression is lost in rat RCC, it was highly elevated in AML. These novel data provide evidence that loss of TSC-2, PTEN, and p53 as well as activation of PI 3-K and mTOR is associated with kidney cancer in the Eker rat, while sustained expression of TSC-2, PTEN, and p53 may prevent progression of kidney cancer in TSC patients.

Novel protective mechanism of reducing renal cell damage in diabetes: Activation AMPK by AICAR increased NRF2/OGG1 proteins and reduced oxidative DNA damage

ABSTRACT Exposure of renal cells to high glucose (HG) during diabetes has been recently proposed to be involved in renal injury. In the present study, we investigated a potential mechanism by which AICAR treatment regulates the DNA repair enzyme, 8-oxoG-DNA glycosylase (OGG1) in renal proximal tubular mouse cells exposed to HG and in kidney of db/db mice. Cells treated with HG for 2 days show inhibition in OGG1 promoter activity as well as OGG1 and Nrf2 protein expression. In addition, activation of AMPK by AICAR resulted in an increase raptor phosphorylation at Ser792 and leads to increase the promoter activity of OGG1 through upregulation of Nrf2. Downregulation of AMPK by DN-AMPK and raptor and Nrf2 by siRNA resulted in significant decease in promoter activity and protein expression of OGG1. On the other hand, downregulation of Akt by DN-Akt and rictor by siRNA resulted in significant increase in promoter activity and protein expression of Nrf2 and OGG1. Moreover, gel shift analysis shows reduction of Nrf2 binding to OGG1 promoter in cells treated with HG while cells treated with AICAR reversed the effect of HG. Furthermore, db/db mice treated with AICAR show significant increased in AMPK and raptor phosphroylation as well as OGG1 and Nrf2 protein expression that associated with significant decrease in oxidative DNA damage (8-oxodG) compared to non-treated mice. In summary, our data provide a novel protective mechanism by which AICAR prevents renal cell damage in diabetes and the consequence complications of hyperglycemia with a specific focus on nephropathy.