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High glutathione and glutathione peroxidase-2 levels mediate cell-type-specific DNA damage protection in human induced pluripotent stem cells.

ABSTRACT: Pluripotent stem cells must strictly maintain genomic integrity to prevent transmission of mutations. In human induced pluripotent stem cells (iPSCs), we found that genome surveillance is achieved via two ways, namely, a hypersensitivity to apoptosis and a very low accumulation of DNA lesions. The low apoptosis threshold was mediated by constitutive p53 expression and a marked upregulation of proapoptotic p53 target genes of the BCL-2 family, ensuring the efficient iPSC removal upon genotoxic insults. Intriguingly, despite the elevated apoptosis sensitivity, both mitochondrial and nuclear DNA lesions induced by genotoxins were less frequent in iPSCs compared to fibroblasts. Gene profiling identified that mRNA expression of several antioxidant proteins was considerably upregulated in iPSCs. Knockdown of glutathione peroxidase-2 and depletion of glutathione impaired protection against DNA lesions. Thus, iPSCs ensure genomic integrity through enhanced apoptosis induction and increased antioxidant defense, contributing to protection against DNA damage.

SUBMITTER: Dannenmann B

PROVIDER: S-EPMC4437487 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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High glutathione and glutathione peroxidase-2 levels mediate cell-type-specific DNA damage protection in human induced pluripotent stem cells.

Dannenmann Benjamin B Lehle Simon S Hildebrand Dominic G DG Kübler Ayline A Grondona Paula P Schmid Vera V Holzer Katharina K Fröschl Mirjam M Essmann Frank F Rothfuss Oliver O Schulze-Osthoff Klaus K

Stem cell reports 20150430 5

Pluripotent stem cells must strictly maintain genomic integrity to prevent transmission of mutations. In human induced pluripotent stem cells (iPSCs), we found that genome surveillance is achieved via two ways, namely, a hypersensitivity to apoptosis and a very low accumulation of DNA lesions. The low apoptosis threshold was mediated by constitutive p53 expression and a marked upregulation of proapoptotic p53 target genes of the BCL-2 family, ensuring the efficient iPSC removal upon genotoxic in ...[more]

PMID: 25937369

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Project description:BackgroundNiemann-Pick type C1 disease (NPC1) is a rare progressive neurodegenerative disorder caused by mutations in the NPC1 gene. In this lysosomal storage disorder the intracellular transport and sequestration of several lipids like cholesterol is severely impaired, resulting in an accumulation of lipids in late endosomes and lysosomes. The neurological manifestation of the disease is caused by dysfunction and cell death in the central nervous system. Several animal models were used to analyze the impaired pathways. However, the underlying pathogenic mechanisms are still not completely understood and the genetic variability in humans cannot be reflected in these models. Therefore, a human model using patient-specific induced pluripotent stem cells provides a promising approach.MethodsWe reprogrammed human fibroblasts from a NPC1 patient and a healthy control by retroviral transduction with Oct4, Klf4, Sox2 and c-Myc. The obtained human induced pluripotent stem cells (hiPSCs) were characterized by immunocytochemical analyses. Neural progenitor cells were generated and patch clamp recordings were performed for a functional analysis of derived neuronal cells. Filipin stainings and the Amplex Red assay were used to demonstrate and quantify cholesterol accumulation.ResultsThe hiPSCs expressed different stem cell markers, e.g. Nanog, Tra-1-81 and SSEA4. Using the embryoid body assay, the cells were differentiated in cells of all three germ layers and induced teratoma in immunodeficient mice, demonstrating their pluripotency. In addition, neural progenitor cells were derived and differentiated into functional neuronal cells. Patch clamp recordings revealed voltage dependent channels, spontaneous action potentials and postsynaptic currents. The accumulation of cholesterol in different tissues is the main hallmark of NPC1. In this study we found an accumulation of cholesterol in fibroblasts of a NPC1 patient, derived hiPSCs, and neural progenitor cells, but not in cells derived from fibroblasts of a healthy individual. These findings were quantified by the Amplex Red assay, demonstrating a significantly elevated cholesterol level in cells derived from fibroblasts of a NPC1 patient.ConclusionsWe generated a neuronal model based on induced pluripotent stem cells derived from patient fibroblasts, providing a human in vitro model to study the pathogenic mechanisms of NPC1 disease.

Project description:BackgroundGlutathione (GSH) is one of the most important agents of the antioxidant defense system of the cell because, in conjunction with the enzymes glutathione peroxidase (GSH-Px) and glutathione S transferase pi (GSTpi), it plays a central role in the detoxification and biotransformation of chemotherapeutic drugs. This study evaluated the expression of GSH and the GSH-Px and GSTpi enzymes by immunohistochemistry in 30 canine mammary tumors, relating the clinicopathological parameters, clinical outcome and survival of the bitches. In an in vitro study, the expression of the genes glutamate cysteine ligase (GCLC) and glutathione synthetase (GSS) that synthesize GSH and GSH-Px gene were verified by qPCR and subjected to treatment with doxorubicin, to check the resistance of cancer cells to chemotherapy.ResultsThe immunohistochemical expression of GSH, GSH-Px and GSTpi was compared with the clinical and pathological characteristics and the clinical outcome in the bitches, including metastasis and death.The results showed that high immunoexpression of GSH was correlated to the absence of tumor ulceration and was present in dogs without metastasis (P < 0.05). There was significant correlation of survival with the increase of GSH (P < 0.05). The expression of the GSH-Px and GSTpi enzymes showed no statistically significant correlation with the analyzed variables (p > 0.05). The analysis of the relative expression of genes responsible for the synthesis of GSH (GCLC and GSS) and GSH-Px by quantitative PCR was done with cultured cells of 10 tumor fragments from dogs with mammary tumors.The culture cells showed a decrease in GCLC and GSS expression when compared with no treated cells (P < 0.05). High GSH immunoexpression was associated with better clinical outcomes.ConclusionTherefore, high expression of the GSH seems to play an important role in the clinical outcome of patients with mammary tumors and suggest its use as prognostic marker. The in vitro doxorubicin treatment significantly reduces the expression of GCLC and GSS genes so we can consider them to be candidates for predictive markers of therapeutic response in mammary cancer.

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High glutathione and glutathione peroxidase-2 levels mediate cell-type-specific DNA damage protection in human induced pluripotent stem cells.

Publications

High glutathione and glutathione peroxidase-2 levels mediate cell-type-specific DNA damage protection in human induced pluripotent stem cells.

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