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Fumarate induces redox-dependent senescence by modifying glutathione metabolism.

Zheng L, Cardaci S, Jerby L, MacKenzie ED, Sciacovelli M, Johnson TI, Gaude E, King A, Leach JD, Edrada-Ebel R, Hedley A, Morrice NA, Kalna G, Blyth K, Ruppin E, Frezza C, Gottlieb E - Nat Commun (2015)

Bottom Line: Here, we show that the accumulation of fumarate caused by the inactivation of FH leads to oxidative stress that is mediated by the formation of succinicGSH, a covalent adduct between fumarate and glutathione.Chronic succination of GSH, caused by the loss of FH, or by exogenous fumarate, leads to persistent oxidative stress and cellular senescence in vitro and in vivo.Importantly, the ablation of p21, a key mediator of senescence, in Fh1-deficient mice resulted in the transformation of benign renal cysts into a hyperplastic lesion, suggesting that fumarate-induced senescence needs to be bypassed for the initiation of renal cancers.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK, Beatson Institute, Switchback Road, Glasgow G61 1BD, UK.

ABSTRACT
Mutations in the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) are associated with a highly malignant form of renal cancer. We combined analytical chemistry and metabolic computational modelling to investigate the metabolic implications of FH loss in immortalized and primary mouse kidney cells. Here, we show that the accumulation of fumarate caused by the inactivation of FH leads to oxidative stress that is mediated by the formation of succinicGSH, a covalent adduct between fumarate and glutathione. Chronic succination of GSH, caused by the loss of FH, or by exogenous fumarate, leads to persistent oxidative stress and cellular senescence in vitro and in vivo. Importantly, the ablation of p21, a key mediator of senescence, in Fh1-deficient mice resulted in the transformation of benign renal cysts into a hyperplastic lesion, suggesting that fumarate-induced senescence needs to be bypassed for the initiation of renal cancers.

No MeSH data available.


Related in: MedlinePlus

GSH succination leads to redox stress-induced senescence in primary kidney cells and human diploid fibroblasts.(a) Primary epithelial kidney cells (1 × 104) from Fh1fl/fl animals were either left uninfected or infected with adeno-CRE and treated with vehicle or the antioxidant trolox or ascorbate. Cell number was measured after 2 days of culture. (b) The ablation of FH in cells described in a resulted in the activation of p16 and p21 transcription. Results are expressed as average fold induction over non-infected cells±s.e.m (representative experiments; n=6 for p21 and n=3 for p16). (c) FH and p21 mRNA levels were assessed by qPCR in non-targeting control (NTC)-transfected cells in FH-silenced cells. (d) Microscopy quantitative analysis of senescence-associated β-galactosidase activity, induced by DMF or adeno-CRE infection, with or without trolox and ascorbate. (e,f) cell growth (e) and p21 mRNA expression levels (f) were assessed in IMR90 cells after DMF treatment, with or without the antioxidant N-acetyl cysteine (NAC). All the results were obtained from three independent cultures and expressed as average±s.e.m.
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f6: GSH succination leads to redox stress-induced senescence in primary kidney cells and human diploid fibroblasts.(a) Primary epithelial kidney cells (1 × 104) from Fh1fl/fl animals were either left uninfected or infected with adeno-CRE and treated with vehicle or the antioxidant trolox or ascorbate. Cell number was measured after 2 days of culture. (b) The ablation of FH in cells described in a resulted in the activation of p16 and p21 transcription. Results are expressed as average fold induction over non-infected cells±s.e.m (representative experiments; n=6 for p21 and n=3 for p16). (c) FH and p21 mRNA levels were assessed by qPCR in non-targeting control (NTC)-transfected cells in FH-silenced cells. (d) Microscopy quantitative analysis of senescence-associated β-galactosidase activity, induced by DMF or adeno-CRE infection, with or without trolox and ascorbate. (e,f) cell growth (e) and p21 mRNA expression levels (f) were assessed in IMR90 cells after DMF treatment, with or without the antioxidant N-acetyl cysteine (NAC). All the results were obtained from three independent cultures and expressed as average±s.e.m.

Mentions: We hypothesized that the observed metabolic reprogramming is a required step for the support of cell transformation. Since both the human and mouse FH-deficient cells used here were immortalized cell lines, we tested the effects of GSH succination on primary kidney cells isolated from Fh1fl/fl mouse pups and on the human diploid fibroblasts IMR90. On infection with adenoviral vector expressing Cre recombinase (Ad-Cre), Fh1fl/fl cells, which lost FH activity, underwent proliferation arrest, whereas the uninfected cells continued to proliferate (Fig. 6a). The proliferation arrest was associated with a robust induction of the cyclin-dependent kinase inhibitor p21CIP1/WAF1 (thereafter referred to as p21) and of the cyclin-dependent kinase inhibitor 2A p16 (Fig. 6b). These results suggested that oxidative stress-mediated cellular senescence18 might be responsible for the observed phenotype. Similarly, the non-transformed non-immortalized human diploid fibroblast cells IMR90 exhibited a significant upregulation of p21 on acute depletion of FH (Fig. 6c). To confirm the involvement of senescence, we tested the senescence-associated β-galactosidase (SA-β-gal) activity in FH-deficient cells. On infection with Ad-Cre, Fh1fl/fl exhibited a strong SA-β-gal staining (Fig. 6d and Supplementary Fig. 5a). Reassuringly, the loss of FH was also associated with the activation of an antioxidant response similar to that observed in Fh1-deficient cell lines (Supplementary Fig 5b). To ascertain the role of oxidative stress in senescence induction, cells were incubated with ascorbate (vitamin C) or Trolox (vitamin E derivative), two well-characterized antioxidants, which are water or lipid soluble respectively. Importantly, both the growth inhibitory effect, and the induction of SA-β-gal activity in Fh1-deficient primary kidney cells were abrogated by these antioxidants (Fig. 6a,d). These results indicate that redox stress induced by FH loss is, at least in part, responsible for the ensuing senescence.


Fumarate induces redox-dependent senescence by modifying glutathione metabolism.

Zheng L, Cardaci S, Jerby L, MacKenzie ED, Sciacovelli M, Johnson TI, Gaude E, King A, Leach JD, Edrada-Ebel R, Hedley A, Morrice NA, Kalna G, Blyth K, Ruppin E, Frezza C, Gottlieb E - Nat Commun (2015)

GSH succination leads to redox stress-induced senescence in primary kidney cells and human diploid fibroblasts.(a) Primary epithelial kidney cells (1 × 104) from Fh1fl/fl animals were either left uninfected or infected with adeno-CRE and treated with vehicle or the antioxidant trolox or ascorbate. Cell number was measured after 2 days of culture. (b) The ablation of FH in cells described in a resulted in the activation of p16 and p21 transcription. Results are expressed as average fold induction over non-infected cells±s.e.m (representative experiments; n=6 for p21 and n=3 for p16). (c) FH and p21 mRNA levels were assessed by qPCR in non-targeting control (NTC)-transfected cells in FH-silenced cells. (d) Microscopy quantitative analysis of senescence-associated β-galactosidase activity, induced by DMF or adeno-CRE infection, with or without trolox and ascorbate. (e,f) cell growth (e) and p21 mRNA expression levels (f) were assessed in IMR90 cells after DMF treatment, with or without the antioxidant N-acetyl cysteine (NAC). All the results were obtained from three independent cultures and expressed as average±s.e.m.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4340546&req=5

f6: GSH succination leads to redox stress-induced senescence in primary kidney cells and human diploid fibroblasts.(a) Primary epithelial kidney cells (1 × 104) from Fh1fl/fl animals were either left uninfected or infected with adeno-CRE and treated with vehicle or the antioxidant trolox or ascorbate. Cell number was measured after 2 days of culture. (b) The ablation of FH in cells described in a resulted in the activation of p16 and p21 transcription. Results are expressed as average fold induction over non-infected cells±s.e.m (representative experiments; n=6 for p21 and n=3 for p16). (c) FH and p21 mRNA levels were assessed by qPCR in non-targeting control (NTC)-transfected cells in FH-silenced cells. (d) Microscopy quantitative analysis of senescence-associated β-galactosidase activity, induced by DMF or adeno-CRE infection, with or without trolox and ascorbate. (e,f) cell growth (e) and p21 mRNA expression levels (f) were assessed in IMR90 cells after DMF treatment, with or without the antioxidant N-acetyl cysteine (NAC). All the results were obtained from three independent cultures and expressed as average±s.e.m.
Mentions: We hypothesized that the observed metabolic reprogramming is a required step for the support of cell transformation. Since both the human and mouse FH-deficient cells used here were immortalized cell lines, we tested the effects of GSH succination on primary kidney cells isolated from Fh1fl/fl mouse pups and on the human diploid fibroblasts IMR90. On infection with adenoviral vector expressing Cre recombinase (Ad-Cre), Fh1fl/fl cells, which lost FH activity, underwent proliferation arrest, whereas the uninfected cells continued to proliferate (Fig. 6a). The proliferation arrest was associated with a robust induction of the cyclin-dependent kinase inhibitor p21CIP1/WAF1 (thereafter referred to as p21) and of the cyclin-dependent kinase inhibitor 2A p16 (Fig. 6b). These results suggested that oxidative stress-mediated cellular senescence18 might be responsible for the observed phenotype. Similarly, the non-transformed non-immortalized human diploid fibroblast cells IMR90 exhibited a significant upregulation of p21 on acute depletion of FH (Fig. 6c). To confirm the involvement of senescence, we tested the senescence-associated β-galactosidase (SA-β-gal) activity in FH-deficient cells. On infection with Ad-Cre, Fh1fl/fl exhibited a strong SA-β-gal staining (Fig. 6d and Supplementary Fig. 5a). Reassuringly, the loss of FH was also associated with the activation of an antioxidant response similar to that observed in Fh1-deficient cell lines (Supplementary Fig 5b). To ascertain the role of oxidative stress in senescence induction, cells were incubated with ascorbate (vitamin C) or Trolox (vitamin E derivative), two well-characterized antioxidants, which are water or lipid soluble respectively. Importantly, both the growth inhibitory effect, and the induction of SA-β-gal activity in Fh1-deficient primary kidney cells were abrogated by these antioxidants (Fig. 6a,d). These results indicate that redox stress induced by FH loss is, at least in part, responsible for the ensuing senescence.

Bottom Line: Here, we show that the accumulation of fumarate caused by the inactivation of FH leads to oxidative stress that is mediated by the formation of succinicGSH, a covalent adduct between fumarate and glutathione.Chronic succination of GSH, caused by the loss of FH, or by exogenous fumarate, leads to persistent oxidative stress and cellular senescence in vitro and in vivo.Importantly, the ablation of p21, a key mediator of senescence, in Fh1-deficient mice resulted in the transformation of benign renal cysts into a hyperplastic lesion, suggesting that fumarate-induced senescence needs to be bypassed for the initiation of renal cancers.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK, Beatson Institute, Switchback Road, Glasgow G61 1BD, UK.

ABSTRACT
Mutations in the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) are associated with a highly malignant form of renal cancer. We combined analytical chemistry and metabolic computational modelling to investigate the metabolic implications of FH loss in immortalized and primary mouse kidney cells. Here, we show that the accumulation of fumarate caused by the inactivation of FH leads to oxidative stress that is mediated by the formation of succinicGSH, a covalent adduct between fumarate and glutathione. Chronic succination of GSH, caused by the loss of FH, or by exogenous fumarate, leads to persistent oxidative stress and cellular senescence in vitro and in vivo. Importantly, the ablation of p21, a key mediator of senescence, in Fh1-deficient mice resulted in the transformation of benign renal cysts into a hyperplastic lesion, suggesting that fumarate-induced senescence needs to be bypassed for the initiation of renal cancers.

No MeSH data available.


Related in: MedlinePlus