Disruption of NAD(P)H:quinone oxidoreductase 1 gene in mice leads to 20S proteasomal degradation of p63 resulting in thinning of epithelium and chemical-induced skin cancer (original) (raw)
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Cancer Research, 2010
Quinone oxidoreductases (NQO1 and NQO2) are cytosolic proteins that detoxify quinones, prevent redox cycling and protect cells against oxidative stress and neoplasia. Double knockout (DKO) mice deficient in both NQO1 and NQO2 proteins were generated in our laboratory. To investigate the combined role of NQO1 and NQO2 in chemical carcinogenesis, the dorsal skin of C57BL/6 wild type and DKO mice were shaved on the back and treated with dimethylbenz(a)anthracene (DMBA) or benzo(a)pyrene followed by twice weekly application of phorbol 12-myristate 13-acetate (TPA). DKO mice exposed to DMBA and BP showed significantly higher skin tumor frequency and multiplicity per mouse as compared with wild type and single knockout mice. One hundred percent DKO mice developed DMBA-induced skin tumors and average tumor multiplicity was greater than 15 per mouse. In contrast, only 30% of wild type mice showed tumor incidence and average tumor multiplicity was less than 3. BP also showed 100% incidence of tumors in DKO mice, as compared to 43% in wild type mice. In related experiments, wild type and DKO mice exposed to BP for 6, 12 and 24 hours were analyzed for growth/differentiation, proliferation and apoptosis factors by immunohistochemical and immunoblot analysis. BP demonstrated delayed activation of p63/p53/ p19 and decreased apoptosis in the skin of DKO mice, as compared with wild type mice. This led to significant increase in sensitivity of DKO mice to BP induced skin tumors and tumor multiplicity.
Oncogene, 2012
Previously, we have shown a role of cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1) in stabilization of p63 against 20S proteasomal degradation resulting in thinning of epithelium and chemical-induced skin cancer [Oncogene (2011[Oncogene ( ) 30,1098[Oncogene ( -1107. Current studies demonstrate that NQO1 control of C/EBPα against20S proteasomal degradation also contributes to the up regulation of p63 expression and protection. Western and immunohistochemistry analysis revealed that disruption of NQO1 gene in mice and mouse keratinocytes led todegradation of C/EBPα and loss of p63 gene expression. p63 promoter mutagenesis, transfection and ChIP assays identified C/ EBPα binding site between nucleotide position −185 to −174 that bound to C/EBPα and up regulated p63 gene expression. Coimmunoprecipitation and immunoblot analysis demonstrated that 20S proteasomes directly interacted and degraded C/EBPα. NQO1 direct interaction with C/ EBPα led to stabilization of C/EBPα against 20S proteasomal degradation. NQO1 protection of C/ EBPα required binding of NADH with NQO1. Exposure of skin and keratinocytes to chemical stress agent benzo(a)pyrene led to induction of NQO1 and stabilization of C/EBPα protein resulting in an increase in p63 RNA and protein in wild type but not in NQO1−/− mice. Collectively, the current data combined with previous suggest that stress-induction of NQO1 through both stabilization of C/EBPα and increase in p63 and direct stabilization of p63 controls keratinocyte differentiation leading to protection against chemical-induced skin carcinogenesis. The studies are significant since 2-4% human individuals are homozygous and 23% are heterozygous for NQO1P187S mutation and might be susceptible to stress-induced skin diseases.
Biochemical and Biophysical Research Communications, 1987
NAD(P)H:quinone reductase, which plays an important role in the detoxification of carcinogenic metabolites as well as oxidative cellular damage, was found to be present in epidermal cytosol where its specific activity far exceeds (140-160%) the corresponding hepatic value. The effect of topical application of crude coal tar, 3-methylcholanthrene and polychlorinated biphenyl Aroclor 1254, on epidermal and hepatic cytosolic NAD(P)H:quinone reductase activities was investigated in neonatal rats, Sencar and athymic nude mice. A single topical application of each agent resulted in significant increases in epidermal (lSS%-389%) and hepatic (150-255%) enzyme activities. This inducible enzyme may play an important role in the detoxification of reactive quinone species during the course of malignant neoplasia and against oxidative cellular damage in skin.
Cancer Research, 2005
NAD(P)H:quinone oxidoreductase 1 (NQO1) is a cytosolic protein that catalyzes metabolic detoxification of quinones and protects cells against redox cycling and oxidative stress. NQO1 -null mice deficient in NQO1 protein showed increased sensitivity to 7,12-dimethylbenz(a)anthraceneand benzo(a)pyrene-induced skin carcinogenesis. In the present studies, we show that benzo(a )pyrene metabolite benzo(a)pyrene-trans -7,8-dihydrodiol-9,10-epoxide and not benzo(a)pyrene quinones contributed to increased benzo(a)pyrene-induced skin tumors in NQO1-null mice. An analysis of untreated skin revealed an altered intracellular redox state due to accumulation of NADH and reduced levels of NAD/NADH in NQO1-null mice as compared with wild-type mice. Treatment with benzo(a)pyrene failed to significantly increase p53 and apoptosis in the skin of NQO1-null mice when compared with wild-type mice. These results led to the conclusion that altered intracellular redox state along with lack of induction of p53 and decreased apoptosis plays a significant role in increased sensitivity of NQO1-null mice to benzo(a) pyrene-induced skin cancer. (Cancer Res 2005; 65(6): 2054-8)
Cancer Research, 2007
NRH:quinone oxidoreductase 2 (NQO2) is a cytosolic flavoprotein that catalyzes the two-electron reduction of quinones and quinoid compounds to hydroquinones. Although the role of a homologue, NAD(P)H:quinone oxidoreductase 1 (NQO1), is well defined in oxidative stress, neoplasia, and carcinogenesis, little is known about the mechanism of actions of NQO2 in these cellular responses. Whether NQO2 has any role in tumor necrosis factor (TNF) signaling was investigated using keratinocytes derived from wild-type and NQO2 knockout (NQO2 À/À ) mice. Although exposure of wild-type cells to TNF led to activation of nuclear factor-KB (NF-KB) and IKBA kinase, IKBA degradation, p65 phosphorylation, and p65 nuclear translocation, this cytokine had no effect on NQO2 À/À cells. Deletion of NQO2 also abolished TNF-induced c-Jun NH 2 -terminal kinase, Akt, p38, and p44/p42 mitogen-activated protein kinase activation. The induction of various antiapoptotic gene products (MMP-9, cyclin D1, COX-2, IAP1, IAP2, Bcl-2, cFLIP, and XIAP) by TNF was also abolished in NQO2 À/À cells. This correlated with potentiation of TNF-induced apoptosis as indicated by cell viability, Annexin V staining, and caspase activation. In agreement with this, we also found that TNF activated NQO2, and NQO2-specific small interfering RNA abrogated the TNF-induced NQO2 activity and NF-KB activation. Overall, our results indicate that deletion of NQO2 plays a differential role in TNF signaling pathway: by suppressing cell survival signals and potentiating TNF-induced apoptosis.
AJP Cell Physiology
The transcription factor nuclear factor-B (NF-B) is constitutively activated in malignancies from enhanced activity of inhibitor of NF-B (IB) kinase, with accelerated IB␣ degradation. We studied whether redox signaling might stimulate these events. Cultured melanoma cells generated superoxide anions (O 2 Ϫ) without serum stimulation. O 2 Ϫ generation was reduced by the NAD(P)H:quinone oxidoreductase (NQO) inhibitor dicumarol and the quinone analog capsaicin, suggesting that electron transfer from NQO through a quinone-mediated pathway may be an important source of endogenous reactive oxygen species (ROS) in tumor cells. Treatment of malignant melanoma cells with the H 2 O 2 scavenger catalase, the sulfhydryl donor N-acetylcysteine, the glutathione peroxidase mimetic ebselen, or dicumarol decreased NF-B activation. Catalase, N-acetylcysteine, ebselen, dicumarol, and capsaicin also inhibited growth of melanoma and other malignant cell lines. These results raise the possibility that ROS produced endogenously by mechanisms involving NQO can constitutively activate NF-B in an autocrine fashion and suggest the potential for new antioxidant strategies for interruption of oxidant signaling of melanoma cell growth.
Journal of Biological Chemistry, 2003
NAD(P)H:quinone oxidoreductase 1 (NQO1) has been proposed to stabilize p53 via a redox mechanism involving oxidation of NAD(P)H as a consequence of the catalytic activity of NQO1. We report that treatment of HCT-116 human colon carcinoma cells with the NQO1 inhibitor ES936 had no effect on the levels of p53 protein. ES936 is a mechanism-based inhibitor of NQO1 that irreversibly blocks the catalytic function of the enzyme. This suggests that a redox mechanism involving NQO1mediated NAD(P)H oxidation is not responsible for the stabilization of p53. We also examined the ability of the NQO1 protein to associate with p53 using co-immunoprecipitation experiments. Results from these experiments demonstrated co-immunoprecipitation of NQO1 with p53 and vice versa. The association between p53 and NQO1 was not affected by treatment of HCT-116 cells with ES936, demonstrating that the association was not dependent on the catalytic activity of NQO1. A comparison of isogenic HCT-116 p53؉/؉ and HCT-116 p53؊/؊ cells demonstrated an interaction of NQO1 and p53 only in the p53؉/؉ cells. Experiments performed in an in vitro transcription/translation system utilizing rabbit reticulocyte lysates confirmed the interaction of NQO1 and p53. In these experiments a full-length p53 coding region was used to express p53 in the presence of recombinant NQO1 protein. An association of p53 and NQO1 was also observed in primary human keratinocytes and mammary epithelial cells. In studies where mdm-2 co-immunoprecipitated with p53, no association of mdm-2 with NQO1 was observed. These data demonstrate an association between p53 and NQO1 that may represent an alternate mechanism of p53 stabilization by NQO1 in a wide variety of human cell types.
Archives of Biochemistry and Biophysics, 2010
NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1) is a widely-distributed FAD-dependent flavoprotein that promotes obligatory 2-electron reductions of quinones, quinoneimines, nitroaromatics, and azo dyes, at rates that are comparable with NADH or NADPH. These reductions depress quinone levels and thereby minimize opportunities for generation of reactive oxygen intermediates by redox cycling, and for depletion of intracellular thiol pools. NQO1 is a highlyinducible enzyme that is regulated by the Keap1/Nrf2/ARE pathway. Evidence for the importance of the antioxidant functions of NQO1 in combating oxidative stress is provided by demonstrations that induction of NQO1 levels or their depletion (knockout, or knockdown) are associated with decreased and increased susceptibilities to oxidative stress, respectively. Furthermore, benzene genotoxicity is markedly enhanced when NQO1 activity is compromised. Not surprisingly, human polymorphisms that suppress NQO1 activities are associated with increased predisposition to disease. Recent studies have uncovered protective roles for NQO1 that apparently are unrelated to its enzymatic activities. NQO1 binds to and thereby stabilizes the important tumor suppressor p53 against proteasomal degradation. Indeed, NQO1 appears to regulate the degradative fate of other proteins. These findings suggest that NQO1 may exercise a selective "gatekeeping" role in regulating the proteasomal degradation of specific proteins, thereby broadening the cytoprotective role of NQO1 far beyond its highly effective antioxidant functions.
EMBO reports, 2008
The tumour suppressor p33 ING1b (ING1b for inhibitor of growth family, member 1b) is important in cellular stress responses, including cell-cycle arrest, apoptosis, chromatin remodelling and DNA repair; however, its degradation pathway is still unknown. Recently, we showed that genotoxic stress induces p33 ING1b phosphorylation at Ser 126, and abolishment of Ser 126 phosphorylation markedly shortened its half-life. Therefore, we suggest that Ser 126 phosphorylation modulates the interaction of p33 ING1b with its degradation machinery, stabilizing this protein. Combining the use of inhibitors of the main degradation pathways in the nucleus (proteasome and calpains), partial isolation of the proteasome complex, and in vitro interaction and degradation assays, we set out to determine the degradation mechanism of p33 ING1b. We found that p33 ING1b is degraded in the 20S proteasome and that NAD(P)H quinone oxidoreductase 1 (NQO1), an oxidoreductase previously shown to modulate the degradation of p53 in the 20S proteasome, inhibits the degradation of p33 ING1b. Furthermore, ultraviolet irradiation induces p33 ING1b phosphorylation at Ser 126, which, in turn, facilitates its interaction with NQO1.