Ascorbate depletion mediates up-regulation of hypoxia-associated proteins by cell density and nickel

Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick, Frederick, Maryland 21702, USA.
Journal of Cellular Biochemistry (Impact Factor: 3.26). 04/2006; 97(5):1025-35. DOI: 10.1002/jcb.20705
Source: PubMed


Exposure of human lung cells to carcinogenic nickel compounds in the presence of oxygen up-regulated carbonic anhydrase IX (CA IX) and NDRG1/Cap43, both known as intrinsic hypoxia markers and cancer-associated genes. This suggests that factors other than a shortage of oxygen may be involved in this induction. Both proteins can also be induced in the presence of oxygen by culturing these cells to a high density without medium change. The intracellular ascorbate measurements revealed its rapid depletion in both metal- and density-exposed cells. Nickel exposure caused strong activation of HIF-1alpha and HIF-2alpha proteins, underscoring activation of HIF-1-dependent transcription. In contrast, cell density-dependent transcription was characterized by minor induction of HIF-1alpha or HIF-2alpha. Moreover, the up-regulation of NDRG1/Cap43 in HIF-1alpha deficient fibroblasts suggested the involvement of different transcription factor(s). The repletion of intracellular ascorbate reversed the induction of CA IX and NDRG1/Cap43 caused by cell density or nickel exposure. Thus, the loss of intracellular ascorbate triggered the induction of both tumor markers by two different conditions in the presence of oxygen. Ascorbate is delivered to lung cells via the SVCT2 ascorbate transporter, which was found to be sensitive to nickel or cell density. Collectively these findings establish the importance of intracellular ascorbate levels for the regulation of expression of CA IX and NDRG1/Cap43. We suggest, that, in addition to low oxygenation, insufficient supply of ascorbate or its excessive oxidation in tumors, can contribute to the induction of hypoxia-associated proteins via both HIF-dependent and independent mechanisms.

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    • "Diverse chemical and cellular signals stimulate the expression of NDRG1, including reducing agents such as tunicamycin [18], metals (cobalt, nickel, calcium, and iron chelators) [19,20], nitric oxide [21], vitamin D [22], vitamin C [23], retinoids [7], androgens and estrogens [24-27], and DNA-damaging compounds (actinomycin D, doxorubicin, geldanamycin) [28-30]. "
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    ABSTRACT: The protein N-Myc downstream-regulated gene 1 (NDRG1) is implicated in the regulation of cell proliferation, differentiation, and cellular stress response. NDRG1 is expressed in primary human trophoblasts, where it promotes cell viability and resistance to hypoxic injury. The mechanism of action of NDRG1 remains unknown. To gain further insight into the intracellular action of NDRG1, we analyzed the expression pattern and cellular localization of endogenous NDRG1 and transfected Myc-tagged NDRG1 in human trophoblasts exposed to diverse injuries. In standard conditions, NDRG1 was diffusely expressed in the cytoplasm at a low level. Hypoxia or the hypoxia mimetic cobalt chloride, but not serum deprivation, ultraviolet (UV) light, or ionizing radiation, induced the expression of NDRG1 in human trophoblasts and the redistribution of NDRG1 into the nucleus and cytoplasmic membranes associated with the endoplasmic reticulum (ER) and microtubules. Mutation of the phosphopantetheine attachment site (PPAS) within NDRG1 abrogated this pattern of redistribution. Our results shed new light on the impact of cell injury on NDRG1 expression patterns, and suggest that the PPAS domain plays a key role in NDRG1's subcellular distribution.
    Preview · Article · Sep 2013 · PLoS ONE
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    • "Under this hypothesis, maintained low levels of AA would trigger the expression of both genes at 2–3 DPH but subsequently would drop at 4–5 DPH as a consequence of first uptakes of AA from diet (the ~5-fold increase in AA content detected in larvae between 3 and 5 DPH matched with a ~ 3.8-fold and a ~ 1.6-fold decrease of slc23a1 and slc23a2 mRNA levels, respectively). Several reports sustain the existence of a feedback mechanism of transcriptional control by AA in both SVCTs genes (MacDonald et al., 2002; Karaczyn et al., 2006; Amano et al., 2010; Darias et al., 2011). However, gene expression patterns in later stages are not compatible with a transcriptional control by AA. "
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    ABSTRACT: Vitamin C (ascorbic acid, AA) is an antioxidant that acts as a free radical scavenger and cofactor for several important enzymatic reactions, thus being important for normal cellular functions, growth and development. Accumulation of AA in cells depends on two types of sodium-dependent vitamin C transporters (SVCTs), designed as SVCT1 and SVCT2. In human, they are the products of SLC23A1 and SLC23A2 genes, respectively. In the present work, the molecular cloning of the cDNAs corresponding to slc23a1 and slc23a2 in a teleost fish, the Senegalese sole (Solea senegalensis Kaup, 1858) is first described. Sequence analysis of the predicted polypeptides revealed a conserved topology with those of mammals with important motifs involved in structure and function, being also present in svct1 and svct2. Phylogenetic analyses including a range of vertebrate SVCTs suggest that both transporters are the result of an ancient gene duplication event that occurred prior to the divergence of tetrapods and teleosts, which took place 450 million years ago. Expression profiles in juvenile tissues and during larval development were analyzed using a real-time PCR approach. In juvenile fish, slc23a1 was strongly expressed in intestine, whereas slc23a2 exhibited a widespread distribution in tissues. Transcripts of both genes were detected at early developmental stages, probably representing mRNAs of maternal origin. A possible regulation by their own substrate was detected after first uptakes of AA from diet in both genes. During metamorphosis, both slc23a1 and slc23a2 were down-regulated, the former in a thyroid hormone (TH) dependent way. This pattern coincided with a significant reduction in the AA content of larvae during metamorphosis. These results are interpreted in a physiological context of general reduction in the metabolism of metamorphic larvae. Data presented here provide the first step toward a better understanding of the physiological role of SVCTs in teleost fish.
    Full-text · Article · Nov 2011 · Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology
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    • "NRDG1 was upregulated within 24 h of HIF-1a stabilization following exposure to NiCl 2 , NiO nanoparticles, and metallic nickel nanoparticles but not to metallic micron-sized particles. After 72 h, increased NDRG1 expression was also induced in control cultures, consistent with previous reports of increased NDRG1 over time in culture (Karaczyn et al., 2006). Taken together, these results indicate that ionic nickel mobilized from nickel oxide and metallic nickel nanoparticles activates the HIF-1a pathway in H460 cells. "
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    ABSTRACT: Micron-sized particles of poorly soluble nickel compounds, but not metallic nickel, are established human and rodent carcinogens. In contrast, little is known about the toxic effects of a growing number of Ni-containing materials in the nano-sized range. Here, we performed physicochemical characterization of NiO and metallic Ni nanoparticles and examined their metal ion bioavailability and toxicological properties in human lung epithelial cells. Cellular uptake of metallic Ni and NiO nanoparticles, but not metallic Ni microparticles, was associated with the release of Ni(II) ions after 24-48 h as determined by Newport Green fluorescence. Similar to soluble NiCl₂, NiO nanoparticles induced stabilization and nuclear translocation of hypoxia-inducible factor 1α (HIF-1α) transcription factor followed by upregulation of its target NRDG1 (Cap43). In contrast to no response to metallic Ni microparticles, nickel nanoparticles caused a rapid and prolonged activation of the HIF-1α pathway that was stronger than that induced by soluble Ni(II). Soluble NiCl₂ and NiO nanoparticles were equally toxic to H460 human lung epithelial cells and primary human bronchial epithelial cells; metallic Ni nanoparticles showed lower toxicity and Ni microparticles were nontoxic. Cytotoxicity induced by all forms of Ni occurred concomitant with activation of an apoptotic response, as determined by dose- and time-dependent cleavage of caspases and poly (ADP-ribose) polymerase. Our results show that metallic Ni nanoparticles, in contrast to micron-sized Ni particles, activate a toxicity pathway characteristic of carcinogenic Ni compounds. Moderate cytotoxicity and sustained activation of the HIF-1α pathway by metallic Ni nanoparticles could promote cell transformation and tumor progression.
    Full-text · Article · Aug 2011 · Toxicological Sciences
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