title: Ascorbate depletion mediates up-regulation of hypoxia-associated proteins by cell density and nickel.
abstract: 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.
authors.affiliation: Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick, Frederick, Maryland 21702, USA.
authors.forename: Aldona
authors.initials: A
authors.lastname: Karaczyn
authors.forename: Sergey
authors.initials: S
authors.lastname: Ivanov
authors.forename: Mindy
authors.initials: M
authors.lastname: Reynolds
authors.forename: Anatoly
authors.initials: A
authors.lastname: Zhitkovich
authors.forename: Kazimierz S
authors.initials: KS
authors.lastname: Kasprzak
authors.forename: Konstantin
authors.initials: K
authors.lastname: Salnikow
mesh_terms: D000951:Antigens, Neoplasm / Q000378:metabolism; D001205:Ascorbic Acid / Q000494:pharmacology*; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases / Q000378:metabolism; D002452:Cell Count; D018797:Cell Cycle Proteins; D015687:Cell Hypoxia; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression; D005786:Gene Expression Regulation; D006801:Humans; D051793:Hypoxia-Inducible Factor 1 / Q000235:genetics / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D007501:Iron / Q000494:pharmacology; D019344:Lactic Acid / Q000494:pharmacology; D009532:Nickel / Q000494:pharmacology*; D029363:Organic Anion Transporters, Sodium-Dependent / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D060347:Sodium-Coupled Vitamin C Transporters; D027981:Symporters / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D015854:Up-Regulation; D000099259:N-myc Downstream-Regulated Gene 1 Protein
chemical_list: D000951:Antigens, Neoplasm; D001205:Ascorbic Acid; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases; D018797:Cell Cycle Proteins; D051793:Hypoxia-Inducible Factor 1; D047908:Intracellular Signaling Peptides and Proteins; D007501:Iron; D019344:Lactic Acid; D009532:Nickel; D029363:Organic Anion Transporters, Sodium-Dependent; D011506:Proteins; D060347:Sodium-Coupled Vitamin C Transporters; D027981:Symporters; D014157:Transcription Factors; D000099259:N-myc Downstream-Regulated Gene 1 Protein; C555163:SLC23A2 protein, human; C089226:CA9 protein, human