No abstract

Potentiometric spectroscopic and X-ray studies of the open chain oxime ligand, N,NЈ-bis(2-hydroxyiminopropionyl)propane-1,3-diamine and its complexes with Ni II and Cu II ions showed a very high efficacy of the ligand studied in the co-ordination of both Cu II and Ni II ions. The metal complexes are very stable and square-planar with four nitrogens involved in metal-ion binding. These complexes may be additionally stabilised by hydrogen bonds between two oxime oxygen atoms.

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.

Hypoxia-inducible factor (HIF-1) regulates the expression of genes that facilitate tumor cell survival by making them more resistant to therapeutic intervention. Recent evidence suggests that the activation of other transcription factors, in cooperation with HIF-1 or acting alone, is involved in the upregulation of hypoxia-inducible genes. Here we report that high cell density, a condition that might mimic the physiologic situation in growing tumor and most probably representing nutritional starvation, upregulates hypoxia-inducible genes. This upregulation can occur in HIF-independent manner since hypoxia-inducible genes carbonic anhydrase 9 (CA9), lysyloxidase like 2 (LOXL2) and n-myc-down regulated 1 (NDRG1)/calcium activated protein (Cap43) can be upregulated by increased cell density under both normoxic and hypoxic conditions in both HIF-1 alpha-proficient and -deficient mouse fibroblasts. Moreover, cell density upregulates the same genes in 1HAEo- and A549 human lung epithelial cells. Searching for other transcription factors involved in the regulation of hypoxia-inducible genes by cell density, we focused our attention on ETS1. As reported previously, members of v-ets erythroblastosis virus E26 oncogene homolog (ETS) family transcription factors participate in the upregulation of hypoxia-inducible genes. Here, we provide evidence that ETS1 protein is upregulated at high cell density in both human and mouse cells. The involvement of ETS1 in the upregulation of hypoxia-inducible genes was further confirmed in a luciferase reporter assay using cotransfection of ETS1 expression vector with NDRG1/Cap43 promoter construct. The downregulation of ETS1 expression with small interfering RNA (siRNA) inhibited the upregulation of CA9 and NDRG1/Cap43 caused by increased cell density. Collectively, our data indicate the involvement of ETS1 along with HIF-1 in regulating hypoxia-inducible genes.