Abstract 727: Does gene transfer of gulono-lactone oxidase into human hepatocellular carcinoma cells restore ascorbate biosynthesis

Abstract

Abstract Humans are unable to synthesise ascorbate (vitamin C) due to a mutation in the gene for gulono-lactone oxidase (Gulo), the terminal enzyme in the synthesis pathway. Ascorbate is a vital micronutrient required for many biological functions, including its action as a vital cofactor for Cu- and Fe-containing enzymes. As enzyme cofactor for HIF-hydroxylases, it is important for the regulation of hypoxia-inducible factor-1 (HIF-1), which governs cancer growth and spread. A lack of ascorbate increases both the level and activity of HIF-1 in cell culture. This study aimed to restore ascorbate synthesis to human cells and to determine the effect of intracellular ascorbate biosynthesis on HIF-1 levels. HepG2 cells were gene modified using Lipofectamine LTX with a plasmid encoding the mouse Gulo cDNA and a control plasmid encoding the green fluorescent protein (GFP). Modified HepG2 cells were tested for genomic incorporation by PCR, for Gulo enzyme production by Western blotting, and ascorbate synthesis by high performance liquid chromatography with electrochemical detection (HPLC-ECD). Protein levels of HIF-1alpha in hypoxic cells were measured by Western blot analysis. Parental HepG2 cells accumulated a mean of 120 nmol ascorbate per 1 million cells when loaded for 24h with 0.5 mM ascorbate. Gene-modified HepG2Gulo cells demonstrated a change in morphology and an increase in adherence, compared to HepG2GFP and parental HepG2 cells. A PCR-positive stable clone was able to synthesise ascorbate but only when the Gulo substrate, L-gulono-1,4-lactone, was supplied. Intracellular ascorbate levels then reached 5% of saturation levels. Hypoxic HIF-1 accumulation was reduced by addition of gulonolactone to this clone. However, gulonolactone also reduced HIF-1 levels in parental HepG2 cells, suggesting that it may similarly act as cofactor for the HIF-hydroxlases. This study indicated that the ascorbate biosynthesis pathway in human cells is significantly modified and may contain numerous non-functional members besides gulono-lactone oxidase. Future biochemical studies will determine which other enzymes are non-functional. Citation Format: Teresa Flett, Elizabeth Campbell, Elisabeth Phillips, Margreet CM Vissers, Gabi U. Dachs. Does gene transfer of gulono-lactone oxidase into human hepatocellular carcinoma cells restore ascorbate biosynthesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 727. doi:10.1158/1538-7445.AM2014-727