Abstract
Choline kinase catalyzes the phosphorylation of choline, the first step of phospholipid synthesis. Increased phosphorylation of choline is a hallmark characteristic of the malignant phenotype in a variety of neoplasms. However, in hypoxic cancer cells, choline phosphorylation is decreased. To understand the mechanism behind this altered metabolic state, we examined the expression and regulation of the major choline kinase isoform, choline kinase α (ChKα), in hypoxic PC-3 human prostate cancer cells. Hypoxia decreased choline phosphorylation, choline kinase activity, and ChKα mRNA and protein levels. Promoter analysis studies revealed a region upstream of the ChKα gene bearing a conserved DNA consensus binding motif, hypoxia response element-7 (HRE7), at position -222 relative to +1 translation start site, for binding the hypoxia dependent master regulator transcription factor, hypoxia-inducible factor 1α (HIF-1α). Electrophoretic mobility shift competition/supershift assay and chromatin immunoprecipitation assay confirmed binding of HIF-1α to HRE7. A putative promoter of ChKα was isolated from PC-3 genomic DNA and cloned into a luciferase-based reporter vector system. In PC-3 cells, hypoxia decreased the expression of luciferase under the control of the ChKα promoter. Mutation of HRE7 abrogated this hypoxia effect, further demonstrating the involvement of HRE7 in hypoxia-sensitive regulation of ChKα. The results strongly suggest that transcriptional control of choline phosphorylation is largely mediated via HIF-1α binding to the newly identified HRE7.
Highlights
Choline kinase catalyzes the phosphorylation of choline, the first step of phospholipid synthesis
The goals of the present study were to utilize PC-3 prostate cancer cells to assess the effects of hypoxia on 1) steady-state levels of choline metabolites, 2) equilibrium status of choline phosphorylation, 3) radiolabeled choline uptake and phosphorylation, 4) ChK␣ mRNA and protein levels, and 5) choline kinase activity
Following exposure to chronic hypoxia, at early incubation times (60 min), choline accumulation saturated with no change in tracer accumulation (Fig. 1B)
Summary
Choline kinase catalyzes the phosphorylation of choline, the first step of phospholipid synthesis. In hypoxic cancer cells, choline phosphorylation is decreased. To understand the mechanism behind this altered metabolic state, we examined the expression and regulation of the major choline kinase isoform, choline kinase ␣ (ChK␣), in hypoxic PC-3 human prostate cancer cells. Choline kinase activity, and ChK␣ mRNA and protein levels. Our previous tracer studies with two human prostate cancer cell lines, PC-3 and LNCaP, showed 15% and 28% decreases in choline accumulation, respectively, after 4 h of anoxia (0% O2) [11]. The goals of the present study were to utilize PC-3 prostate cancer cells to assess the effects of hypoxia on 1) steady-state levels of choline metabolites, 2) equilibrium status of choline phosphorylation, 3) radiolabeled choline uptake and phosphorylation, 4) ChK␣ mRNA and protein levels, and 5) choline kinase activity. We demonstrate that choline phosphorylation is not at equilibrium
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