Choline availability modulates human neuroblastoma cell proliferation and alters the methylation of the promoter region of the cyclin-dependent kinase inhibitor 3 gene.
Choline is an important methyl donor and a component of membrane phospholipids. In this study, we tested the hypothesis that choline availability can modulate cell proliferation and the methylation of genes that regulate cell cycling. In several other model systems, hypomethylation of cytosine bases that are followed by a guanosine (CpG) sites in the promoter region of a gene is associated with increased gene expression. We found that in choline-deficient IMR-32 neuroblastoma cells, the promoter of the cyclin-dependent kinase inhibitor 3 gene (CDKN3) was hypomethylated. This change was associated with increased expression of CDKN3 and increased levels of its gene product, kinase-associated phosphatase (KAP), which inhibits the G(1)/S transition of the cell cycle by dephosphorylating cyclin-dependent kinases. Choline deficiency also reduced global DNA methylation. The percentage of cells that accumulated bromodeoxyuridine (proportional to cell proliferation) was 1.8 times lower in the choline-deficient cells than in the control cells. Phosphorylated retinoblastoma (p110) levels were 3 times lower in the choline-deficient cells than in control cells. These findings suggest that the mechanism whereby choline deficiency inhibits cell proliferation involves hypomethylation of key genes regulating cell cycling. This may be a mechanism for our previously reported observation that stem cell proliferation in hippocampus neuroepithelium is decreased in choline-deficient rat and mouse fetuses.
Niculescu
Mihai D
MD
Department of Nutrition, School of Public Health, University of North Carolina at Chapel Hill, NC 27599-7461, USA.
Yamamuro
Yutaka
Y
Zeisel
Steven H
SH
D001483:Base Sequence; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000235:genetics*; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D002794:Choline / Q000494:pharmacology*; D018899:CpG Islands / Q000187:drug effects; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D019175:DNA Methylation / Q000187:drug effects*; D054637:Dual-Specificity Phosphatases; D006801:Humans; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000188:drug therapy* / Q000378:metabolism* / Q000473:pathology; D020411:Oligonucleotide Array Sequence Analysis; D010766:Phosphorylation / Q000187:drug effects; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D017027:Protein Tyrosine Phosphatases; D016160:Retinoblastoma Protein / Q000187:drug effects / Q000378:metabolism
D018797:Cell Cycle Proteins; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D016160:Retinoblastoma Protein; C084429:CDKN3 protein, human; D054637:Dual-Specificity Phosphatases; D017027:Protein Tyrosine Phosphatases; D002794:Choline