Choline availability modulates human neuroblastoma cell proliferation and alters the methylation of the promoter region of the cyclin‐dependent kinase inhibitor 3 gene

Abstract

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.

The fetal origins of memory: The role of dietary choline in optimal brain development

“…70 In cholinedeficient cells in culture, methylation of the cyclindependent kinase inhibitor 3 gene promoter is decreased, resulting in overexpression of this gene, which inhibits cell proliferation. 72 We replicated this observation in brains of fetuses from choline-deficient mothers and found that cyclin-dependent kinase inhibitor 3 was hypomethylated and overexpressed in the neuroepithelium of the fetal hippocampus (submitted for publication); we suggest that this is the likely molecular mechanism for decreased stem cell proliferation in brains of these fetuses. This is not an outlandish hypothesis, because we already know that dietary intake of methyl donors in pregnant mice can permanently alter the expression of genes that control coat color in their pups.…”

Prenatal choline supplementation attenuates neuropathological response to status epilepticus in the adult rat hippocampus

“…Prenatal choline availability during ED12-18 determines ED 18 brain betaine concentration (Garner et al, 1995), alters both global and gene-specific DNA methylation (Niculescu et al, 2004(Niculescu et al, , 2006Kovacheva et al, in press), and leads to changes in adult expression of genes relevant for hippocampal plasticity . Enhanced hippocampal plasticity likely plays a significant role in both the memory-enhancing effects of prenatal choline supplementation (Meck & Williams, 2003;McCann et al, 2006) and its neuroprotective actions.…”

Choline: Critical Role During Fetal Development and Dietary Requirements in Adults

“…When this modification occurs in promoter regions, gene expression is altered (9); increased methylation is associated with gene silencing or reduced gene expression (62). In choline-deficient cells in culture, and in fetal rodent brains from mothers fed choline-deficient diets, methylation of the CDKN3 gene promoter is decreased, resulting in overexpression of this gene, which inhibits cell proliferation (93,94). In choline-deficient liver, there is hypomethylation of specific CCGG sites within several genes for which mRNA levels were increased, including c-myc, c-fos, and c-Ha-ras (24).…”