Possible involvement of NF-kappaB in the response of cells to folate deficiency

Folate, a water soluble B vitamin, serves as a methyl donor for the synthesis of nucleotides and methionine. Folate deficiency has been linked to a variety of health problems that include developmental defects, cancer and neurological diseases. The relation between folate deficiency and human health problems has not been fully explained. Our preliminary results and limited published data have shown that the activity of NF-kappaB increases in folate deficient conditions. NF-kappaB is a cytosolic dimeric transcription factor that responds to a variety of stimuli by regulating genes involved in immune response, cell survival and cell growth. The purpose of the study was to determine if folate deficiency leads to altered NF-kappaB activity, protein levels and nuclear localization, and to determine the possible mechanisms behind this alteration. NIH3T3 cells containing a NF-kappaB reporter construct (NF-kappaB-LUC-3T3 cells) were grown for 7 and 11 days in folate sufficient and folate deficient DMEM medium either with or without TNF-alpha (stimulator of NF-kappaB). NF-kappaB activity was higher in folate deficient medium at days 7 and 11 in both unstimulated and stimulated cells, as measured by luciferase assay per unit DNA. NF-kappaB protein levels analyzed by Western Blot showed no difference between folate sufficient and deficient cells. Immunofluorescence by confocal microscopy showed that there was no significant change in the nuclear localization of NF-kappaB in folate deficient conditions even when stimulated with TNF-alpha. Analysis of reactive oxygen species (ROS) using a fluorescent assay with flow cytometry revealed that there was more fluorescence per cell in folate deficient cells at day 11. This increase in ROS could be one of the mechanisms behind the increase in NF-kappaB activity in folate deficient cells.