Opposing regulation of choline deficiency-induced apoptosis by p53 and nuclear factor kappaB.

We have previously shown that fetal rat brain cells, preneuronal (PC12), and hepatocyte (CWSV-1) cells undergo apoptosis during choline deficiency (CD). The PC12 and epithelial cell culture models were used to determine the molecular mechanism by which CD induces apoptosis. Our data indicate that CD leads to both growth arrest and apoptosis in a subpopulation of cells, which correlate with the up-regulation of the tumor suppressor protein p53 and concurrent up-regulation of the cyclin-dependent kinase-inhibitor p21(WAF1/CIP1). Additionally, CD induced both a G1/S and a G2/M arrest. Transient transfection of a dominant negative p53 (p53DN) construct into PC12 cells, which inhibited endogenous p53 activation, significantly reduced the induction of apoptosis associated with CD. Interestingly, CD also induced the persistent activation of the transcription factor NF-kappaB. Activation of NF-kappaB has been shown to promote cell survival and proposed to antagonize p53. Consistent with this, expression of a super-repressor form of IkappaBalpha (SR-IkappaBalpha) that functions to strongly inhibit NF-kappaB activation, profoundly enhanced cell death during CD. In summary, these results suggest that the effects of CD on apoptosis and subsequent cell survival are mediated through two different signaling pathways, p53 and NF-kappaB, respectively. Taken together, our data demonstrates the induction of opposing mechanisms associated with nutrient deficiency that may provide a molecular mechanism by which CD promotes carcinogenesis.

Apoptosis is induced by choline deficiency in fetal brain and in PC12 cells.

Treatment of rats with choline during critical periods in brain development results in long-lasting enhancement of spatial memory in their offspring. Apoptosis is a normal process during brain development, and, in some tissues, is modulated by the availability of the nutrient choline. In these studies, we examined whether availability of choline influences apoptosis in fetal brain and in the PC12 cell line derived from a rat pheochromocytoma. Timed-bred Sprague Dawley rats were fed a choline-deficient (CD), choline-control, or choline-supplemented (CS) diet for 6 days and, on embryonic day 18, fetal brain slices were prepared and apoptosis was assessed using terminal dUTP nucleotide end labeling (TUNEL) to detect DNA strand breaks and by counting of apoptotic bodies. TUNEL-positive cells were detected in 15.9% (P < 0.01), 8.7% and 7.2% of hippocampal cells from fetuses of dams fed the CD, control or CS diets, respectively. A similar inverse relationship between dietary intake of choline and TUNEL positive cells was detected in an area of cerebral cortex from these fetal brain slices. Counts of apoptotic bodies in fetal brain slices correlated inversely with choline intake of the mothers (6.2% (P < 0.01), 2.5% and 1.9% of hippocampal cells had apoptotic bodies in fetuses of dams fed the CD, control and CS diets, respectively). PC12 cells were grown in DMEM/F12 media supplemented with 70 microM choline or with 0 microM choline. The number of apoptotic bodies in PC12 cells increased when cells were grown in 0 microM choline medium (1.5%; P < 0.05) compared to 70 microM choline medium (0.55%). In PC12 cells, TUNEL labeling (DNA strand breaks) increased in choline deficient (13.5%, P < 0.05) compared to sufficient medium (5.0%). In addition, cleavage of genomic DNA-into 200 bp internucleosomal fragments was detected in choline-deficient cells. These results show that choline deficiency induces-apoptotic cell death in neuronal-type cells and in whole brain. We suggest that variations in choline availability to brain modulate apoptosis rates during development.

Choline and choline esters in human and rat milk and in infant formulas.

Large amounts of choline are required in neonates for rapid organ growth and membrane biosynthesis. Human infants derive much of their choline from milk. In our study, mature human milk contained more phosphocholine and glycerophosphocholine than choline, phosphatidylcholine, or sphingomyelin (P < 0.01). Previous studies have not recognized that phosphocholine and glycerophosphocholine exist in human milk. Concentrations of choline compounds in mature milk of mothers giving birth to preterm or full-term infants were not significantly different. Infant formulas also contained choline and choline-containing compounds. In infant formulas derived from soy or bovine milk, unesterified choline, phosphocholine, glycerophosphocholine, phosphatidylcholine, and sphingomyelin concentrations varied greatly. All infant formulas contained significantly less phosphocholine than did human milk. Soy-derived formulas contained significantly less glycerophosphocholine (P < 0.01) and phosphocholine (P < 0.01) and more phosphatidylcholine (P < 0.01) than did human or bovine milk or bovine milk-derived infant formulas. Rat milk contained greater amounts of glycerophosphocholine (almost 75% of the total choline moiety in milk) and phosphocholine than did human milk. When dams were provided with either a control, choline-deficient, or choline-supplemented diet, milk composition reflected the choline content of the diet. Because there are competing demands for choline in neonates, it is important to ensure adequate availability through proper infant nutrition. Although the free choline moiety is adequately provided by infant formulas and bovine milk, reevaluation of the concentrations of other choline esters, in particular glycerophosphocholine and phosphocholine, may be warranted.