In the posting in which I discussed the article by Mashiyama et al. (2004) (http://www.ncbi.nlm.nih.gov/pubmed/15183762), I was going to note that a 0 nM extracellular folate concentration in a culture medium, as used by Mashiyama et al. (2004), generally does not produce a complete depletion of intracellular total folate. When Mashiyama et al. (2004) showed that uracil misincorporation into DNA had been less in cells grown in 0 nM than in 20 nM extracellular folate, this just meant that the cells in 0 nM folate were not growing and dividing rapidly (if at all) and were therefore able to exhibit lower degrees of uracil misincorporation. Courtemanche et al. (2004) [(http://www.jimmunol.org/cgi/content/full/173/5/3186) (http://www.ncbi.nlm.nih.gov/pubmed/15322179?dopt=Abstract)] found essentially the same thing. Uracil misincorporation was higher at 6 and 12 nM extracellular folate than at 0 and was the lowest, among the various concentrations of extracellular folate that had been used, at 3,000 nM. But Stempak et al. (2005) (http://www.ncbi.nlm.nih.gov/pubmed/15695236?dopt=Abstract) found that cells cultured in 0 nM extracellular folate did contain some intracellular folate and were able to divide at low rates. I'm adding the conversions to nM intracellular total folates, for those cells grown at 0 nM extracellular folate, at the end of this posting.
It's interesting that Koury and Horne (1994) (http://www.ncbi.nlm.nih.gov/pubmed/8171036?dopt=Abstract) found a biphasic influence of exogenous thymidine on the rate of apoptosis in cultured, folate-depleted proerythroblasts. Koury and Horne (1994) found that exogenous thymidine, in the culture medium, up to about 10 uM increased the percentage of cell survival. As the concentration was increased past 10 uM, however, the rate of apoptosis began to increase gradually. This might be explained by the general mechanism that Mashiyama et al. (2004) proposed. Although it's conceivable that hypoxanthine depletion, in response to depletion of intracellular folate, could have played a role in the concentration-dependence of the effect of extracellular thymidine, thymidine availability, which is increased by the folate-coenzyme-dependent de novo biosynthesis of thymidine, is generally known to be "rate-limiting" for DNA replication. Thus, as the extracellular thymidine concentration was increased past a certain level, the rate of cell proliferation may have increased to a point that, as proposed by Mashiyama et al. (2004), would actually increase the degree of uracil misincorporation and initiate apoptosis. There might be intracellular purine depletion that cannot be compensated for by thymidine alone, but it's interesting to note that exogenous hypoxanthine, alone and without thymidine, has frequently been shown to partially prevent apoptosis in folate-depleted, cultured cells [(Kane et al., 1987: http://www.ncbi.nlm.nih.gov/pubmed/2445472) (Koury et al., 2000: http://www.ncbi.nlm.nih.gov/pubmed/11050010)]. The pro-apoptotic effect of >10 uM thymidine could, therefore, have been mediated by a thymidine-induced purine-depletion that had, in turn, produced an increase in uracil misincorporation. Thymidine may upregulate 5'-nucleotidase activity or produce an imbalance of nucleotides by some other mechanism.
Here are the conversions (based on: http://hardcorephysiologyfun.blogspot.com/2008/12/cell-biology-conversion-factors-for-ngg.html) for the intracellular folate levels in cells grown in 0 nM extracellular folate (from Stempak et al., 2005):
NIH/3T3 cells [murine (mouse-derived) embryonic fibroblasts]:
(1.91 ng intracellular folates/5 x 10^6 cells) x (1 nmol/459.44 ng) x (1.9 x 10^8 cells/mL intracellular water) x (1000 mL water/1 L water) = ~158 nM
CHO-K1 (chinese hamster ovary cells): 1.28 ng folates/5 x 10^6 cells = ~106 nM
HCT116 cells (human colonic epithelial carcinoma cells): 2.40 ng folates/5 x 10^6 cells = ~199 nM
Caco-2 (human epithelial colorectal carcinoma cells): 3.50 ng folates/5 x 10^6 cells = ~289 nM
No comments:
Post a Comment