| Popis: |
Although production of formate has not been previously reported in cell culture, we have measured formate concentrations in culture supernatants as high as 6 mM by ion chromatography. To determine if the generation of formate is folate dependent, folate was titrated in 60 mm dishes with concentrations from 0 to 14.8 RM. Cultures were evaluated for cell growth by measuring lactate dehydrogenase (LDH) and for formate concentration by liquid chromotography. While media folate concentration had no effect on cell growth, formate levels were reduced in the absence of folate for one passage. Multiple passaging of cells in folate-free medium in order to deplete intracellular levels of folate resulted in no production of formate. This implies that folate is required as a cofactor for formate production. Formate is one of several one carbon units in the cell. A major source of one-carbon units is serine. To determine whether serine is the source of formate, media serine levels were varied in the presence of 14.7uM folate. Formate synthesis increased in direct proportion to serine concentration. Formate was not produced in the absence of serine. This indicates that exogenous serine is the source for formate in these cells. To determine formate toxicity, cells were seeded at low density (20,000 cells/ml) or high density (600,000 cells/ml) with 0 to 20mM formate, and measured for cell growth over 8 days. At low cell densities, cultures were growth-inhibited by 20% with 4 mM formate and by 60% with 10 mM formate. Inhibitory effects on cell growth were also seen in cultures inoculated at high cell density. It is therefore possible that formate is toxic in cell culture production systems. |
