Abstract
Two human hepatoma cell lines, Hep G2 and Hep 3B, were screened for vitamin D3-25-hydroxylase enzyme activity by incubation with radioactive vitamin D3. A compound co-chromatographing with 25-OH-D3 was synthesized in both cell lines but its rate of synthesis was tenfold greater in Hep 3B than in Hep G2 cells. The identity of the compound was confirmed by comparing its chromatographic properties with authentic 25-OH-D3 on three different high pressure liquid chromatography systems. Its production was suppressed by adding fetal calf serum (10%), lipoprotein-deficient fetal calf serum, or pure vitamin D-binding globulin to the medium. The mechanism of action of these plasma proteins appears to involve retardation of uptake of the substrate. These two cell lines offer considerable potential as defined in vitro models for studying the effects of physiological factors on the 25-hydroxylation of vitamin D3.
Highlights
Two human hepatoma cell lines, Hep G2 and Hep 3B, were screened for vitamin Ds-25-hydroxyIase enzyme activity by incubation with radioactive vitamin DS. h compound co-chromatographing with 25-aH-D3 was synthesized in both cell lines but its rate of synthesis was tenfold greater in Hep 3B than in Hep G2 cells
D3 was incubated with 2 ml of MEM containing 10% fetal calf serum (FCS) in the absence of cells for 2 days, little radioactivity was recovered in the fractions eluting in the position of authentic 25-OH-D3 (Table 1).Small amounts of radioactivity migrating in this region of the chromatogram were not significantly above background and were attributable to minute amounts of impurities in the original substrate and to formation of nonenzymatic products during incubation
Further chromatography of the material produced by Hep 3B and Hep G2 cells revealed that it comigrated with authentic, standard 25-OH-D3 on three different high pressure liquid chromatography (HPLC) systems (Fig. 1)
Summary
Experiments were carried out using two human hepatoma-derived cell lines, Hep G2 and Hep 3B, obtained from American Tissu?, Culture Collection. Cells were grown in T75 flasks con’taining 30 ml of Earle’s minimal essential medium (MEMfsupplemented with L-glutamine (4 mM), penicillin (100 I.u./ml), and streptomycin (100 pg/ml) containing 10% fetal calf serum (FCS), and incubated at 37OC in a humidified atmosphere of 95% air, 5% COP.Fresh medium was added every 2 days. Cells were plated in T25 flasks and grown to late log phase. Hep G2 and Hep 3B cells were incubated for 48 hr in one of the followingfive media: I ) MEM; 2) MEM + 10%. FCS; 3) MEM + 10% FCS + 1 pM estradiol; 4)MEM + 10% lipoprotein-depleted serum (LPDS); 5)MEM + DBP (25 pg/ml).
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