Effect of triamcinolone acetonide on the growth of NEL-M1 human melanoma cells cultured in the presence and absence of growth stimulatory agents.

The human melanoma cell line NEL-M1 proliferates in Ham's F-12 medium in the absence of serum, hormones, and growth factors. Culturing NEL-M1 cells in defined medium supplemented with insulin (5 micrograms/ml) and transferrin (5 micrograms/ml) results in a 94% increase in [3H]thymidine incorporation after 24 h and a 6- to 8-fold increase in cell number after 5 days. The addition of 17 beta-estradiol, testosterone, and progesterone to defined medium, either as single agents or in combination with insulin and transferrin, had no effect on cell growth. No specific estrogen, androgen, or progesterone receptor proteins were detected in NEL-M1 cells. In contrast, the synthetic glucocorticoid triamcinolone acetonide (10 nM) inhibited growth of NEL-M1 cells in serum-free Ham's F-12 medium by 50%. The 6- to 8-fold stimulation of cell growth by insulin and transferrin was reduced to 1.75-fold when triamcinolone acetonide (10 nM) was added to the medium. Additional studies show that medium conditioned by NEL-M1 cells stimulated [3H]thymidine incorporation into cellular DNA of NEL-M1 cells and increased the growth of NEL-M1 cells 3-fold over cells maintained in defined medium. The addition of triamcinolone acetonide (10 nM) to defined medium supplemented with conditioned medium resulted in only a 1.48-fold increase in cell number. These results show that triamcinolone acetonide inhibits growth of NEL-M1 human melanoma cells in serum-free defined medium, medium supplemented with insulin and transferrin, and medium supplemented with an endogenous growth stimulatory factor(s). Thus, NEL-M1 cells are an excellent model system to study the mechanism of action of glucocorticoids and also the interplay between exogenous hormones and growth factors and endogenous growth factors.

In vitro growth inhibition of human malignant melanoma cells by glucocorticoids.

The human malignant melanoma cell line, NEL, was found to contain glucocorticoid receptors. When the binding data were analyzed according to the method of Scatchard, results indicated a ligand binding capacity of 247 fmol/mg protein and a Kd of 1 X 10(-9) M. Additional studies show that the continuous incubation of NEL cells with triamcinolone acetonide (TA) for 72 hr results in a 30% inhibition in cell growth. To ascertain the mechanism by which glucocorticoids inhibit the growth of NEL cells, uptake and incorporation studies were carried out using various 3H precursors. Results indicate that, after 4 hr of TA treatment, a modest inhibition in [3H]thymidine uptake was observed, while stimulation of [3H]thymidine incorporation was noted at all steroid concentrations tested. However, cells incubated for 18 hr with TA (concentration, greater than or equal to 10(-8) M) showed a 30% decrease in the amount of [3H]thymidine incorporated into DNA. TA had no effect on [3H]leucine or [3H]glucose uptake after 4 hr of treatment but did inhibit [3H]glucose (42%) uptake after 18 hr of treatment. A slight stimulation (9%) in [3H]leucine incorporation was observed at this time point. When NEL cells were incubated with TA and the antiglucocorticoid, progesterone, the inhibition in [3H]thymidine incorporation was negated. These findings indicate that glucocorticoids exert some influence on the growth of human melanoma cells, and this effect is mediated through the glucocorticoid receptor.

Effect of triamcinolone acetonide on tyrosinase activity in a human melanoma cell line.

The synthetic glucocorticoid, triamcinolone acetonide, was found to increase melanogenesis in the human melanoma cell line NEL. Treatment of NEL cells for 24 hr with triamcinolone acetonide (1 X 10(-7) M) increased the activity of the enzyme tyrosinase by 43% and the incorporation of the melanin precursor, L-3,4-dihydroxyphenylalanine, by 23%. Additional studies revealed no change in cyclic AMP levels over an 18-hr test period. A 2-hr preincubation of NEL cells with actinomycin D (10 micrograms/ml) prevented the increase in tyrosinase activity by triamcinolone acetonide. When triamcinolone acetonide was added to a synchronized population of NEL cells, an increase in tyrosinase activity was observed at 16 hr, coinciding with the late S phase of the cell cycle. These results suggest that glucocorticoids are involved in the regulation of melanogenesis in NEL cells by increasing the activity of the rate-controlling enzyme tyrosinase.

Suppression of DNA synthesis in NEL-M1 human melanoma cells by triamcinolone acetonide.

The present study characterizes the biological response of a cloned human melanotic melanoma cell line (NEL-M1) to glucocorticoid treatment. Scatchard analysis of the binding of [3H]-triamcinolone acetonide to the glucocorticoid receptor showed a binding capacity of 170 fmol/mg protein and a dissociation constant (KD) of 1.76 X 10(-9) M. When the 3H-labeled cytosol was warmed to 25 degrees C for 30 min and then incubated with DNA-cellulose at 4 degrees C for 45 min, 32% of the specific glucocorticoid-receptor complexes were bound to DNA-cellulose. Additional studies showed that when NEL-M1 cells were cultured for 72 h with 1 X 10(-7) M triamcinolone acetonide, a 36% reduction in cellular growth was observed compared to the control cultures. The calculated population doubling time for the control cells was 17.5 h compared to 20.3 h for the triamcinolone acetonide-treated cells. Analysis of the effect of triamcinolone acetonide on macromolecular synthesis revealed that, over a 24-h incubation period, triamcinolone acetonide (a) inhibited [3H]thymidine incorporation by 51%; (b) increased the incorporation of the melanin precursor, L-3,4-dihydroxy[3H]phenylalanine, by 59%; and (c) had essentially no effect on [3H]leucine or [3H]uridine incorporation. During this same incubation period, triamcinolone acetonide inhibited [3H]glucose uptake by 19%. Further studies using synchronized NEL-M1 cells clearly show that the earliest detectable action of triamcinolone acetonide was the inhibition [3H]thymidine incorporation during the S phase of the cell cycle. Thus, these findings show that the human melanoma cell line, NEL-M1, is biologically responsive to glucocorticoid treatment. Continued studies using NEL-M1 cells may eventually lead to ascertaining the exact mechanism by which glucocorticoids regulate DNA synthesis.

Pyridoxine resistance in a rat hepatoma cell line.

The natural vitamin, pyridoxine, in the millimolar range is toxic to cultured rat hepatoma cells. A pyridoxine-resistant Fu5-5 rat hepatoma cell line was established by a stepwise increase in the concentration of pyridoxine in the medium. The newly established cell line, referred to as clone 10 (Cl.10), is resistant to killing by pyridoxine in concentrations up to 5 mM. Saturation kinetics for the uptake of [3H]pyridoxine into Fu5-5 and Cl.10 cells revealed that Fu5-5 cells take up 10 times more [3H]pyridoxine than do Cl.10 cells. Whereas the Vmax value for the uptake of [3H]pyridoxine was the same for both cell lines, the apparent Km for the Cl.10 cells was 12.5 microM compared to 0.71 microM for the Fu5-5 cells. However, intracellular levels of pyridoxal 5'-phosphate were 69% higher in Cl.10 cells than in the parental line. The resistant line is neither a permeability mutant nor deficient in pyridoxal kinase. Cl.10 cells contain 37% more adenosine 5'-triphosphate than do Fu5-5 cells and have a mitochondrial volume that is 50% greater than that of the parental line. In the absence of pyridoxine in the medium, Cl.10 cells revert to parental type with respect to pyridoxine uptake but not with respect to resistance to killing. They also maintain an enlarged mitochondrial volume. Thus, increased mitochondrial volume may be related to the development of resistance to high levels of pyridoxine.

High-dose pyridoxal supplemented culture medium inhibits the growth of a human malignant melanoma cell line.

The in vitro growth characteristics of a human malignant melanoma cell line cultured in 0.5 mM pyridoxal supplemented medium were studied. Experimentation revealed that the high-dose pyridoxal supplemented medium severely inhibited the growth of melanoma cells over a 72-hour growth period. Additional experimentation showed that cells cultured for 6 hours in the pyridoxal supplemented medium took up 25% less and incorporated 20% less [3H]uridine than control cultures. [3H]Glucose uptake was reduced by 23% at this time point. [3H]Thymidine uptake was inhibited by 12%, but no inhibition was detected in [3H]thymidine incorporation. When the vitamin B6 antagonist 4-deoxypyridoxine (which competes with pyridoxal for pyridoxal kinase) was added to the pyridoxal supplemented medium, the inhibition in [3H]uridine incorporation was reduced from 19% to 6%. However, 4-deoxypyridoxine did not reverse the inhibition of [3H]uridine uptake. These results indicate that pyridoxal and its metabolite, pyridoxal 5'-phosphate, may be involved in the growth regulation of a human malignant melanoma cell line.

Vitamin B6 kills hepatoma cells in culture.

Data are presented which indicate that vitamin B6 (pyridoxine) can retard and eventually kill Fu5-5 rat hepatoma cells in culture. Additional studies indicate that the human kidney cell line 293-31, and a rat glial cell strain. C6, display growth characteristics similar to those of hepatoma cells when cultured in 5 mM pyridoxine-supplemented medium. However, the pyridoxine-supplemented culture medium had little effect on the growth of the human mammary cancer cell line MCF-7. The resistance of the MCF-7 cells to growth inhibition by vitamin B6 suggests that the vitamer pyridoxine must be metabolized by pyridoxal before it can act as a growth inhibitor. These findings suggest the potential use of vitamin B6 as an antineoplastic agent. Possible mechanisms by which vitamin B6 promotes this effect are presented.

In vivo and in vitro inhibition of B16 melanoma growth by vitamin B6.

The effect of vitamin B6 on the growth of B16 melanoma cells in vivo and in vitro was studied. B16 melanoma cells grown for three days in medium supplemented with 5.0 mM pyridoxine or 0.5 mM pyridoxal showed an 80% reduction in cell proliferation compared with control culture. Cells cultured for six hours in medium supplemented with 0.5 mM pyridoxal took up and incorporated 13 and 32% less [3H]thymidine, respectively, than did control cultures. A 17% reduction in [3H]glucose uptake was observed at this time point. When the incubation time was decreased to three hours, an inhibition of cellular uptake of [3H]thymidine (22%), [3H]uridine (14%), and [3H]glucose (15%) was observed; however, little or no inhibition in incorporation was detected. In in vivo studies, mice pretreated with pyridoxal for two weeks and then injected with B16 melanoma cells had a 62% reduction in tumor weight compared with controls at the end of a three-week period. If tumors were first established in mice and then treated with pyridoxal for six days, a 39% reduction in tumor growth was observed. There were no differences observed in body weights or liver weights in any of the animal groups. These results indicate that supraphysiological doses of vitamin B6 can inhibit the growth of B16 melanoma cells both in vitro and in vivo. The exact mechanism by which pyridoxal exerts its inhibitory effect was not ascertained, but experiments suggest that the vitamer may be acting on the plasma membrane to reduce precursor transport into the cell.

Effect of pyridoxal phosphate on the DNA binding site of activated hepatic glucocorticoid receptor.

The binding of rat liver glucocorticoid.receptor complexes to DNA-cellulose and nuclei has been studied after activation of the complexes by heating. Subsequent exposure to pyridoxal 5'-phosphate or pyridoxal markedly inhibited this binding. In one system 0.75 mM pyridoxal 5'-phosphate or 6.5 mM pyridoxal gave 50% inhibition. Pyridoxamine 5'-phosphate, pyridoxamine, and pyridoxine did not inhibit significantly. The inhibition by pyridoxal 5'-phosphate is competitive with respect to DNA suggesting that its effect is directly on the DNA binding site of the activated receptor. The inhibition of DNA-cellulose binding by pyridoxal 5'-phosphate can be reversed by treatment with dithiothreitol or by gel filtration, but not if the modified receptor is first reduced using sodium borohydride. These results suggest that pyridoxal 5'-phosphate acts by forming a Schiff base of an epsilon-NH2 of a lysine which may be 1 residue appearing on the surface of the steroid.receptor complex upon activation. However, since pretreatment of the DNA-cellulose with the intercalating drug ethidium bormide also inhibits activated receptor binding, we conclude that the binding of the receptor involves more than electrostatic interactions between receptor positive charges and DNA phosphate groups.

Dual effects f pyridoxal 5'-phosphate on glucocorticoid-receptor complexes.

The ability of pyridoxal 5'-phosphate to inhibit DNA-cellulose binding of activated glucocorticoid-receptor complexes is pH and protein concentration dependent. At the tested pHs, all of the inhibitory activity of pyridoxal 5'- phosphate appears to be due to its ability to form a Schiff base. 2-Amino-2-(hydroxymethyl)-1,3-propanediol (100 mM) is unable to prevent or reverse the pyridoxal 5'-phosphate mediated inhibition of DNA-cellulose binding, while the same concentration of lysine is partially effective. Pyridoxal 5'-phosphate does not alter the elution profile of glucocorticoid-receptor complexes as ascertained by diethylaminoethyl (DEAE)-cellulose of DEAE-Sephadex chromatography. This observation permitted the use of these resins in detecting the previously unreported stimulation of glucocorticoid-receptor complex activation by pyridoxal 5'-phosphate. This stimulation is specific for pyridoxal 5'-phosphate and appears to be mediated via a Schiff base formation. Additionally, glucocorticoid-receptor complexes activated by pyridoxal 5'-phosphate treatment at low temperatures do not differ in size from thermally activated complexes. Thus, in vitro, pyridoxal 5'-phosphate can exert both a stimulatory effect on activation as well as an inhibitory effect on the binding of activated complexes to DNA-cellulose.

Epidermal growth factor stimulation of ornithine decarboxylase activity in a human hepatoma cell line.

Epidermal growth factor (EGF) bound specifically to the human hepatoma cell line PLC/PRF/5. Treatment of these cells with nanomolar concentrations of EGF for 4-6 hr resulted in a 2- to 6-fold increase in ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity. 12-O-Tetradecanoylphorbol 13-acetate also produced an increase in enzyme activity in these cells and exhibited an additive effect with EGF. It did not inhibit the binding of 125I-labeled EGF to these cells. The stimulation of enzyme activity by EGF was not inhibited by cycloheximide or actinomycin D, although these agents did cause a significant decrease in enzyme levels when added without EGF. Also, colchicine, chloroquine, ammonium chloride, and methylamine, compounds that inhibit EGF degradation in various cells types, did not interfere with the ability of EGF to elevate enzyme levels in the human hepatoma cells.