Density‐dependent inhibition of expression of syncytiotrophoblastic markers by cultured human choriocarcinoma (BeWo) cells

Abstract

In the presence of methotrexate, cultured human choriocarcinoma (BeWo) cells undergo a differentiative response that resembles normal trophoblastic development. In the current study, the effects of cell number and population density on drug-induced conversion of BeWo cells from the cytotrophoblastlike to the syncytiotrophoblastlike phenotype were investigated using as markers of differentiation formation of "giant" cells, a process shown to require exogenous purines, and expression of placental (heat-stable) alkaline phosphatase. Giant cell formation, assessed by determination of cell volumes, was reduced in crowded cultures, and addition of hypoxanthine to growth media partially restored methotrexate-induced cell enlargement. Cellular uptake of methotrexate, assessed by following the loss of methotrexate from cell culture fluids during drug exposures, was two-threefold greater in sparsely populated than in densely populated cultures. Although the concentration of methotrexate in culture fluids of crowded cultures declined during exposures of 48 hr, the amount of extracellular drug remaining at 48 hr was well above the threshold for induction of the differentiative response. When culture population was held constant and population density was manipulated by varying the substratum available to cells, methotrexate-induced cell enlargement was inversely related to population density. Expression of placental alkaline phosphatase, salvage of exogenous hypoxanthine, and synthesis of RNA were also reduced at high population densities. These results indicate that expression of markers of methotrexate-induced differentiation of BeWo cells was inhibited in a density-dependent manner that may have been related to reduced cellular uptake of the inducing agent and of exogenous nutrients (purines) from culture fluids.

Demonstration of Equilibrative Nucleoside Transporters (hENT1 and hENT2) in Nuclear Envelopes of Cultured Human Choriocarcinoma (BeWo) Cells by Functional Reconstitution in Proteoliposomes

“…Cell Culture-The origin and characteristics of BeWo cells have been described (24,25). Cultures were initiated from mycoplasma-free frozen stocks and grown at 37°C in a humidified atmosphere of 5% CO 2 in air as described previously (25).…”

“…Cultures were initiated from mycoplasma-free frozen stocks and grown at 37°C in a humidified atmosphere of 5% CO 2 in air as described previously (25). Cells were harvested at weekly intervals from proliferating cultures by trypsin treatment and maintained in plastic culture flasks in Roswell Park Memorial Institute (RPMI) 1640 basal medium supplemented with 5% fetal bovine serum and 5% NUserum type 1V.…”

Characterization of the Na + /H + exchanger in human choriocarcinoma (BeWo) cells

“…We examined heatstable alkaline phosphatase activity that has been used as a marker of BeWo cell differentiation [8]. The results ( Table 1) show that alkaline phosphatase activity increased over 11-fold when cells were treated with methotrexate to induce differentiation.…”

“…To confirm that cells treated with methotrexate underwent differentiation, we examined the effects on cell size and proliferation. During exposure to methotrexate BeWo cells have been shown to stop proliferating and fuse to form greatly enlarged "giant" multinucleate cells [8,17]. We examined the effect of 3 days of treatment with methotrexate on these cells (Fig.…”

“…As a further check of the differentiative response we examined the effect of methotrexate on BeWo cell proliferation. BeWo cells have been shown to stop proliferating when treated with methotrexate [8,17]. Replicate cultures of cells were grown and experimental cells were treated with 1 µM methotrexate for the times indicated.…”