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Inhibition of Methotrexate-induced Differentiation of Cultured Human Choriocarcinoma (BeWo) Cells by Thymidine¹

Cancer Research Group (McEachern Laboratory) and Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7

During exposure to methotrexate, cultured human choriocarcinoma (BeWo) cells stop proliferating, enlarge, and undergo a complex differentiative response that resembles in utero development of quiescent syncytiotrophoblasts. In the present work, complete inhibition of proliferation and maximal cell enlargement required exposure to 1 µM methotrexate, whereas colony-forming ability, determined after transfer of cells to drug-free medium, was unaffected over a wide range of concentrations (10^-12–10^-5 M). BeWo cells were sensitive to the antifolate effects of methotrexate since thymidylate synthase activity and incorporation of [¹⁴C]formate into DNA, RNA, and protein were reduced by >90% after short drug exposures, and progression of cells through S phase of the cell cycle was blocked by prolonged drug exposures. When methotrexate was coadministered with hypoxanthine and thymidine or leucovorin, its antiproliferative and differentiative effects were blocked. When methotrexate was coadministered with either hypoxanthine or thymidine, its antiproliferative activity was unaffected, whereas expression of syncytiotrophoblastic markers was blocked in the presence of thymidine but not in the presence of hypoxanthine. Exposure of BeWo cells to fluorodeoxyuridine also stimulated cell enlargement and expression of syncytiotrophoblastic markers, and these effects were blocked by coadministration of thymidine. Thus BeWo cells, which were sensitive to the antifolate effects of methotrexate, were not killed during cytostasis but instead entered a reversible differentiated state, apparently resulting from thymidylate starvation and consequent inhibition of DNA synthesis.

¹ Supported by an operating grant from the National Cancer Institute of Canada. The flow cytometry was supported by a program grant from the Alberta Heritage Savings Trust Fund-Applied Research, Cancer. N.S.B. was a recipient of a studentship award from the Alberta Heritage Foundation for Medical Research.

² Present address: Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06510.

³ Research Associate of the National Cancer Institute of Canada. To whom requests for reprints should be addressed.

Received 8/19/85. Revised 4/21/86. Revised 4/22/87. Accepted 7/ 2/87.

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