Nitrogen Mustard Sensitivity and Choline Transport in Walker 256 Carcinosarcoma Cells in Vitro

Summary

Single cell suspension cultures of Walker 256 carcinosarcoma were established in vitro in Fischer's medium supplemented with 10% horse serum. The cells grew exponentially with a doubling time of 16 to 17 hours, had a cell volume (mean ± S.E.) of 2313 ± 89 cu µm, and were cloned in soft agar with a cloning efficiency of 68.7 ± 6.3%. Sensitivity to the alkylating agent nitrogen mustard (HN2) was comparable to that observed previously with L5178Y murine lymphoblasts; the D₀ (the dose of drug reducing survival to 37% of the initial cell population) was 5.35 ng/ml, and the extrapolation number, n, was 1.27. An investigation of the interaction of hydrolyzed nitrogen mustard and choline transport revealed that uptake of choline-¹⁴C obeyed simple Michaelis-Menten kinetics, proceeded “uphill” against a concentration gradient of over 30-fold, and showed a decrease in distribution ratio as choline concentration increased and choline transport was competitively inhibited by hydrolyzed NH2. The Michaelis constant, K_m, for choline transport was 2.90 × 10⁻⁵ m, the transport capacity, V_max, was 4.83 × 10^-17 moles/min/cell, and the inhibition constant, K₁, with hydrolyzed HN2 as inhibitor, was 7.83 × 10⁻⁵ m. These findings indicate that choline transport by Walker carcinosarcoma cells is an active carrier-mediated process and that choline and hydrolyzed HN2 compete for the same transport mechanism.