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, Km, for choline transport was 2.90 × 10−5 m, the transport capacity, Vmax, 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.
Footnotes
-
↵1 This work was supported by a grant from the National Cancer Institute of Canada.
- Received June 5, 1973.
- Accepted July 9, 1973.
- ©1973 American Association for Cancer Research.