Membrane transport in synchronized ehrlich ascites tumor cells: Uptake of amino acids by the A and L system during the cell cycle

Abstract

Using the double thymidine block technique. Ehrlich ascites tumor cells (ELD) carried in continuous spinner culture have been synchronized. Simultaneous monitoring of ³H-thymidine incorporation, cell number and mitotic index yielded a cell cycle time of approximately 13.5 hours. This is composed of an S period of 3–4 hours, G₂ of 6–8 hours and M of 1–2 hours. No appreciable G₁ is present. Ehrlich cells synchronized in this manner were used to investigate the characteristics of two neutral amino acid transport systems during progression through the cell cycle. Unidirectional influx via the Na-dependent system A was studied using C¹⁴-alpha-aminoisobutyrate (AIB) as substrate. The Na-independent system L was monitored using ³H-leucine and ¹⁴C-cycloleucine as substrates. Transport by the A system was minimal in M and early S. It underwent a three-fold increase during late S and early G₂. In mid G₂ the transport via this system rapidly dropped and remained low again through M and early S. The intracellular/extracellular ratios of AIB indicate that the system is actively transporting AIB throughout the cell cycle. The minimum ratios of approximately 3 were achieved during early M and the maximum ratios of approximately 9 were achieved in late S, early G₂. The uptake of leucine and cycloleucine by the L system was quite different during the cell cycle. Maximal unidirectional influx by this system occurred during early and mid S period. Upon progression into G₂ the transport rate dropped and remained reduced throughout M. Intracellular/extracellular ratios of leucine or cycloleucine were near unity at the peak of the transport activity (early S) and dropped to values of 0.5 to 0.6 throughout the remainder of the cycle. This result indicates that inward transport by the L system is, for the most part, non-active in growing cells.