Protection of B lymphocyte hybridoma against starvation-induced apoptosis: survival-signal role of some amino acids.

The phenomenon of starvation-induced apoptosis was studied in cultures of a mouse B lymphocyte hybridoma. In a continuous culture the limitation of nutrients was modelled by dilution of a protein-free medium with saline to 15%. Surprisingly, the hybridoma clone did not die out under extreme starvation conditions. A steady state was established in which the cells continued to grow at very low viable cell concentration, concomitantly with an enhanced rate of apoptotic death. Suppression of the death rate, and increase of steady-state viable cell concentration, could be achieved by additions of L-alanine, L-asparagine or L-glutamine, but not by addition of L-phenylalanine. This specificity pattern is in agreement with previous screening experiments that have identified a set of apoptosis-preventing amino acids (glycine, L-alanine, L-serine, L-threonine, L-proline, L-asparagine, L-glutamine, L-histidine). The analysis of amino acid consumption and production showed a consistent production of alanine and serine both in standard medium and in diluted media. When alanine was added at a final concentration of 2 mM to media diluted either to 40 or 20%, apoptosis was partly suppressed. A limited production of alanine was observed also in alanine-enriched diluted media. It is concluded that the apoptosis-preventing amino acids act as signal molecules, besides their nutritive function, and that the signal has a character of a survival factor. The observed phenomena are interpreted in terms of a survival-control mechanism that regulates the viable cell number of a lymphocyte clone in an adequate proportion with the level of available nutrients.