Complete amino acid deprivation in mammalian cells causes a significant enhancement in gene expression for a number of important cellular activities; among these is asparagine synthetase (AS). The data presented demonstrate that, in both nonleukemic (rat Fao hepatoma cells) and human leukemia cells (MOLT-4, NALL-1, and BALL-1), AS mRNA levels, protein content, and enzymatic activity are induced after incubation in an otherwise complete tissue culture medium that is deficient in a single amino acid or in medium that has been depleted of the amino acid asparagine by the addition of asparaginase. Complete amino acid deprivation results in a concerted increase in AS mRNA, protein, and enzymatic activity, which, in conjunction with previously published research, suggests that the mechanism of this cellular response involves transcriptional control of the AS gene. Asparaginase treatment is a standard component of acute lymphoblastic leukemia therapy for which the effectiveness is related to the inability of these cells to upregulate AS activity to a sufficient level. With regard to the asparaginase sensitivity of the three human leukemia cell lines, there was a trend toward an inverse relation to the degree of AS expression. Selection for asparaginase-resistant MOLT-4 sublines resulted in enhanced AS mRNA and protein content regardless of whether the cells had been selected by asparaginase treatment directly or asparagine was removed from the culture medium. Collectively, the data illustrate that further advances in asparaginase therapy will require additional knowledge of amino acid-dependent regulation of AS gene expression and, conversely, that asparaginase resistance represents a model system for investigating metabolite control in a clinically relevant setting.

A full-length cDNA clone for rat asparagine synthetase (AS) was isolated from a cDNA library enriched for amino acid-regulated sequences. The AS cDNA was used to investigate the amino acid-dependent repression of AS mRNA content in rat Fao hepatoma cells. In response to complete amino acid starvation, there was an approximately 10-fold increase in the level of AS mRNA. Three species of mRNA, of approx. sizes 2.0, 2.5 and 4.0 kb, were detected and each was simultaneously regulated to the same degree. The expression of AS mRNA increased by 6 h after removal of amino acids, reached a plateau after 9 h, and was blocked by either actinomycin D or cycloheximide. Partial repression of the AS mRNA content was maintained by the presence of a single amino acid in the culture medium, but the degree of effectiveness for each one varied widely. Glutamine showed the greatest ability to repress the AS mRNA content, even at an extracellular concentration 10 times below its plasma level. Other effective repressors included the amino acids asparagine, histidine and leucine, as well as ammonia. Depletion of selected single amino acids from an otherwise complete culture medium also caused up-regulation. In particular, removal of histidine, threonine or tryptophan from the medium, or the addition of histidinol to inhibit histidinyl-tRNA synthetase, resulted in a significant increase in AS mRNA content. The data indicate that nutrient regulation of AS mRNA occurs by a general control mechanism that is responsive to a spectrum of amino acids.

Given the central role of protein synthesis in cellular function, it is likely that intricate mechanisms exist to detect and respond to amino acid deprivation. However, the current understanding of amino acid-dependent control of gene expression in mammalian cells is limited. A few examples of enzyme, transporters, and unidentified mRNA species subject to amino acid availability have been reported and some examples are summarized here. Each example chosen-asparagine synthetase, system A transport activity, and ribosomal protein L17--are associated with different aspects of amino acid metabolism, and therefore reflect the spectrum of metabolic pathways influenced by substrate control. Most of the data accumulated thus far suggest that a general control response exists such that these various activities are induced when any one of several amino acids becomes limiting. Consistent with observations in yeast, it appears that the degree of tRNA acylation and its resultant effect on protein synthesis may play an important role in initiating the starvation signal. De novo protein synthesis is required for starvation-dependent increases in several mRNA species, which suggests that the amino acid signaling pathway is composed of a series of intermediate steps before activation of specific structural genes.