Glutamate Oxidation in 6C3HED Lymphoma: Effects of L-Asparaginase on Sensitive and Resistant Lines

Abstract

L-Asparaginase (EC 3.5.1.1) inhibited respiration in sensitive, but not resistant, lines of mukine lymphoma 6C3HED. Glucose, in these tumor lines, was principally converted to lactate, and very little was oxidized in the citric acid cycle or hexose monophosphate shunt. The oncolytic effects of L-asparaginase (L-asparagine aminohydrolase; EC 3.5.1.1) have been ascribed to the deamination of asparagine, leading to depletion of intracellular asparagine in tumor cells that lack asparagine synthetase (1, 2). The resulting deficiency is ultimately seen as a general reduction of macromolecular syntheses (3-6). However, asparaginase has diverse effects that may not be completely explained on this basis: cells lacking asparagine synthetase, when cultured in asparagine-deficient tissue culture media, show a marked delay in proliferation, but do not die (7); asparaginase inhibits lectin-induced blast transformation of lymphocytes, and addition of exogenous asparagine does not prevent this effect (5, 8); asparaginase has immunosuppressive properties that are not consistently reversed by asparagine (4, 5); tumor cells in culture show marked cytotoxic changes within an hour of exposure to asparaginase (9), and it seems unlikely that deprivation of asparagine alone would have such a rapid effect on cell structure; asparaginase delays mitotic events in regenerating rat liver, a tissue in which asparagine synthetase deficiency has not been demonstrated (10).Inhibition of blast transformation by asparaginase and its immunosuppressive property suggest an effect of the enzyme on the cell surface. The production of energy may also be affected, since a number of tumors and cell lines derive much of their respiratory energy from glutamine and glutamate (11-13). Aspartate and asparagine are also linked to the citric acid cycle, and this adds another argument for complexity. Asparaginase, by depleting asparagine with a resulting increase in aspartate levels, might lead to accumulation of oxalo- Studies with Isotopes. Cell suspensions were incubated with labeled substrates (New England Nuclear Corp.) at 370 for 45 min in sealed, 25-ml Erlenmeyer flasks equipped with disposable tubes in center wells. Ten minutes before the end of incubation, 0.5 ml of hyamine hydroxide, a CO2 absorbant, was injected into the center-well tubes. After incubation, flasks were put into an ice-bath to stop metabolic activity.CO2 absorption was allowed to continue for an additional 30 min. The center-well tubes, containing hyamine, were then placed directly into liquid scintillation counting vials containing 15 ml of 0.4% 2,5-diphenyloxazole (PPO) and 0.1% 1,

Glucose‐limited chemostat culture of chinese hamster ovary cells producing recombinant human interferon‐γ

“…In wild-type CHO cells, it has been suggested that the oxidation of glutamine may provide up to 40% of the energy requirements in the presence of glucose.8 This phenomenon has also observed in a number of other cell lines. 24,35,47 The predominant mechanism for the metabolism of glutamine appears to be via phosphate dependent glutaminase, which generates glutamate and ammonia, while conversion of glutamate to a-ketoglutarate occurs mainly by transamination reactions.26 Thus, the synthesis of alanine and serine by these cells may account for a proportion of the nitrogen derived from glutamate. In the chemostat the yield of ammonia on glutamine declined with time, suggesting a more efficient nitrogen assimilation by these cells in the latter stages of the culture.…”

Effects of L‐glutamine on Tyrosinase and γ‐glutamyl Transpeptidase of B‐16 Melanoma Cells in Culture

“…Gin in tissue culture media is unique among other amino acids because it can be a major source of energy for cultured cells such as human diploid fibroblasts (9), lymphoma cells (10), and L-Mmouse cells (11). Its yield ratio (p,g cell dry weight produced per fig of amino acid or glucose utilized) is significantly lower than those of other amino acids and glucose in the human diploid cell, WI-38 (12), and it is necessary for the survival of cultured 6C3HED lymphoma cells and human leukemic cells (10,13). Surprisingly, melanoma cells can be grown in medium free of GIn; depletion of this amino acid seems to be a favorable factor in initiation of human malignant melanoma cell lines and perhaps is related to increased cloning efficiency (8).…”

Isolation of tumor-secreted products from human carcinoma cells maintained in a defined protein-free medium.

“…Other studies provide evidence that glutamine serves as a major energy source not only for normal cells in vivo but also for tumor cells cultivated in vitro. Energy provision through glutamine utilization has been described for several tumor types which include HeLa, lymphoma, and myeloma cells (27)(28)(29). Moreover, increased glutamine utilization has been shown to correlate with malignant growth in vivo (30,31).…”