Asparagine incorporation into the DNA of hepatic and 6C3HED murine lymphosarcoma cells.

It was first demonstrated by Kidd (4) in 1953 that the growth of certain lymphosarcomas of rats and mice could be suppressed by treatment with guinea pig serum. In 1961, Broome (1) identified the lymphosarcoma-inhibitory factor as the enzyme L-asparagine aminohydrolase, EC 3.5.1.1 (L-asparaginase). Recently, Kidd and Sobin (5) showed that lymphosarcoma 6C3HED cells growing intraperitoneally in susceptible C3H mice take up L-asparagine readily from cutaneous depots. Such incorporation is almost completely prevented by treatment with guinea pig serum containing L-asparaginase. L-Aspartic acid, on the other hand, a require ment of normal cells, is incorporated only sparingly, if at all, by the sensitive tumor cells. While some fraction of the L-asparagine is used by the cells to synthesize protein, another route of incorporation was suggested by a recent report by Handschumacher et aL (2). He reported that, when L-asparagine-requiring cells were incubated in the presence of L-asparagine and its antimetabo lite 5-diazo-4-oxo-L-norvaline, the uptake of the amino acid into cellular proteins was not affected, but the growth of cells in tissue culture was inhibited 87%. Such activity might be explained in terms of inhibition of the incorporation of L-asparagine by the tumor cells in the biosynthesis of pyrimidines via a modification of the pathway taken by L-aspartic acid in normal tissue. This route, the so-called orotic acid pathway, is a lengthy and circuitous cellular process, which could be considerably shortened if L-aspara gine, instead of L-aspartate, were to condense with carbamyl phosphate in the initial steps of the process. CMP would then be produced in approximately one-half the steps required by the aspartate-utiizing pathway in normal tissues, because the terminal amide group of L-asparagine would permit by passing the steps required for producing the uridine deriva tives prior to CMP production. For a test of this hypothesis, 40 male C3H mice, each weighing 20 to 25 g, were inoculated with one million 6C3HED L-asparaginase-sensitive lymphosarcoma cells. After ascites tumors had developed, the mice were divided into 2 groups of 20 each and were treated individually with 2-MCi doses of either L-aspartic acid or L-asparagine, both uniform ly labeled with †@̃C(Nuclear-Chicago Corp., Des Plaines, Ill.). The purity of the amino acids was confirmed by paper