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The Rate of Mutation of L5178Y Asparagine-dependent Mouse Leukemia Cells to Asparagine Independence and Its Biological Consequences¹

Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510

L5178Y mouse lymphoblastic leukemia cells normally require asparagine (ASN) for growth, but phenotypically stable ASN-independent cells are detectable at low frequency by direct cloning, in ASN-free soft-agar medium, of cells from cultures that have been grown nonselectively in medium containing ASN. The rate of mutation in vitro to ASN independence was determined by the Luria-Delbrück fluctuation test to be approximately 9 x 10^-7/cell/generation. This test also demonstrated that the conversion from ASN dependence to independence has the properties of a spontaneous and random mutational event. New ASN-independent mutants isolated in this way and maintained in ASN-containing medium have significant levels of ASN synthesis but are slightly stimulated by ASN when subcultured in ASN-free medium. However, further selection by growth of these mutant cells in ASN-free medium gives cell lines that grow identically with or without ASN and produce ASN in excess of their demands for growth. A substrain of L5178Y cells resistant to arabinosylcytosine had a lower rate of mutation to ASN independence in vitro. The mutation to ASN independence also occurs in vivo in mice with L5178Y ascites tumors. The frequency of ASN independent cells observed in vivo with L5178Y cells is highly dependent on the regimen of tumor transfer and is a determinant of the effectiveness of therapy with L-asparaginase in this model tumor system.

¹ This work was supported by Grants CA10748 from the National Cancer Institute and IC-64L and PR27 (Lifetime Professorship) from the American Cancer Society.

² Present address: Radiobiology laboratories, Yale University School of Medicine, New Haven, Conn. 06510.

Received 1/10/73. Accepted 4/ 9/73.