Asparagine depletion in the serum during L-asparaginase treatment causes the the death of lymphoblasts that lack ability to synthesize asparagine. Cellular levels of asparagine synthetase (AS) inversely correlate with cellular sensitivity to L-asparaginase. Human leukemia cells that do not express AS in detectable quantities are hypersensitive to the effects of L-asparaginase. In the present studies, we have established a flow cytometry for AS protein detection assay with the monoclonal anti-AS antibody, 3G6. We compared AS expression in single-cell suspensions with standard biochemical and Western blot assays to validate the fluorescence-activated cell sorting (FACS) method. FACS yielded a linear relationship between the mean fluorescence intensity and AS activity. Using this standard curve, FACS-analyzed AS activity in leukemia cells ranged from 25.8 to 436 pmol asparagine formed/mg protein /min, similar to those obtained by Western blot analyse and even to AS mRNA levels by RT-PCR. Thus, AS-FACS can rapidly assess the heterogeneity of steady-state AS in single-cell suspensions and may be useful for assay in peripheral blood cells, bone marrow cells and leukemic cells. This quantitative assay system should be developed as a potential application of AS estimation for fresh leukemia cells.
