The effect of N-phosphonacetyl-l-aspartate (PALA) pretreatment on the metabolism and cytotoxicity of 5-azacytidine (5-aza-Cyd) was studied in two murine leukemic cell lines. Exposure of P388 and L1210 cells to 3 mm PALA for 3 hr before adding 5-aza-Cyd at 75 µm was accompanied by a two-fold increment in acid-soluble and 3-fold increment in acid-insoluble incorporation of 5-aza-Cyd in both cell lines. RNA incorporation of 5-aza-Cyd increased from 97.5 ± 3.4 pmol 5-aza-Cyd per µg d-ribose in control cells to 299.2 ± 4.2 pmol 5-aza-Cyd per µg d-ribose in PALA-treated cells; a smaller increment in DNA incorporation of 5-aza-Cyd was also noted. Sequential treatment of cells with PALA and 5-aza-Cyd was associated with a 40% reduction in protein synthesis compared to only a 2 and 8% reduction, respectively, produced by the drugs given alone. Sequential administration of PALA and 5-aza-Cyd resulted in greater than additive cytotoxicity as measured by both growth inhibition and in vitro soft-agar cloning assays. Exposure of both cell lines to 3 mm PALA for 3 hr produced 50 and 65% reductions in intracellular levels of cytidine triphosphate and uridine triphosphate; intracellular accumulation of 5-azacytidine triphosphate, the lethal metabolite of 5-aza-Cyd, increased from 43.4 ± 2.1 pmol/106 cells to 92.4 ± 3.3 pmol/106 cells in PALA-treated cells. PALA was able to augment the metabolism and cytotoxicity of 5-aza-Cyd in a uridine-cytidine kinase-mutant 5-aza-Cyd-resistant L5178Y subline. This sequential drug combination has a rational biochemical basis and may offer significant advantages over either drug when administered alone, especially in cells which are resistant to 5-aza-Cyd.
Supported by Young Investigator Grant CA-24187, Grant CA-27130 from the National Cancer Institute, Grant CH-145 from the American Cancer Society, and a Swebilius Award from the Yale Comprehensive Cancer Center.