APOPTOSIS INDUCED IN ADVANCED CD8F1-MURINE MAMMARY-TUMORS BY THE COMBINATION OF PALA, MMPR AND 6AN PRECEDES TUMOR-REGRESSION AND IS PRECEDED BY ATP DEPLETION

The drug combination N-(phosphonacetyl)-L-aspartic acid (PALA). methyl mercaptopurine riboside (MMPR) and 6-aminonicotinamide (6AN). referred to as PMA. induces regressions of advanced CD8F1 murine mammary carci nomas in vivo. We demonstrated that CD8F1 tumor regressions were prece ded by the appearance of apoptotic bodies, as observed by microscopic examination of morphology and TUNEL end-labeling, and fragmentation of DNA into nucleosomal ''ladder'' patterns. These indications of apopto sis were present as early as 6 h after simultaneous administration of MMPR and 6AN and further increased by over fivefold during the next 3 to 6 h. then remained at 7 to 12.8% (0.6 to 2.4% in saline-treated con trols) of the cell population for at least 24 h after MMPR + 6AN admin istration. The 5'-phosphate derivative of MMRP. MMPR-5P, which inhibit s de novo purine biosynthesis. was present at a ''steady-state'' level . and significant (40%) depletion of ATP had occurred by 3 h and both of these events preceded the onset of apoptosis. In addition. MMPR-SP was retained in CD8F1 tumors at a high level over a prolonged period ( >96 h) even as rumors were undergoing regression. The prolonged presen ce of MMPR-5P was important for optimal chemotherapeutic effect. since treatment with iodotubercidin (Iodo T), an inhibitor of MMPR, adenosi ne kinase, 6 h after MMPR + 6AN administration prevented the prolonged accumulation of MMPR-5P and reversed the regression of CDSF1 tumors. In addition. compared to the PMA-treated group, there was a significan t restoration of ATP levels after treatment with Iodo T, In individual PMA-treated CD8F1 tumors the degree of ATP depletion was found to cor relate with the degree of tumor shrinkage at 24 h, after tumors had su fficient time to respond to treatment, These results define the timeco urse of drug-induced apoptosis in CD8F1 tumors, show that ATP depletio n occurs prior to apoptosis and demonstrate that prolonged retention o f MMPR-5P is associated with optimal chemotherapy. Collectively, these results suggest that the depletion of ATP by PMA treatment may be a c omponent of the biochemical apoptotic cascade in the CDSF1 tumor.