Abstract
The intracellular metabolism of 6-mercaptopurine (6-MP) was studied in a murine leukemia cell line, WEHI-3b. Cells were incubated 3 to 24 h with 10 nm to 50 μm 6-MP. Nucleotides were extracted with perchloric acid, and the 6-thiopurine nucleotides were isolated on mercurial cellulose. The endogenous ribonucleotides in the perchloric acid extracts as well as 6-thiopurine nucleotides were separated and quantified with anion exchange high-performance liquid chromatography. The concentration of 6-thioinosinate (6-TIMP) and 6-thioxantinate (6-TXMP) increased with an increasing 6-MP dose. The concentration of the 6-thioguanosine nucleotides (6-TGN) increased with 6-MP concentrations between 10 nm and 1 μm. However, further increase in 6-MP concentration led to a decrease in the formation of 6-TGN. At 50 μm 6-MP, the concentration of 6-thioguanosine 5′-triphosphate was one fifth of that seen at 1 μm. The incorporation of 6-[35S]mercaptopurine into DNA was also slightly higher at 1 μm compared with 50 μm. The cytocidal effect on clonogenic cells was one log greater at 1 μm 6-MP compared with 50 μm 6-MP. The decrease of 6-TGN was accompanied not only by an increased 6-TIMP concentration but also by an inhibition of the purine de novo synthesis and consequently by a decrease of the cellular ATP concentration. The ATP concentration in the cells treated with 1 μm 6-MP could be reduced to the level seen in cells treated with 50 μm 6-MP by simultaneous incubation with 0.3 μm antimycin A. This decrease of ATP concentration was accompanied by a reduction of 6-TGN and to a lesser extent of 6-TXMP. These experiments suggest that the “self-limiting” phenomenon in the metabolism of 6-MP might be caused by a depletion of ATP by inhibition of purine de novo synthesis presumably by 6-TIMP.
Supported by grants from the Swedish Cancer Society, the Cancer Society in Stockholm, the Swedish Medical Research Council (Project No. 08640), the Swedish Medical Society, and the Swedish Child Cancer Foundation.