6-mercaptopurine(6-MP), the hypoxanthine analog, has been found to be a potent inhibitor of human leukemia cells. For this reason, the biochemical mechanism of action of this compound has been the subject of many investigations. It became apparent from early studies that 6-MP was catalyzed by hypoxanthine guanine phosphoribosyltransferase (HGPRTase) to 6-thioinosine 5'-phosphate(6-TIMP) which inhibited the enzymes, PRPP amidotransferase, IMP dehydrogenase, adenylosuccinate synthetase and adenylosuccinate lyase. Such metabolic blockade would be expected to decrease the concentration of purine nucleotides in target cells, thereby resulting in inhibition of cell-growth. An increasing production of 6-TIMP in leukemia cells leads to enhance the inhibitory effect of 6-MP.
HGPRTase activity and PRPP content in leukemia cells were already reported, but the mode of transport of this compound to cell-membrane was not reported because of difficulty to investigate on account of the possibility of efflux of metabolites during wash-out of the incubation medium. We made the quantitative investigation of incorporation of this compound possible by use of new method of Wohlhueter et al using silicone oil. In this method, cell component could be separated from the incubation medium without washing.
The result showed that the radioactivity of incorporated 6-MP reached at its maximum approximately at 10 min. of incubation, thereafter reduced rapidly. This result suggests the possibility of efflux of 6-MP or metabolites of 6-MP such as 6-TX and 6-TUA. Especially, efflux of 6-TUA may affect remarkably the concentration of 6-MP in the cells. The kinetic examination of intracellular metabolites of 6-MP supported this efflux.
After this rapid increasing and decreasing phase, the radioactivity in the cells showed gradual increment. This increment is supposed to reflect the phosphorylation of incorporated compound. The mode of transport of Ara-C to cell-membrane was also investigated, Under the condition that most incorporated Ara-C was converted to Ara-C nucleotide, the radioactivity in the cells showed linear increment until 30 min. of incubation. It is apparent, as compared 6-MP with Ara-C, that the mode of transport to cell-membrane is affected markedly by intracellular phosphorylation of these compounds.
The present investigation suggests that 6-MP should be contacted to cells for a longer period for its effective administration even if a low dose.
