6-MERCAPTOPURINE - CYTOTOXICITY AND BIOCHEMICAL PHARMACOLOGY IN HUMAN-MALIGNANT T-LYMPHOBLASTS

The effects of prolonged exposure to 2 and 10 muM 6-mercaptopurine (6M P) in the human lymphoblastic T-cell line MOLT-4 were studied with res pect to cell-kinetic parameters, phosphoribosyl pyrophosphate (PRPP) a nd purine ribonucleotide levels, formation of 6MP-nucleotides, especia lly methyl-thio-IMP (Me-tIMP), DNA and RNA synthesis ([P-32] incorpora tion), and [8-C-14]6MP incorporation into newly synthesized DNA and RN A. The results provided new insights into the complex mechanism of act ion of 6MP in human malignant lymphoblasts. Exposure to 2 muM 6MP resu lted in a rapid inhibition of purine de novo synthesis (PDNS) by incre ased levels of Me-tIMP, resulting in increased PRPP levels and decreas ed purine ribonucleotides, affecting cell growth and clonal growth, an d less cell death. DNA synthesis decreased, associated with an increas ing delay of cells in S phase. Incorporation of thioguanine nucleotide s into newly synthesized DNA resulted in an increasing arrest of cells in G2 + M phase. RNA synthesis, initially decreased, recovered partia lly, associated with a recovery of purine ribonucleotides. New formati on of 6MP-nucleotides (tIMP) was only detected within the first 24 hr, and 6MP levels in the culture medium were already undetectable after 6 hr of exposure to 2 muM, indicating a high rate of incorporation and complete conversion of 6MP within this period. Incorporation of 6MP-n ucleotides into DNA was 5 times as high as incorporation into RNA. Exp osure to 10 muM 6MP resulted in early cytotoxicity at 24 hr, associate d with a complete inhibition of PDNS by a large pool of Me-tIMP and lo wer levels of purine ribonucleotides as compared to 2 muM 6MP. A more severe delay of cells in S phase was associated with an inhibition of DNA synthesis to 14% of control within the first 24 hr, and an arrest in G2 + M phase. Further increasing levels of Me-tIMP caused an arrest of cells and late cytotoxicity in S phase at 48 hr, preventing furthe r progression into G2 + M Phase. Our data suggest that inhibition of P DNS due to Me-tIMP is a crucial event in the mechanism of 6MP cytotoxi city. It is responsible for decreased RNA synthesis and decreased avai lability of natural deoxyribonucleotides, causing a delay of DNA synth esis in S phase. This enhances incorporation of 6MP as thioguanine nuc leotides into DNA in the S phase and subsequent late cytotoxicity in t he G2 phase. However, with high concentrations of 6MP, the large pool of Me-tIMP causes severe reduction of natural deoxyribonucleotides in lymphoblasts with an active PDNS. This is responsible for pronounced i nhibition of DNA synthesis and early cytotoxicity in the S phase.