6-Mercaptopurine: cytotoxicity and biochemical pharmacology in human malignant T-lymphoblasts.
The effects of prolonged exposure to 2 and 10 microM 6-mercaptopurine (6MP) in the human lymphoblastic T-cell line MOLT-4 were studied with respect to cell-kinetic parameters, phosphoribosyl pyrophosphate (PRPP) and purine ribonucleotide levels, formation of 6MP-nucleotides, especially methyl-thio-IMP (Me-tIMP), DNA and RNA synthesis ([32P] incorporation), and [8-14C]6MP incorporation into newly synthesized DNA and RNA. The results provided new insights into the complex mechanism of action of 6MP in human malignant lymphoblasts. Exposure to 2 microM 6MP resulted in a rapid inhibition of purine de novo synthesis (PDNS) by increased levels of Me-tIMP, resulting in increased PRPP levels and decreased purine ribonucleotides, affecting cell growth and clonal growth, and less cell death. DNA synthesis decreased, associated with an increasing delay of cells in S phase. Incorporation of thioguanine nucleotides into newly synthesized DNA resulted in an increasing arrest of cells in G2 + M phase. RNA synthesis, initially decreased, recovered partially, associated with a recovery of purine ribonucleotides. New formation 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 microM, indicating a high rate of incorporation and complete conversion of 6MP within this period. Incorporation of 6MP-nucleotides into DNA was 5 times as high as incorporation into RNA. Exposure to 10 microM 6MP resulted in early cytotoxicity at 24 hr, associated with a complete inhibition of PDNS by a large pool of Me-tIMP and lower levels of purine ribonucleotides as compared to 2 microM 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 further progression into G2 + M phase. Our data suggest that inhibition of PDNS due to Me-tIMP is a crucial event in the mechanism of 6MP cytotoxicity. It is responsible for decreased RNA synthesis and decreased availability of natural deoxyribonucleotides, causing a delay of DNA synthesis in S phase. This enhances incorporation of 6MP as thioguanine nucleotides into DNA in the S phase and subsequent late cytotoxicity in the 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.(ABSTRACT TRUNCATED AT 400 WORDS)
Bökkerink
J P
JP
Department of Pediatrics, St. Radboud University Hospital of Nijmegen, The Netherlands.
Stet
E H
EH
De Abreu
R A
RA
Damen
F J
FJ
Hulscher
T W
TW
Bakker
M A
MA
van Baal
J A
JA
D002250:Carbon Radioisotopes; D002453:Cell Cycle / Q000187:drug effects; D004247:DNA / Q000096:biosynthesis; D006801:Humans; D007291:Inosine Monophosphate / Q000031:analogs & derivatives / Q000032:analysis; D007945:Leukemia, Lymphoid / Q000188:drug therapy / Q000378:metabolism; D015122:Mercaptopurine / Q000494:pharmacology*; D010754:Phosphoribosyl Pyrophosphate / Q000032:analysis; D010761:Phosphorus Radioisotopes; D012313:RNA / Q000096:biosynthesis; D012265:Ribonucleotides / Q000032:analysis; D013234:Stem Cells / Q000187:drug effects; D013601:T-Lymphocytes / Q000187:drug effects* / Q000378:metabolism; D013873:Thionucleotides / Q000032:analysis
D002250:Carbon Radioisotopes; D010761:Phosphorus Radioisotopes; D012265:Ribonucleotides; D013873:Thionucleotides; D007291:Inosine Monophosphate; D012313:RNA; D010754:Phosphoribosyl Pyrophosphate; C003449:thioinosinic acid; D004247:DNA; D015122:Mercaptopurine