Thioguanine and mercaptopurine are prodrugs requiring conversion into thiopurine nucleotides to exert cytotoxicity. Thiopurine S-methyltransferase (TPMT), an enzyme subject to genetic polymorphism, catabolizes thiopurines into inactive methylated bases, but also produces methylthioguanine nucleotides and methylmercaptopurine nucleotides from thioguanine and mercaptopurine nucleotides, respectively. To study the effect of TPMT on activation versus inactivation of mercaptopurine and thioguanine, we used a retroviral gene transfer technique to develop human CCRF-CEM cell lines that did (TPMT+) and did not (MOCK) overexpress TPMT. After transduction, TPMT activities were 14-fold higher in the TPMT+ versus the MOCK cell lines (P < 0.001). TPMT+ cells were less sensitive to thioguanine than MOCK cells (IC50 = 1.10± 0.12 μm versus 0.55 ± 0.19 μm; P = 0.02); in contrast, TPMT+ cells were more sensitive to mercaptopurine than MOCK cells (IC50 = 0.52 ± 0.20 μm versus 1.50 ± 0.23 μm; P < 0.01). The lower sensitivity of TPMT+ versus MOCK cells to thioguanine was associated with lower thioguanine nucleotide concentrations (917 ± 282 versus 1515 ± 183 pmol/5 × 106 cells; P = 0.01), higher methylthioguanine nucleotide concentrations (252 ± 34 versus 27 ± 10 pmol/5 × 106 cells; P = 0.01), less inhibition of de novo purine synthesis (13 versus 95%; P < 0.01), and lower deoxythioguanosine incorporation into DNA (2.0 ± 0.6% versus 7.2 ± 2.0%; P < 0.001). The higher sensitivity of TPMT+ cells to mercaptopurine was associated with higher concentrations of methylmercaptopurine nucleotide (2601 ± 1055 versus 174 ± 77 pmol/5 × 106 cells; P = 0.01) and greater inhibition of de novo purine synthesis (>99% versus 74%; P < 0.01) compared with MOCK cells. We conclude that methylation of mercaptopurine contributes to the antiproliferative properties of the drug, probably through inhibition of de novo purine synthesis by methylmercaptopurine nucleotides, whereas thioguanine is inactivated primarily by TPMT.
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↵1 This work was supported by Grants CA51001, CA36401, CA21765, and CA56819 from the NIH; by a Center of Excellence grant from the State of Tennessee; by American Lebanese Syrian Associated Charities (ALSAC); and by the Aventis Oncology fellowship from the American College of Clinical Pharmacy.
↵2 To whom requests for reprints should be addressed, at Department of Pharmaceutical Sciences, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105. Phone: (901) 495-2348; Fax: (901) 525-6869; E-mail:
- Received February 23, 2001.
- Accepted May 31, 2001.
- ©2001 American Association for Cancer Research.