TPMT is a cytosolic enzyme that catalyzes the S-methylation of aromatic and heterocyclic sulfhydryl compounds, including medications such as mercaptopurine and thioguanine. TPMT activity exhibits autosomal codominant genetic polymorphism, and patients inheriting TPMT deficiency are at high risk of potentially fatal hematopoietic toxicity. The most prevalent mutant alleles associated with TPMT deficiency in humans have been cloned and characterized (TPMT‫2ء‬ and TPMT‫3ء‬A), but the mechanisms for loss of catalytic activity have not been elucidated. In the present study, we established that erythrocyte TPMT activity was significantly related to the amount of TPMT protein on Western blots of erythrocytes from patients with TPMT activities of 0.4-23 units͞ml pRBC (r s ‫؍‬ 0.99; P < 0.001). Similarly, heterologous expression of wild-type (TPMT‫)1ء‬ and mutant (TPMT‫2ء‬ and TPMT‫3ء‬A) human cDNAs in yeast and COS-1 cells demonstrated comparable levels of TPMT mRNA but significantly lower TPMT protein with the mutant cDNAs. Rates of protein synthesis were comparable for wild-type and mutant proteins expressed in yeast and with in vitro translation in rabbit reticulocyte lysates. In contrast, pulse-chase experiments revealed significantly shorter degradation halflives for TPMT‫2ء‬ and TPMT‫3ء‬A (ϳ0.25 hr) compared with wild-type TPMT‫1ء‬ (18 hr). The degradation of mutant proteins was impaired by ATP depletion and in yeast with mutant proteasomes (pre-1 strain) but unaffected by the lysosomal inhibitor chloroquine. These studies establish enhanced degradation of TPMT proteins encoded by TPMT‫2ء‬ and TPMT‫3ء‬A as mechanisms for lower TPMT protein and catalytic activity inherited by the predominant mutant alleles at the human TPMT locus.TPMT (S-adenosyl L-methionine:thiopurine S-methyltransferase; EC 2.1.1.67) is a cytosolic protein that catalyzes the S-methylation of mercaptopurine, mercaptopurine formed from the prodrug azathioprine, 6-thioguanine, and other aromatic and heterocyclic sulfhydryl compounds. TPMT activity exhibits codominant polymorphism in Caucasian and AfricanAmerican populations (1, 2), with about 10% having intermediate activity due to heterozygosity at the TPMT locus and approximately 1 in 300 inheriting TPMT deficiency. Decreased S-methylation in TPMT-deficient and heterozygous patients leads to more extensive accumulation of thioguanine nucleotides (3), the principal cytotoxic metabolites (4), thereby producing greater toxicity. Patients inheriting TPMT deficiency have a pronounced risk of potentially life-threatening hematopoietic toxicity when treated with conventional doses of these medications (5-8), and patients with heterozygosity at the TPMT locus have lower activity and an intermediate risk of toxicity.The inherited polymorphism of TPMT activity in humans has now been elucidated at the molecular level with the identification of inactivating mutations in the human TPMT gene (9, 10). The initial molecular defect to be discovered was a single G238 3 C transversion mutation leading to an...

Etoposide, an effective agent for acute lymphoblastic leukemia (ALL), can cause secondary acute myeloid leukemia (AML) in a subset of patients. Our objectives were to determine whether patients who develop secondary AML displayed altered etoposide pharmacokinetics or other pharmacologic characteristics compared to identically treated patients who did not develop AML. Children with newly diagnosed ALL were treated according to a protocol which included etoposide 300 mg/m 2 given three times over 8 days during remission induction and once every 2-4 weeks during 120 weeks of continuation therapy. Characteristic 11q23 rearrangements were documented in seven of the eight patients with AML. Etoposide clearance, time that etoposide concentrations exceeded 10 M, etoposide or etoposide catechol area-under-the-plasma-concentration vs time curve (AUC), serum albumin, and average methotrexate concentration did not differ significantly between the two groups; thiopurine methyltransferase (TPMT) activity tended to be lower in the eight children who did vs the 23 matched control children who did not develop AML (P = 0.16). Further regression analyses likewise indicated that lower TPMT activity tended to be associated with shorter onset of secondary AML (P = 0.11); it also tended to be lower among the eight index cases compared to the entire unmatched cohort of 105 identically treated children with ALL (P = 0.10). We observed no statistically significant differences in etoposide disposition and antimetabolite pharmacology between patients who did and did not develop secondary AML.

SUMMARYCellular accumulation of methotrexate polyglutamates (MTXPGs) is recognized as an important determinant of the cytotoxicity and selectivity of methotrexate in acute lymphoblastic leukemia (ALL). We identified a significantly lower cellular accumulation of MTXPGs in T-lineage versus B-lineage lymphoblasts in children with ALL, which is consistent with the worse prognosis of T-lineage ALL when treated with conventional antimetabolite-based therapy. Maximum MTXPG accumulation in leukemic blasts in vivo was 3-fold greater in lymphoblasts of children with B-lineage ALL (129 children) compared with those with T-lineage ALL (20 children) ( p Ͻ 0.01) and was characterized by a saturable (E max ) model in both groups. The human leukemia cell lines NALM6 (B-lineage) and CCRF/CEM (T-lineage) were used to assess potential mechanisms for these lineage differences in MTX accumulation, revealing i) greater total and long-chain MTXPG accumulation in NALM6 over a wide range of methotrexate concentrations (0.2-100 M), ii) saturation of MTXPG accumulation in both cell lines, with a higher maximum (E max ) in NALM6, iii) 3-fold higher constitutive FPGS mRNA expression and enzyme activity in NALM6 cells, iv) 2-fold lower levels of DHFR mRNA and protein in NALM6 cells, and v) 4 -6 fold lower extracellular MTX concentration and 2-fold lower intracellular MTXPG concentration to produce equivalent cytotoxicity (LC 50 ) in NALM6 versus CEM. There was a significant relationship between FPGS mRNA and enzyme activity in lymphoblasts from children with newly diagnosed ALL, and blast FPGS mRNA and activity increased after methotrexate treatment. These data indicate higher FPGS and lower DHFR levels as potential mechanisms contributing to greater MTXPG accumulation and cytotoxicity in B-lineage lymphoblasts.