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1.
FASEB J ; 15(11): 1902-8, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11532970

ABSTRACT

Inhibition of HIV-1 reverse transcriptase (RT) and HIV protease are effective mechanisms for anti-retroviral agents, and the combined use of mechanistically different medications has markedly improved the treatment of HIV infected patients. The active metabolite of mercaptopurine and thioguanine (TG), deoxythioguanosine triphosphate, was shown to be incorporated into DNA by the polymerase function of HIV-1 RT and then to abrogate RNA cleavage by HIV-1 RNaseH. Treatment of human lymphocyte cultures with thioguanine produced substantial inhibition of HIV replication (IC(50)=0.035 microM, IC(95)=15.4 microM), with minimal toxicity to host lymphocytes (<10% at 15.4 microM TG, P<0.000005). Furthermore, low concentrations of TG and zidovudine were synergistic in inhibiting HIV replication in human lymphocytes (synergy volume=19 microM(2)%), without additive cytotoxicity to host lymphocytes. Thus, thiopurines are novel anti-retroviral agents that alter the DNA-RNA substrates for HIV RNaseH, thereby abrogating early stages of HIV replication.


Subject(s)
Anti-HIV Agents/pharmacology , Deoxyguanine Nucleotides/metabolism , HIV Reverse Transcriptase/metabolism , HIV-1/drug effects , Mercaptopurine/pharmacology , Nucleic Acid Synthesis Inhibitors/pharmacology , Thioguanine/pharmacology , Thionucleotides/metabolism , Virus Replication/drug effects , Base Sequence , Cells, Cultured , DNA/metabolism , HIV-1/metabolism , HIV-1/physiology , HeLa Cells , Humans , Molecular Sequence Data , Nucleic Acid Heteroduplexes , Polymers , RNA/metabolism , Ribonuclease H/metabolism
2.
Cancer Res ; 61(15): 5810-6, 2001 Aug 01.
Article in English | MEDLINE | ID: mdl-11479220

ABSTRACT

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 (IC(50) = 1.10+/- 0.12 microM versus 0.55 +/- 0.19 microM; P = 0.02); in contrast, TPMT+ cells were more sensitive to mercaptopurine than MOCK cells (IC(50) = 0.52 +/- 0.20 microM versus 1.50 +/- 0.23 microM; 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 x 10(6) cells; P = 0.01), higher methylthioguanine nucleotide concentrations (252 +/- 34 versus 27 +/- 10 pmol/5 x 10(6) 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 x 10(6) 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.


Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Leukemia-Lymphoma, Adult T-Cell/drug therapy , Leukemia-Lymphoma, Adult T-Cell/enzymology , Mercaptopurine/analogs & derivatives , Mercaptopurine/pharmacology , Methyltransferases/metabolism , Thioguanine/pharmacology , 3T3 Cells , Animals , Antimetabolites, Antineoplastic/pharmacokinetics , Biotransformation , Cytosol/metabolism , DNA, Neoplasm/metabolism , Deoxyguanosine/metabolism , Gene Transfer Techniques , HeLa Cells , Humans , Leukemia-Lymphoma, Adult T-Cell/genetics , Mercaptopurine/pharmacokinetics , Methyltransferases/biosynthesis , Methyltransferases/genetics , Mice , Purine Nucleotides/metabolism , Purines/biosynthesis , Retroviridae/genetics , Thioguanine/pharmacokinetics , Thionucleosides/metabolism , Thionucleotides/metabolism , Tumor Cells, Cultured
3.
J Clin Oncol ; 19(8): 2293-301, 2001 Apr 15.
Article in English | MEDLINE | ID: mdl-11304783

ABSTRACT

PURPOSE: To assess thiopurine S-methyltransferase (TPMT) phenotype and genotype in patients who were intolerant to treatment with mercaptopurine (MP) or azathioprine (AZA), and to evaluate their clinical management. PATIENTS AND METHODS: TPMT phenotype and thiopurine metabolism were assessed in all patients referred between 1994 and 1999 for evaluation of excessive toxicity while receiving MP or AZA. TPMT activity was measured by radiochemical analysis, TPMT genotype was determined by mutation-specific polymerase chain reaction restriction fragment length polymorphism analyses for the TPMT*2, *3A, *3B, and *3C alleles, and thiopurine metabolites were measured by high-performance liquid chromatography. RESULTS: Of 23 patients evaluated, six had TPMT deficiency (activity < 5 U/mL of packed RBCs [pRBCs]; homozygous mutant), nine had intermediate TPMT activity (5 to 13 U/mL of pRBCs; heterozygotes), and eight had high TPMT activity (> 13.5 U/mL of pRBCs; homozygous wildtype). The 65.2% frequency of TPMT-deficient and heterozygous individuals among these toxic patients is significantly greater than the expected 10% frequency in the general population (P <.001, chi(2)). TPMT phenotype and genotype were concordant in all TPMT-deficient and all homozygous-wildtype patients, whereas five patients with heterozygous phenotypes did not have a TPMT mutation detected. Before thiopurine dosage adjustments, TPMT-deficient patients experienced more frequent hospitalization, more platelet transfusions, and more missed doses of chemotherapy. Hematologic toxicity occurred in more than 90% of patients, whereas hepatotoxicity occurred in six patients (26%). Both patients who presented with only hepatic toxicity had a homozygous-wildtype TPMT phenotype. After adjustment of thiopurine dosages, the TPMT-deficient and heterozygous patients tolerated therapy without acute toxicity. CONCLUSION: There is a significant (> six-fold) overrepresentation of TPMT deficiency or heterozygosity among patients developing dose-limiting hematopoietic toxicity from therapy containing thiopurines. However, with appropriate dosage adjustments, TPMT-deficient and heterozygous patients can be treated with thiopurines, without acute dose-limiting toxicity.


Subject(s)
Antimetabolites, Antineoplastic/adverse effects , Azathioprine/adverse effects , Mercaptopurine/adverse effects , Methyltransferases/deficiency , Methyltransferases/genetics , Polymorphism, Restriction Fragment Length , Thrombocytopenia/chemically induced , Adolescent , Adult , Child , Child, Preschool , Female , Genotype , Hospitalization , Humans , Infant , Male , Methyltransferases/metabolism , Neoplasms/drug therapy , Phenotype , Platelet Transfusion , Risk Factors , Thrombocytopenia/genetics
4.
Mol Pharmacol ; 59(2): 367-74, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11160874

ABSTRACT

To elucidate molecular mechanism(s) of cellular response to mercaptopurine, a widely used antileukemic agent, we assessed mercaptopurine (MP) sensitivity in mismatch repair (MMR) proficient and MMR deficient human acute lymphoblastic leukemia (ALL) cells. Sensitivity to thiopurine cytotoxicity was not dependent on MMR (i.e., MutSalpha) competence among six cell lines tested. Using electrophoretic mobility shift assay analysis, we found that the incubation of nuclear extracts from ALL cells with synthetic 34-mer DNA duplexes containing deoxythioguanosine (G(S)) within either G(S).T or G(S).C pairs, resulted in formation of a DNA-protein complex distinct from the DNA-MutSalpha complex and unaffected by ATP. Isolation and sequence analysis of proteins involved in this DNA-protein complex identified glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a component. Western blot analysis of nuclear extracts from a panel of human lymphoblastic leukemia cell lines revealed markedly different basal levels of GAPDH in nuclei, which was significantly related to thiopurine sensitivity (p = 0.001). Confocal analysis revealed markedly different intracellular distribution of GAPDH between nucleus and cytosol in six human ALL cell lines. Redistribution of GAPDH from cytosol to nucleus was evident after MP treatment. These findings indicate that a new DNA-protein complex containing GAPDH and distinct from known MMR protein-DNA complexes binds directly to thioguanylated DNA, suggesting that this may act as a sensor of structural alterations in DNA and serve as an interface between these DNA modifications and apoptosis.


Subject(s)
Base Pair Mismatch , DNA Repair/physiology , DNA-Binding Proteins/isolation & purification , Thionucleosides/metabolism , Antimetabolites, Antineoplastic/pharmacology , Cell Nucleus/metabolism , DNA Primers/chemistry , DNA-Binding Proteins/metabolism , Drug Screening Assays, Antitumor , Glyceraldehyde-3-Phosphate Dehydrogenases/isolation & purification , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Humans , Mercaptopurine/pharmacology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/enzymology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Thioguanine/chemistry , Tumor Cells, Cultured
5.
FASEB J ; 14(14): 2339-44, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11053256

ABSTRACT

Thiopurines and topoisomerase II-targeted drugs (e.g., etoposide) are widely used anticancer drugs. However, topoisomerase II-targeted drugs can cause acute myeloid leukemia, with the risk of this secondary leukemia linked to a genetic defect in thiopurine catabolism. Chronic thiopurines result in thioguanine substitution in DNA. The effect of these substitutions on DNA topoisomerase II activity is not known. Our goal was to determine whether deoxythioguanosine substitution alters DNA cleavage stabilized by human topoisomerase II. We studied four variations of a 40 mer oligonucleotide with a topoisomerase II cleavage site, each with a single deoxythioguanosine in a different position relative to the cleavage site (-1 or +2 in the top and +2 or +4 in the bottom strand). Deoxythioguanosine substitution caused position-dependent quantitative effects on cleavage. With the -1 or +2 top and +2 or +4 bottom substitutions, mean topoisomerase II-induced cleavage was 0.6-, 2.0-, 1.1-, and 3.3-fold that with the wild-type substrate (P=0. 011, < 0.008, 0.51, and < 0.001, respectively). In the presence of 100 microM etoposide, cleavage was enhanced for wild-type and all thioguanosine-modified substrates relative to no etoposide, with the +4 bottom substitution showing greater etoposide-induced cleavage than the wild-type substrate (P=0.015). We conclude that thioguanine incorporation alters the DNA cleavage induced by topoisomerase II in the presence and absence of etoposide, providing new insights to the mechanism of thiopurine effect and on the leukemogenesis of thiopurines, with or without topoisomerase inhibitors.


Subject(s)
DNA Topoisomerases, Type II/metabolism , DNA/metabolism , Thioguanine/metabolism , Base Sequence , DNA/chemistry , DNA/genetics , Electrophoresis, Polyacrylamide Gel , Models, Molecular , Oligonucleotides/genetics , Oligonucleotides/metabolism , Substrate Specificity , Thioguanine/chemistry
6.
Pharmacology ; 61(3): 136-46, 2000 Sep.
Article in English | MEDLINE | ID: mdl-10971199

ABSTRACT

The activity of thiopurine S-methyltransferase (TPMT) is inherited as an autosomal co-dominant trait. In most large world populations studied to date, approximately 10% of the population have intermediate activity due to heterozygosity at the TPMT locus, and about 0.33% is TPMT deficient. TPMT is now one of the most well characterized genetic polymorphisms of drug metabolism, with the genetic basis having been well defined in most populations, providing molecular strategies for studying this genetic polymorphism in human and experimental models. Three mutant alleles, TPMT(*)2, TPMT(*)3A and TPMT(*)3C, account for the great majority of mutant alleles in all human populations studied to date. Significant ethnic differences occur in the frequencies of these mutant alleles. Progress in DNA analysis has made it practical to use genotyping techniques for the molecular diagnosis of TPMT deficiency and heterozygosity, thereby avoiding adverse effects that are more prevalent in TPMT-deficient and heterozygous patients prescribed thiopurine medications.


Subject(s)
Methyltransferases/genetics , Polymorphism, Genetic/genetics , Animals , Biological Evolution , Humans , Methyltransferases/metabolism , Species Specificity
7.
Leukemia ; 14(4): 567-72, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10764140

ABSTRACT

Thiopurine methyltransferase (TPMT) catalyses the S-methylation of thiopurines, including 6-mercaptopurine and 6-thioguanine. TPMT activity exhibits genetic polymorphism, with about 1/300 inheriting TPMT deficiency as an autosomal recessive trait. If treated with standard doses of thiopurines, TPMTdeficient patients accumulate excessive thioguanine nucleotides in hematopoietic tissues, leading to severe hematological toxicity that can be fatal. However, TPMT-deficient patients can be successfully treated with a 10- to 15-fold lower dosage of these medications. The molecular basis for altered TPMT activity has been defined, with rapid and inexpensive assays available for the three signature mutations which account for the majority of mutant alleles. TPMT genotype correlates well with in vivo enzyme activity within erythrocytes and leukemic blast cells and is clearly associated with risk of toxicity. The impact of 6-mercaptopurine dose intensity is also being clarified as an important determinate of event-free survival in childhood leukemia. In addition, there are emerging data that TPMT genotype may influence the risk of secondary malignancies, including brain tumors and acute myelogenous leukemia. Ongoing studies aim to clarify the influence of TPMT on thiopurine efficacy, acute toxicity, and risk for delayed toxicity. Together, these advances hold the promise of improving the safety and efficacy of thiopurine therapy.


Subject(s)
Antimetabolites, Antineoplastic/pharmacokinetics , Inactivation, Metabolic/genetics , Mercaptopurine/pharmacokinetics , Methyltransferases/genetics , Neoplasm Proteins/genetics , Polymorphism, Genetic , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Antimetabolites, Antineoplastic/adverse effects , Asia , Black People/genetics , Child , Child, Preschool , Codon/genetics , Disease-Free Survival , Drug Resistance, Neoplasm/genetics , England , Ethnicity/genetics , France , Gene Frequency , Genotype , Humans , India , Infant , Mercaptopurine/adverse effects , Neoplasms, Second Primary/chemically induced , Point Mutation , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/enzymology , Recombinant Fusion Proteins/genetics , Risk , Safety , Treatment Outcome , White People/genetics
8.
J Natl Cancer Inst ; 91(23): 2001-8, 1999 Dec 01.
Article in English | MEDLINE | ID: mdl-10580024

ABSTRACT

BACKGROUND: Patients with acute lymphoblastic leukemia are often treated with 6-mercaptopurine, and those with homozygous deficiency in thiopurine S-methyltransferase (TPMT) enzyme activity have an extreme sensitivity to this drug as a result of the accumulation of higher cellular concentrations of thioguanine nucleotides. We studied the metabolism, dose requirements, and tolerance of 6-mercaptopurine among patients with different TPMT phenotypes. METHODS: We compared, by use of statistical modeling, 6-mercaptopurine pharmacology and tolerance in 180 patients who achieved remission on St. Jude Children's Research Hospital Protocol Total XII composed of weekly methotrexate (40 mg/m(2)) and daily oral 6-mercaptopurine (75 mg/m(2)) given for 2.5 years, interrupted every 6 weeks during the first year for treatment with either high-dose methotrexate or teniposide plus cytarabine. Statistical tests were two-sided. RESULTS: Erythrocyte concentrations of thioguanine nucleotides (pmol/8 x 10(8) erythrocytes) were inversely related to TPMT enzyme activity (P<.01), with averages (+/- standard deviations) of 417 (+/-179), 963 (+/-752), and 3565 (+/-1282) in TPMT homozygous wild-type (n = 161), heterozygous (n = 17), and homozygous-deficient (n = 2) patients, respectively. There was complete concordance between TPMT genotype and phenotype in a subset of 28 patients for whom TPMT genotype was determined. There were no sex differences in thioguanine nucleotide concentrations (P =.24), TPMT enzyme activity (P =.22), or average weekly prescribed dose of 6-mercaptopurine (P=.49). The cumulative incidence of 6-mercaptopurine dose reductions due to toxicity was highest among patients homozygous for mutant TPMT (100%), intermediate among heterozygous patients (35%), and lowest among wild-type patients (7%) (P<.001), with average (+/- standard deviation) final weekly 6-mercaptopurine doses of 72 (+/-60), 449 (+/-160), and 528 (+/-90) mg/m(2), respectively. Lowering doses of 6-mercaptopurine in TPMT heterozygotes and in deficient patients allowed administration of full protocol doses of other chemotherapy while maintaining high thioguanine nucleotide concentrations. CONCLUSION: We conclude that genetic polymorphism in TPMT is an important determinant of mercaptopurine toxicity, even among patients who are heterozygous for this trait.


Subject(s)
Antimetabolites, Antineoplastic/administration & dosage , Antimetabolites, Antineoplastic/pharmacokinetics , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Mercaptopurine/administration & dosage , Mercaptopurine/pharmacokinetics , Methyltransferases/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Prodrugs/administration & dosage , Prodrugs/pharmacokinetics , Antimetabolites, Antineoplastic/adverse effects , Area Under Curve , Child , Erythrocytes/metabolism , Female , Guanine Nucleotides/metabolism , Heterozygote , Humans , Male , Mercaptopurine/adverse effects , Methylation , Methyltransferases/metabolism , Phenotype , Polymorphism, Genetic , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Prodrugs/adverse effects , Thionucleotides/metabolism
9.
Pharmacogenetics ; 9(5): 641-50, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10591545

ABSTRACT

Inheritance of the TPMT*2, TPMT*3A and TPMT*3C mutant alleles is associated with deficiency of thiopurine S-methyltransferase (TPMT) activity in humans. However, unlike TPMT*2 and TPMT*3A, the catalytically active protein coded by TPMT*3C does not undergo enhanced proteolysis when heterologously expressed in yeast, making it unclear why this common mutant allele should be associated with inheritance of TPMT-deficiency. To further elucidate the mechanism for TPMT deficiency associated with these alleles, we characterized TPMT proteolysis following heterologous expression of wild-type and mutant proteins in mammalian cells. When expressed in COS-1 cells, proteins encoded by TPMT*2, TPMT*3A, and TPMT*3C cDNAs had significantly reduced steady-state levels and shorter degradation half-lives compared with the wild-type protein. Similarly, in rabbit reticulocyte lysate (RRL), these mutant TPMT proteins were degraded significantly faster than the wild-type protein. Thus, enhanced proteolysis of TPMT*3C protein in mammalian cells is in contrast to its stability in yeast, but consistent with TPMT-deficiency in humans. Proteolysis was ATP-dependent and sensitive to proteasomal inhibitors MG115, MG132 and lactacystin, but not to calpain inhibitor II. We conclude that all of these mutant TPMT proteins undergo enhanced proteolysis in mammalian cells, through an ATP-dependent proteasomal pathway, leading to low or undetectable levels of TPMT protein in humans who inherit these mutant alleles.


Subject(s)
Cysteine Endopeptidases/metabolism , Methyltransferases/genetics , Methyltransferases/metabolism , Multienzyme Complexes/metabolism , Mutation , Adenosine Triphosphate/metabolism , Alleles , Animals , COS Cells , DNA, Complementary/genetics , Humans , In Vitro Techniques , Kinetics , Methyltransferases/deficiency , Proteasome Endopeptidase Complex , Rabbits , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Reticulocytes/metabolism , S-Adenosylmethionine/pharmacology , Transfection
10.
Mol Pharmacol ; 56(4): 841-8, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10496969

ABSTRACT

Mercaptopurine and thioguanine are anticancer and immunosuppressive agents that exert their primary cytotoxic effects via incorporation of deoxythioguanosine (dG(s)) into DNA, but the precise mechanism(s) by which this causes cytotoxicity remains unknown. We initially determined that the level of dG(s) incorporation into DNA of human T- and B-lineage leukemia cell lines did not correlate significantly with the extent of cytotoxicity (IC(50)), except that there was no cytotoxicity in the absence of dG(s) incorporation. To elucidate biological processes perturbed by dG(s) incorporation into DNA, we chemically synthesized oligodeoxyribonucleotides containing a single dG(s) (11 mer and 19 mer), which decreased the melting temperature (T(m)) of DNA-DNA duplexes without major structural changes, as evidenced by circular dichroism spectra. Using nuclear extracts from human lymphoblastic leukemia cells (CCRF-CEM, NALM6, and Molt4), we documented that dG(s) incorporation into the DNA strand of DNA-RNA heteroduplexes significantly inhibited human RNase H-catalyzed RNA cleavage (80-90% inhibition) and that a similar inhibition was evident with bacterial RNase H. These data provide the first evidence that thiopurines inhibit the function of RNase H, indicating that their mechanism of cytotoxicity may involve interference with this component of the replication machinery.


Subject(s)
DNA, Neoplasm/metabolism , Deoxyguanosine/analogs & derivatives , Nucleic Acid Heteroduplexes/metabolism , RNA, Neoplasm/metabolism , Ribonuclease H/metabolism , Thionucleosides/metabolism , Cell Survival/drug effects , DNA Replication , DNA, Neoplasm/biosynthesis , DNA, Neoplasm/drug effects , Deoxyguanosine/metabolism , Deoxyguanosine/pharmacology , Humans , Hydrolysis/drug effects , RNA, Neoplasm/drug effects , Ribonuclease H/antagonists & inhibitors , Substrate Specificity , Thionucleosides/pharmacology , Tumor Cells, Cultured
11.
Nucleosides Nucleotides ; 18(6-7): 1747-8, 1999.
Article in English | MEDLINE | ID: mdl-10474258

ABSTRACT

Synthesis of a number of photoactive thiopurine-containing nucleosides was described. S-methylation of the synthesized compounds in the course of the reaction catalyzed by recombinant human thiopurine S-methyltransferase was studied by UV-spectroscopy.


Subject(s)
Methyltransferases/chemistry , Purine Nucleosides/chemical synthesis , Humans , Protein Conformation , Recombinant Proteins/chemistry
12.
Pharm Res ; 16(3): 342-9, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10213363

ABSTRACT

Genetic polymorphism of drug metabolizing enzymes can be the major determinant of inter-individual differences in drug disposition and effects. In this mini-review, the evolution of pharmacogenetic studies, from the recognition of phenotypic polymorphisms to the discovery of genetic mutations responsible for these inherited traits, is illustrated by the genetic polymorphism of thiopurine S-methyltransferase (TPMT). TPMT, which exhibits autosomal co-dominant polymorphism, plays an important role in metabolism of the antileukemic and immunosuppressive medications, mercaptopurine, thioguanine, and azathioprine. The genetic polymorphism of TPMT activity in humans was first reported in 1980, and in the last five years the genetic basis for this polymorphism has been elucidated. Isolation and cloning of mutant alleles from humans with TPMT deficiency has identified the major mutant alleles, established the basis for loss of TPMT activity and permitted development of PCR-based genotyping assays to make a molecular diagnosis of TPMT-deficiency and heterozygosity. These studies illustrate the potential clinical benefits of elucidating the molecular basis of inherited differences in drug metabolism and disposition, and future automation of molecular diagnostics will make it feasible to more precisely select the optimal drug and dosage for individual patients.


Subject(s)
Mercaptopurine/metabolism , Methyltransferases/genetics , Pharmacogenetics , Amino Acid Sequence , Antimetabolites, Antineoplastic/metabolism , Antimetabolites, Antineoplastic/toxicity , Humans , Mercaptopurine/toxicity , Methyltransferases/deficiency , Methyltransferases/metabolism , Molecular Sequence Data , Mutation , Polymorphism, Genetic , Sequence Homology, Amino Acid
13.
Hum Mol Genet ; 8(2): 371-6, 1999 Feb.
Article in English | MEDLINE | ID: mdl-9931346

ABSTRACT

The molecular basis for the genetic polymorphism of thiopurine S -methyltransferase (TPMT) has been estab-lished for Caucasians, but it remains to be elucidated in African populations. In the current study, we determined TPMT genotypes in a population of 248 African-Americans and compared it with allele frequencies in 282 Caucasian Americans. TPMT genotype was determined in all individuals with TPMT activity indicative of a heterozygous genotype (10.2 U/ml pRBC, n = 23 African-Americans, n = 21 Caucasians). No mutant alleles were found in the high activity control groups. The overall mutant allele frequencies were similar in African-Americans and Caucasians (4.6 and 3.7% of alleles, respectively). However, while TPMT*3C was the most prevalent mutant allele in African-Americans (52.2% of mutant alleles), it represented only 4.8% of mutant alleles in Caucasians ( P < 0.001). In contrast, TPMT*3A and TPMT*2 were less common in African-Americans (17.4 and 8.7% of mutant alleles), whereas TPMT*3A was the most prevalent mutant allele in Caucasians (85.7% of mutant alleles). A novel allele ( TPMT*8 ), containing a single nucleotide transition (G644A), leading to an amino acid change at codon 215 (Arg-->His), was found in one African-American with intermediate activity. These data indicate that the same TPMT mutant alleles are found in American black and white populations, but that the predominant mutant alleles differ in these two ethnic groups.


Subject(s)
Black People/genetics , Genes/genetics , Methyltransferases/genetics , Alleles , DNA/analysis , DNA/genetics , Gene Frequency , Genotype , Heterozygote , Homozygote , Humans , Mutation , Phenotype , Polymorphism, Genetic , Polymorphism, Restriction Fragment Length , White People/genetics
14.
Blood ; 93(5): 1643-50, 1999 Mar 01.
Article in English | MEDLINE | ID: mdl-10029593

ABSTRACT

Methotrexate (MTX) is one of the most active and widely used agents for the treatment of acute lymphoblastic leukemia (ALL). To elucidate the mechanism for higher accumulation of MTX polyglutamates (MTX-PG) in hyperdiploid ALL and lower accumulation in T-lineage ALL, expression of the reduced folate carrier (RFC) was assessed by reverse transcription-polymerase chain reaction in ALL blasts isolated from newly diagnosed patients. RFC expression exhibited a 60-fold range among 29 children, with significantly higher expression in hyperdiploid B-lineage ALL (median, 11.3) compared with nonhyperdiploid ALL (median, 2.1; P <.0006), but no significant difference between nonhyperdiploid B-lineage and T-lineage ALL. Furthermore, mRNA levels of RFC (mapped by FISH to chromosome 21) were significantly related to chromosome 21 copy number (P =.0013), with the highest expression in hyperdiploid ALL blasts with 4 copies of chromosome 21. To assess the functional significance of gene copy number, MTX-PG accumulation was compared in ALL blasts isolated from 121 patients treated with either low-dose MTX (LDMTX; n = 60) or high-dose MTX (HDMTX; n = 61). After LDMTX, MTX-PG accumulation was highest in hyperdiploid B-lineage ALL with 4 copies of chromosome 21 (P =.011), but MTX-PG accumulation was not significantly related to chromosome 21 copy number after HDMTX (P =.24). These data show higher RFC expression as a mechanism for greater MTX accumulation in hyperdiploid B-lineage ALL and indicate that lineage differences in MTX-PG accumulation are not due to lower RFC expression in T-lineage ALL.


Subject(s)
Carrier Proteins/biosynthesis , Membrane Proteins , Membrane Transport Proteins , Methotrexate/analogs & derivatives , Methotrexate/administration & dosage , Polyglutamic Acid/analogs & derivatives , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Cell Lineage , Child , Child, Preschool , Chromosomes, Human, Pair 21 , Humans , Methotrexate/analysis , Methotrexate/metabolism , Methotrexate/pharmacokinetics , Ploidies , Polyglutamic Acid/analysis , Polyglutamic Acid/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Reduced Folate Carrier Protein
15.
Eur J Biochem ; 256(3): 510-7, 1998 Sep 15.
Article in English | MEDLINE | ID: mdl-9780226

ABSTRACT

Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme that catalyzes S-methylation of aromatic and heterocyclic sulfhydryl compounds, including anticancer and immunosuppressive thiopurines. We recently isolated the human TPMT promoter, which does not contain TATA box or CCAAT element consensus sequences, but is GC rich with multiple GC boxes and other putative cis-regulatory elements. Here, we report the functional characterization of the TPMT promoter, revealing several positive regulatory elements and identifying stimulating protein 1 (Sp1) as an important trans-activator essential for constitutive activity in cell culture. One major and two closely located minor transcription start points were identified in HepG2 cells. Deletion analysis revealed positive cis-regulatory elements located in the regions -85 to -75, -68 to -58, -58 to -51 and +34 to +60 relative to the transcription start site. DNaseI footprinting analysis and cotransfection in Drosophila Schneider SL2 cells documented that Sp1 binds to the TPMT promoter and is important for constitutive activity. We conclude that constitutive transcription of the TPMT gene involves a limited upstream GC-rich DNA sequence, containing multiple GC boxes, and that transcription factor Sp1 [or related protein(s)] is an important trans-activator of this TATA-less promoter.


Subject(s)
Methyltransferases/genetics , Promoter Regions, Genetic , Animals , Base Sequence , Cells, Cultured , Drosophila , Genes, Regulator , Humans , Molecular Sequence Data , Sp1 Transcription Factor/metabolism , Structure-Activity Relationship , Transcriptional Activation
16.
Clin Pharmacol Ther ; 64(1): 46-51, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9695718

ABSTRACT

OBJECTIVE: Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme that catalyzes the S-methylation of mercaptopurine, azathioprine, thioguanine and most of their nucleotide metabolites. TPMT exhibits genetic polymorphism, with about 10% of individuals having intermediate TPMT activity because of heterozygosity at the TPMT locus and about 1 in 300 inheriting TPMT deficiency as an autosomal recessive trait. Although several mutant alleles have now been associated with inheritance of TPMT deficiency in humans, the expression of only TPMT*2 and TPMT*3A has been established by isolation and characterization of complementary DNA (cDNA) from individuals with low TPMT activity. METHODS: Radiochemical assay, Western blot analysis, polymerase chain reaction (PCR) genotyping, and cDNA sequencing were used to analyze TPMT activity and protein levels in erythrocytes and to determine TPMT genotype. RESULTS: We established expression of another common mutant allele, TPMT*3C (containing only the A719G mutation), by sequence analysis of cDNA isolated from an individual with a heterozygous TPMT phenotype (7 units/ml packed erythrocytes). The TPMT*3C allele was also confirmed in an unrelated individual by sequencing TPMT coding exons after PCR amplification of genomic DNA. Moreover, Western blot analysis of erythrocytes obtained from five heterozygous individuals with the TPMT*3C allele (i.e., TPMT*1/TPMT*3C) exhibited about 50% less immunodetectable TPMT protein compared with homozygous wild-type individuals, and a TPMT-deficient individual with a TPMT*3A/TPMT*3C genotype had no immunodetectable TPMT protein. CONCLUSION: These data establish that the TPMT*3C allele is expressed in humans and is associated with lower immunodetectable TPMT protein and catalytic activity.


Subject(s)
DNA, Complementary/isolation & purification , Methyltransferases/genetics , Point Mutation/genetics , Adult , Blotting, Western , Child , Child, Preschool , DNA, Complementary/genetics , Genotype , Humans , Introns/genetics , Male , Methyltransferases/metabolism , Polymerase Chain Reaction
19.
FEBS Lett ; 424(3): 143-5, 1998 Mar 13.
Article in English | MEDLINE | ID: mdl-9539138

ABSTRACT

Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme that catalyzes S-methylation of aromatic and heterocyclic sulfhydryl compounds, including anticancer and immunosuppressive thiopurines. Here we report the isolation and functional characterization of the murine TPMT cDNA. The screening of expressed sequence tags database led to isolation of a murine cDNA clone containing an uninterrupted ORF encoding the protein with an amino acid sequence that is 82% similar and 78% identical to the human TPMT. The expression product of the murine cDNA in rabbit reticulocyte and wheat germ lysate coupled transcription-translation systems showed TPMT enzymatic activity. We conclude that the isolated cDNA clone represents the murine TPMT cDNA.


Subject(s)
Methyltransferases/genetics , Methyltransferases/metabolism , Amino Acid Sequence , Animals , Cloning, Molecular , DNA, Complementary/isolation & purification , Humans , Mice , Molecular Sequence Data , Rabbits , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid
20.
Proc Natl Acad Sci U S A ; 94(12): 6444-9, 1997 Jun 10.
Article in English | MEDLINE | ID: mdl-9177237

ABSTRACT

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 (TPMT*2 and TPMT*3A), 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 units/ml pRBC (rs = 0.99; P < 0.001). Similarly, heterologous expression of wild-type (TPMT*1) and mutant (TPMT*2 and TPMT*3A) 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 half-lives for TPMT*2 and TPMT*3A ( approximately 0.25 hr) compared with wild-type TPMT*1 (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 TPMT*2 and TPMT*3A as mechanisms for lower TPMT protein and catalytic activity inherited by the predominant mutant alleles at the human TPMT locus.


Subject(s)
Methyltransferases/genetics , Methyltransferases/metabolism , Polymorphism, Genetic , Alleles , Animals , COS Cells , Cloning, Molecular , Endopeptidases/metabolism , Erythrocytes/enzymology , Humans , Kinetics , Methyltransferases/biosynthesis , Protein Biosynthesis , Rabbits , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Saccharomyces cerevisiae , Transfection
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