RESUMO
Helicobacter pylori strains from 299 patients were tested in six laboratories in different countries. Macrolide susceptibility of the strains was determined by agar dilution (17.4%) or the epsilometer test (82.6%). Mutations in the 23S ribosomal DNA (rDNA) that are associated with macrolide resistance were analyzed by PCR and reverse hybridization (PCR-line probe assay [LiPA]). This method identifies A2115G, G2141A, A2142G, A2142C, A2142T, A2143G, and A2143C mutations in the 23S rDNA. vacA s-region (s1a, s1b, s1c, and s2) and m-region (m1, m2a, and m2b) genotypes and cagA status were also determined using another PCR-LiPA system. Of the 299 strains investigated by MIC testing, 130 (43.5%) were resistant and 169 (56.5%) were susceptible to clarithromycin. Of the 130 resistant strains, 127 (97.7%) contained 23S rDNA mutations, whereas 167 (98.8%) of the 169 susceptible strains contained wild-type sequences. The predominant mutations were A2143G (45.2%) and A2142G (33.3%). Twenty-eight (19.8%) strains contained multiple 23S rDNA mutations. Only five resistant strains contained the A2142C mutation (three of these in combination with the A2142G mutation), and the A2115G, G2141A, A2142T, and A2143C mutations were not found. MICs of clarithromycin for the A2142G mutant strains were significantly higher than MICs for the A2143G strains. Although there was no significant association between 23S rDNA mutations and the vacA and cagA status, clarithromycin-susceptible strains more often contained mixed vacA genotypes, indicating the presence of multiple H. pylori strains. In conclusion, our data confirmed the very strong association between 23S rDNA mutations and macrolide resistance and showed that the PCR-LiPA permits accurate and reliable diagnosis of macrolide resistance in H. pylori.
Assuntos
Antibacterianos/farmacologia , Antígenos de Bactérias , Claritromicina/farmacologia , Helicobacter pylori/genética , RNA Ribossômico 23S/genética , Proteínas de Bactérias/genética , Análise Mutacional de DNA , Resistência Microbiana a Medicamentos/genética , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/patogenicidade , Humanos , Mutação , Reação em Cadeia da Polimerase/métodos , Virulência/genéticaAssuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Leucemia/tratamento farmacológico , Mercaptopurina/uso terapêutico , Metiltransferases/metabolismo , Tioguanina/uso terapêutico , Antimetabólitos Antineoplásicos/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Criança , Cromossomos Humanos Par 6 , Humanos , Leucemia/metabolismo , Mercaptopurina/administração & dosagem , Mercaptopurina/farmacocinética , Metotrexato/administração & dosagem , Metiltransferases/deficiência , Metiltransferases/genética , Polimorfismo Genético , Tioguanina/farmacocinéticaRESUMO
Mycophenolic acid, an inhibitor of the enzyme IMP dehydrogenase, antagonizes the CTP synthetase inhibitor 3-deazauridine in its anti-proliferative effects on MOLT-3 human T leukemia cells. No depletion of CTP occurred, and decreased amounts of 3-deazuridine-triphosphate were measured in cells incubated with mycophenolic acid and 3-deazuridine. Most probably, these phenomena are related to the increased amounts of PRPP observed, which can result in an increased pyrimidine biosynthesis de novo and, as a consequence, a decreased metabolism of 3-deazauridine via the salvage pathway.
Assuntos
3-Desazauridina/farmacologia , Carbono-Nitrogênio Ligases , Inibidores Enzimáticos/farmacologia , IMP Desidrogenase/antagonistas & inibidores , Ligases/antagonistas & inibidores , Ácido Micofenólico/farmacologia , Fosforribosil Pirofosfato/metabolismo , 3-Desazauridina/antagonistas & inibidores , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Leucemia , Ribonucleotídeos/biossíntese , Células Tumorais CultivadasRESUMO
6-Methylmercaptopurine ribonucleoside-5'-phosphate (MeSPuRMP), the sole metabolite of 6-methylmercaptopurine ribonucleoside (MeSPuRib), is a strong inhibitor of purine de novo synthesis, inducing depletion of intracellular purine nucleotides and subsequent cell death in several tumor cell lines. In this study prevention of MeSPuRib cytotoxicity by compounds of the purine salvage pathway was studied in Molt F4 human malignant T-lymphoblasts. Adenosine, adenine and inosine were able to prevent depletion of the adenine nucleotide pool when used in combination with 0.5 microM MeSPuRib, but had virtually no effect on depletion of guanine nucleotides. Nevertheless, these three purine compounds were able to reduce the cytotoxic effects induced by MeSPuRib. Addition of guanosine to cells treated with 0.5 microM MeSPuRib normalized the guanine nucleotide pool, but adenine nucleotides remained depleted. Under these conditions, inhibition of cell growth was significantly decreased. With the combination of guanosine and 10 microM MeSPuRib, cytotoxicity was increased compared to 10 microM MeSPuRib alone, associated with a depletion of adenine nucleotides to 9% of untreated cells. Since cell growth and cell viability of Molt F4 cells are less inhibited by MeSPuRib under conditions where adenine nucleotide depletion is prevented by purine compounds (and where the other nucleotides are depleted) we conclude that depletion of adenine nucleotides is an important factor in MeSPuRib cytotoxicity.
Assuntos
Adenina/farmacologia , Mercaptopurina/análogos & derivados , Metiltioinosina/antagonistas & inibidores , Metiltioinosina/toxicidade , Nucleosídeos de Purina/farmacologia , Ribonucleosídeos/antagonistas & inibidores , Ribonucleosídeos/toxicidade , Adenosina/farmacologia , Antineoplásicos/toxicidade , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Guanosina/farmacologia , Humanos , Inosina/farmacologia , Mercaptopurina/antagonistas & inibidores , Mercaptopurina/toxicidade , Nucleosídeos/metabolismo , Nucleotídeos/metabolismo , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Tioinosina/análogos & derivados , Tioinosina/toxicidade , Tionucleotídeos/toxicidadeRESUMO
The studies described indicate that me-t-IMP formation is an important pathway, contributing to cytotoxicity in Molt F4 cells, which exhibit a highly active de novo purine synthesis. On three levels cytotoxicity is induced during methylation of thiopurines. 1. Purine synthesis de novo is inhibited during formation of me-t-IMP. Inhibition of PDNS results in depletion of purine nucleotides, with subsequently diminishing DNA and RNA synthesis. 2. The increased PRPP levels, induced by me-T-IMP, induce increased pyrimidine biosynthesis and cause an imbalance in purine nucleotides. This imbalance may lead to inhibition of cell growth and after prolonged exposure, to cell death. 3. The observed depletion of SAM and the decrease of the SAM/SAH ratio may be an additional mechanism by which 6MP and me-MPR exert their effects on cell growth and cell viability. Changes in SAM/SAH ratio may directly influence methylation reactions. The significant decrease of DNA methylation by 6MP and me-t-IMP may influence gene regulation and tumor progression. Administration of SAM leads to chemoprevention of rat liver carcinogenesis, indicating a role of DNA methylation in tumor progression. Besides the effects on methylation of DNA, a decrease of SAM/SAH ratio may also affect other processes, such as methylation of RNA, proteins and phospholipids, thereby disturbing their functionality. In conclusion, decrease of the SAM/SAH ratio resulting from treatment with 6MP and me-MPR may exert many effects in these cells. This may open a new field of research, possibly contributing to a deeper understanding of the complex mechanisms by which 6MP provokes cytotoxicity.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , DNA de Neoplasias/metabolismo , Mercaptopurina/farmacologia , Tioinosina/análogos & derivados , Tionucleotídeos/farmacologia , Antimetabólitos Antineoplásicos/metabolismo , Morte Celular/efeitos dos fármacos , Humanos , Mercaptopurina/metabolismo , Metilação , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Tioinosina/farmacologia , Células Tumorais CultivadasRESUMO
6-Mercaptopurine (6-MP) and methylmercaptopurine ribonucleoside (Me-MPR) are purine anti-metabolites which are both metabolized to methylthio-IMP (Me-tIMP), a strong inhibitor of purine synthesis de novo. Me-MPR is converted directly into Me-tIMP by adenosine kinase. 6-MP is converted into tIMP, and thereafter it is methylated to Me-tIMP by thiopurine methyltransferase, an S-adenosylmethionine (S-Ado-Met)-dependent conversion. S-Ado-Met is formed from methionine and ATP by methionine adenosyltransferase, and is a universal methyl donor, involved in methylation of several macromolecules, e.g. DNA and RNA. Therefore, depletion of S-Ado-Met could result in an altered methylation state of these macromolecules, thereby affecting their functionality, leading to dysregulation of cellular processes and cytotoxicity. In this study the effects of 6-MP and Me-MPR on S-Ado-Met, S-adenosylhomocysteine (S-Ado-Hcy), homocysteine and methionine concentrations are determined. Both drugs cause a decrease in intracellular S-Ado-Met concentrations and an increase in S-Ado-Hcy and methionine concentrations in Molt F4 human malignant lymphoblasts. The effects of both 6-MP and Me-MPR can be ascribed to a decreased conversion of methionine into S-Ado-Met, due to the ATP depletion induced by the inhibition of purine synthesis de novo by Me-tIMP. Both 6-MP and Me-MPR thus affect the methylation state of the cells, and this may result in dysregulation of cellular processes and may be an additional mechanism of cytotoxicity for 6-MP and Me-MPR.
Assuntos
Mercaptopurina/farmacologia , S-Adenosilmetionina/biossíntese , Tionucleosídeos/farmacologia , Trifosfato de Adenosina/biossíntese , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Leucemia-Linfoma de Células T do Adulto/metabolismo , Leucemia-Linfoma de Células T do Adulto/patologia , Células Tumorais CultivadasRESUMO
The effects of inhibition of inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in guanine nucleotide de novo synthesis, on cell growth, cell viability, endogenous nucleotide concentrations and concentrations of extracellular nucleosides and bases were studied in Molt F4 human malignant lymphoblasts. Mycophenolic acid (MPA) was used as a specific inhibitor of the enzyme activity. IMPDH activity was maximally inhibited with 0.5 microM MPA. After a 2 h exposure of the cells to 0.5 microM MPA, guanine nucleotides were depleted to approximately 50% of control values, whereas 5-phosphoribosyl-1-pyrophosphate levels increased to approximately 200%. Under these conditions, cytotoxicity became obvious after 24 h. Depletion of guanine nucleotides and cytotoxicity were prevented by addition of guanosine to MPA treatment. Daily supplements of guanosine were required to prevent MPA cytotoxicity during the entire incubation period of 72 h. We conclude that depletion of guanine nucleotides, induced by treatment with MPA, induces a severe and rapid cytotoxicity in Molt F4 cells.
Assuntos
IMP Desidrogenase/antagonistas & inibidores , Ácido Micofenólico/farmacologia , Linfócitos T/enzimologia , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Nucleosídeos/metabolismo , Nucleotídeos de Purina/metabolismo , Nucleotídeos de Pirimidina/metabolismo , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Cytotoxicity of 6-mercaptopurine (6MP) and 6-methylmercaptopurine ribonucleoside (Me-MPR) was studied in Molt F4 human malignant lymphoblasts. Both drugs are converted into methylthioIMP (Me-tIMP), which inhibits purine de novo synthesis. Addition of amidoimidazole carboxamide ribonucleoside (AICAR) circumvented inhibition of purine de novo synthesis, and thus partly prevented 6MP and Me-MPR cytotoxicity. Purine nucleotides, and especially adenine nucleotides, were recovered by addition of AICAR. Under these conditions, Me-tIMP formation decreased. The results of this study indicate that formation of Me-tIMP may be important for 6MP cytotoxicity in Molt F4 cells. These data suggest that depletion of adenine nucleotides is the main cause for Me-tIMP cytotoxicity.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Mercaptopurina/análogos & derivados , Mercaptopurina/antagonistas & inibidores , Ribonucleosídeos/farmacologia , Tioinosina/análogos & derivados , Tionucleosídeos/antagonistas & inibidores , Células Tumorais Cultivadas/efeitos dos fármacos , Nucleotídeos de Adenina/metabolismo , Aminoimidazol Carboxamida/farmacologia , Contagem de Células , Morte Celular/efeitos dos fármacos , Interações Medicamentosas , Nucleotídeos de Guanina/metabolismo , Humanos , Metiltioinosina/análogos & derivados , Metiltioinosina/metabolismo , Tionucleotídeos/metabolismo , Fatores de TempoRESUMO
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)
Assuntos
Mercaptopurina/farmacologia , Linfócitos T/efeitos dos fármacos , Radioisótopos de Carbono , Ciclo Celular/efeitos dos fármacos , DNA/biossíntese , Humanos , Inosina Monofosfato/análogos & derivados , Inosina Monofosfato/análise , Leucemia Linfoide/tratamento farmacológico , Leucemia Linfoide/metabolismo , Fosforribosil Pirofosfato/análise , Radioisótopos de Fósforo , RNA/biossíntese , Ribonucleotídeos/análise , Células-Tronco/efeitos dos fármacos , Linfócitos T/metabolismo , Tionucleotídeos/análiseRESUMO
The importance of methyl-thioIMP (Me-tIMP) formation for methylmercaptopurine ribonucleoside (Me-MPR) cytotoxicity was studied in Molt F4 cells. Cytotoxicity of Me-MPR is caused by Me-tIMP formation with concomitant inhibition of purine de novo synthesis. Inhibition of purine de novo synthesis resulted in decreased purine nucleotide levels and enhanced 5-phosphoribosyl-1-pyrophosphate (PRPP) levels, with concurrent increased pyrimidine nucleotide levels. The Me-tIMP concentration increased proportionally with the concentration of Me-MPR. High Me-tIMP concentration also caused inhibition of PRPP synthesis. Maximal accumulation of PRPP thus occurred at low Me-MPR concentrations. As little as 0.2 microM Me-MPR resulted already after 2 h in maximal inhibition of formation of adenine and guanine nucleotides, caused by inhibition of purine de novo synthesis by Me-tIMP. Under these circumstances increased intracellular PRPP concentrations could be demonstrated, resulting in increased levels of pyrimidine nucleotides. So, in Molt F4 cells, formation of Me-tIMP from Me-MPR results in cytotoxicity by inhibition of purine de novo synthesis.
Assuntos
Inosina Monofosfato/análogos & derivados , Mercaptopurina/análogos & derivados , Metiltioinosina/análogos & derivados , Linfócitos T/efeitos dos fármacos , Tioinosina/análogos & derivados , Tionucleosídeos/farmacologia , Tionucleotídeos/metabolismo , Tionucleotídeos/farmacologia , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Mercaptopurina/metabolismo , Mercaptopurina/farmacologia , Mercaptopurina/toxicidade , Metiltioinosina/metabolismo , Fosforribosil Pirofosfato/biossíntese , Nucleotídeos de Purina/metabolismo , Tionucleosídeos/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
6-Mercaptopurine (6MP) cytotoxicity was studied in Molt F4 cells, a T-cell acute lymphoblastic leukemia (ALL) cell line. The effects on cytotoxicity were concentration-dependent. Measurements of intracellular thionucleotide intermediates of 6MP demonstrated a rapid rise of thio-IMP (tIMP) levels, and subsequently a rapid decrease. Thio-GMP (tGMP) and methyl-thio-IMP (Me-tIMP) appeared later in time, and persisted longer. Mycophenolic acid (MPA), a specific inhibitor of IMP dehydrogenase (IMPDH), was used to inhibit the conversion of tIMP into tGMP, thereby decreasing the incorporation of 6MP into DNA. A synergistic effect on cell viability and cell growth was observed when cells were treated with a combination of 2 microM 6MP and 0.5 microM MPA. Also, intracellular Me-tIMP increased 5 times with the combination. Based on the increase of Me-tIMP concentration and the observed synergism between 6MP and MPA, we conclude that methylation of tIMP into Me-tIMP is an important alternative route for 6MP cytotoxicity.
Assuntos
Citotoxinas/toxicidade , Mercaptopurina/toxicidade , Ácido Micofenólico/toxicidade , Linfócitos T/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras , Células Tumorais CultivadasRESUMO
The cell-kinetics and biochemical pharmacology of simultaneous and sequential combination treatment with 0.02 microM methotrexate (MTX) and 2 microM 6-mercaptopurine (6MP) were studied in MOLT-4 malignant T-lymphoblasts. The results were compared with our data from earlier studies of separate treatment with these antimetabolites. A synergistic effect of combination treatment could be demonstrated, based on the inhibition of purine de novo synthesis by both agents, on DNA and RNA synthesis, on the incorporation of 6-thioguanine nucleotides into DNA and RNA, and on inhibition of cell growth and clonal growth. The synergistic effects of combination treatment with MTX and 6MP will only be available in malignant lymphoblasts, and will be absent in normal bone marrow cells and normal lymphocytes, because the activity of purine de novo synthesis in these cells is absent or low. Based on the synergistic effects of MTX and 6MP and the good penetration of both agents in the cerebrospinal fluid, the Dutch Childhood Leukemia Study Group presently performs a randomized study during protocol M of the BFM/DCLSG-ALL-90 protocol comparing the results of 4 times each two weeks 24 hr intravenous administration of MTX (5 g/m2) versus intravenous MTX, immediately followed by 24 hr intravenous administration of 6MP (1.3 g/m2). The pharmacokinetics and intracellular biochemical pharmacology of 6MP in lymphocytes will be studied, comparing intravenous administration and low dose oral administration.