RESUMEN
Thiopurine S-methyltransferase (TPMT) activity exhibits genetic polymorphism. The purpose of this investigation was to identify TPMT mutant alleles in the Saami population as a basis of developing genotyping tests for prediction of TPMT activity. The most predominant allele in Saamis (n = 194) was the TPMT*3C allele (A719G mutation) representing 92% of the mutant alleles, with an estimated allelic frequency of 3.3%. The most frequent allele in Caucasians (n = 66) living in the same geographic area was the TPMT*3A (A719G and G460A mutations) representing 91% of the mutant alleles, with an estimated allelic frequency of 3.4%. A test for one mutation, A719G, may prospectively identify more than 90% of the Saami individuals who require reduction in thiopurine dose to avoid hematopoietic toxicity. In a Norwegian population, comprising both the major Caucasian population and a minor Saami population, the same genotyping tests (eg, tests for the A719G and G460A mutations) may be used.
Asunto(s)
Metiltransferasas/genética , Mutación , Población Blanca/genética , Adulto , Alelos , Femenino , Genotipo , Humanos , Masculino , Metiltransferasas/metabolismo , Noruega/etnología , Fenotipo , Polimorfismo Genético , Análisis de Secuencia de ADNRESUMEN
The knowledge about the structure and function of the protein families responsible for cGMP synthesis and metabolic conversion has grown vastly the last years, whereas little is known about proteins that account for the cellular export of cGMP. In the present study, we have employed a model with inside-out vesicles prepared from human erythrocytes to characterize modulation and regulation of cellular cGMP extrusion. The active transport was saturable (Km of 2.4 +/- 0.2 microM, mean +/- SEM, n = 3) and coupled to ATP hydrolysis since no accumulation was detected in the presence of ATP-gamma-S and AMP-PNP. The observation that 100 microM of cAMP caused a minimal inhibition (14.4 +/- 0.3%) of active cGMP transport showed that the extrusion system for cGMP was not shared with cAMP, but a competitive interaction occurred for the ATP-independent association to the inside out vesicles. In contrast, the lowest, but physiological relevant cAMP concentrations (0.1-5 microM) stimulated the active cGMP transport with 30-35%, an observation that suggests cAMP as an allosteric regulator of the cGMP transporter. Several well-known modulators of other energy-requiring membrane transport systems caused a competitive and concentration-dependent inhibition, including verapamil (Ki = 13.0 +/- 2.4 microM), forskolin (Ki = 13.5 +/- 1.4 microM) and probenecid (Ki = 27.0 +/- 1.3 microM). Progesterone, which was the most potent inhibitor (Ki = 2.2 +/- 0.3 microM), interacted with the active cGMP transport in a noncompetitive manner. The highest concentration (100 microM) of IBMX and theophylline reduced the active cGMP uptake with 29.5 +/- 1.9% and 21.6 +/- 2.1%, respectively. None of these substances interfered with the association of cGMP to the vesicles in absence of ATP. The present results show that human erythrocytes possess a cell membrane cGMP transporter which is coupled to an ATPase. Its activity is regulated by cAMP in an apparent allosteric manner and inhibited by substances previously known to interact with other membrane transport systems.
Asunto(s)
AMP Cíclico/farmacología , GMP Cíclico/metabolismo , Membrana Eritrocítica/metabolismo , Bombas Iónicas/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Transporte Biológico Activo/efectos de los fármacos , Colforsina/farmacología , Dimetilsulfóxido/farmacología , Membrana Eritrocítica/efectos de los fármacos , Humanos , Bombas Iónicas/efectos de los fármacos , Probenecid/farmacología , Progesterona/farmacología , Teofilina/farmacología , Verapamilo/farmacologíaRESUMEN
The effect of the transmethylation inhibitor 3-deazaadenosine on transcription levels of genes associated with apoptosis was investigated in HL-60 cells. After incubation of HL-60 cells with 100 microM 3-deazaadenosine for 45 min., a schedule known to perturb transmethylation metabolites and initiate apoptosis in these cells, a 50% decrease in c-myc and a 50% increase in bcl-2 RNA steady-state levels compared to control cells were observed. Transcription levels of c-myc continued to decrease after extended exposure to 3-deazaadenosine, while bcl-2 mRNA levels dropped to 25% and 30% below those in control cells after 1.5 hr and 3 hr, respectively. The expression levels of the bcl-2 related bax gene, showed a similar pattern as bcl-2; a 60% increase was initially measured, but after 1.5 and 3 hr, bax transcripts were 80% and 70% respectively, of those found in untreated cells. Another bcl-2 related gene, bcl-x, was previously reported to generate two transcripts in human cells. The long variant bcl-x1 acts as bcl-2, while the short form bcl-xs induces apoptosis. We were unable to detect bcl-xs transcripts in untreated and 3-deazaadenosine treated cells by the highly sensitive reverse transcriptase polymerase chain reaction method. This suggests that this gene product may not be involved in 3-deazaadenosine induced apoptosis in HL-60 cells. Bcl-x1 mRNA levels, however, slowly decreased with about 50% after 1.5 or 3 hr 3-deazaadenosine treatment. It is concluded that 3-deazaadenosine initiated apoptosis affects c-myc, bcl-2, bax and bcl-x1 mRNA levels.
Asunto(s)
Apoptosis/genética , Expresión Génica/efectos de los fármacos , Genes bcl-2/efectos de los fármacos , Genes myc/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2 , Tubercidina/farmacología , Células HL-60/efectos de los fármacos , Células HL-60/metabolismo , Humanos , Proteínas Proto-Oncogénicas/genética , ARN Mensajero , Proteína X Asociada a bcl-2RESUMEN
To investigate the role of transmethylation metabolites in initiation of apoptosis in human leukemia HL-60 cells exposed to 3-deazaadenosine (c3 Ado) as single agent and c3Ado plus homocysteine thiolactone (Hcy), S-adenosylmethionine (AdoMet), S-adenosylhomocysteine (AdoHcy) and 3-deazaadenosylhomocysteine (c3AdoHcy), were measured by HPLC in HL-60 cells exposed to 1-100 microM c3Ado as single agent and 1 to 100 microM c3Ado plus 1 mM Hcy. 3-deaza-(+/-)-aristeromycin (c3Ari), a more specific and potent S-adenosylhomocysteine hydrolase inhibitor compared with c3Ado, was used to inhibit synthesis of c3AdoHcy. AdoMet increased to maximum values 1.5- and 2.3-fold control in cultures treated with c3Ado as single agent and c3Ado plus Hcy, respectively. AdoHcy did not change in cultures treated with c3Ado as single agent, but increased to maximum values 2.0-fold control when Hcy was added. The synthesis of c3AdoHcy was favored by Hcy, and 35- to 70-fold higher levels of c3AdoHcy were found in cultures treated with c3Ado plus Hcy vs. c3Ado as single agent. The amounts of c3AdoHcy in cultures treated with c3Ado at concentrations initiating apoptosis did not exceed c3AdoHcy levels in cultures treated with c3Ado plus Hcy at concentrations not initiating apoptosis. Pretreatment of c3Ado plus Hcy cultures with c3Ari diminished c3AdoHcy and almost completely abrogated apoptosis. Exposure of cells to 100 microM c3Ari as single agent resulted in an increase in AdoHcy to 8.4-fold control but no changes in AdoMet and no initiation of apoptosis. Our findings indicate that c3Ado as single agent exerts its apoptosis-initiating effect through a nontransmethylase-related bio-chemical action. c3AdoHcy seems to be related to biochemical events initiating apoptotic cell death of HL-60 cells when c3Ado and Hcy are combined.