RESUMEN
Deoxycytidine kinase (dCK), a rate-limiting enzyme in the cytosolic deoxyribonucleoside (dN) salvage pathway, is an important therapeutic and positron emission tomography (PET) imaging target in cancer. PET probes for dCK have been developed and are effective in mice but have suboptimal specificity and sensitivity in humans. To identify a more suitable probe for clinical dCK PET imaging, we compared the selectivity of two candidate compounds-[(18)F]Clofarabine; 2-chloro-2'-deoxy-2'-[(18)F]fluoro-9-ß-d-arabinofuranosyl-adenine ([(18)F]CFA) and 2'-deoxy-2'-[(18)F]fluoro-9-ß-d-arabinofuranosyl-guanine ([(18)F]F-AraG)-for dCK and deoxyguanosine kinase (dGK), a dCK-related mitochondrial enzyme. We demonstrate that, in the tracer concentration range used for PET imaging, [(18)F]CFA is primarily a substrate for dCK, with minimal cross-reactivity. In contrast, [(18)F]F-AraG is a better substrate for dGK than for dCK. [(18)F]CFA accumulation in leukemia cells correlated with dCK expression and was abrogated by treatment with a dCK inhibitor. Although [(18)F]CFA uptake was reduced by deoxycytidine (dC) competition, this inhibition required high dC concentrations present in murine, but not human, plasma. Expression of cytidine deaminase, a dC-catabolizing enzyme, in leukemia cells both in cell culture and in mice reduced the competition between dC and [(18)F]CFA, leading to increased dCK-dependent probe accumulation. First-in-human, to our knowledge, [(18)F]CFA PET/CT studies showed probe accumulation in tissues with high dCK expression: e.g., hematopoietic bone marrow and secondary lymphoid organs. The selectivity of [(18)F]CFA for dCK and its favorable biodistribution in humans justify further studies to validate [(18)F]CFA PET as a new cancer biomarker for treatment stratification and monitoring.
Asunto(s)
Nucleótidos de Adenina/química , Arabinonucleósidos/química , Biomarcadores de Tumor/química , Desoxicitidina Quinasa/análisis , Desoxicitidina Quinasa/metabolismo , Tomografía de Emisión de Positrones/métodos , Animales , Antineoplásicos/química , Línea Celular Tumoral , Clofarabina , Medios de Contraste/química , Desoxicitidina Quinasa/antagonistas & inhibidores , Humanos , Leucemia/enzimología , Ratones , Neoplasias/tratamiento farmacológico , Profármacos/química , RatasRESUMEN
Adoptive transfer of tumor-reactive T cells can successfully reduce tumor burden; however, in rare cases, lethal on-target/off-tumor effects have been reported. A noninvasive method to track engineered cells with high sensitivity and resolution would allow observation of correct cell homing and/or identification of dangerous off-target locations in preclinical and clinical applications. Human deoxycytidine kinase triple mutant (hdCK3mut) is a nonimmunogenic PET reporter that was previously shown to be an effective tool to monitor whole-body hematopoiesis. Here, we engineered a construct in which hdCK3mut is coexpressed with the anti-melanoma T cell receptor F5, introduced this construct into human CD34 cells or PBMCs, and evaluated this approach in multiple immunotherapy models. Expression of hdCK3mut allowed engrafted cells to be visualized within recipient bone marrow, while accumulation of [18F]-L-FMAU in hdCK3mut-expressing T cells permitted detection of intratumoral homing. Animals that received T cells coexpressing hdCK3mut and the anti-melanoma T cell receptor had demonstrably higher signals in HLA-matched tumors compared with those in animals that received cells solely expressing hdCK3mut. Engineered T cells caused cytotoxicity in HLA/antigen-matched tumors and induced IFN-γ production and activation. Moreover, hdCK3mut permitted simultaneous monitoring of engraftment and tumor infiltration, without affecting T cell function. Our findings suggest that hdCK3mut reporter imaging can be applied in clinical immunotherapies for whole-body detection of engineered cell locations.
Asunto(s)
Desoxicitidina Quinasa/análisis , Genes Reporteros , Inmunoterapia/métodos , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma Experimental/terapia , Tomografía de Emisión de Positrones , Linfocitos T/inmunología , Animales , Médula Ósea/diagnóstico por imagen , Quimiotaxis de Leucocito , Pruebas Inmunológicas de Citotoxicidad , Desoxicitidina Quinasa/genética , Genes Sintéticos , Vectores Genéticos/genética , Supervivencia de Injerto , Antígeno HLA-A2/inmunología , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/química , Células Madre Hematopoyéticas/fisiología , Humanos , Inmunoterapia Adoptiva , Ensayos de Liberación de Interferón gamma , Lentivirus/genética , Leucocitos Mononucleares/química , Leucocitos Mononucleares/fisiología , Leucocitos Mononucleares/trasplante , Antígeno MART-1/inmunología , Melanoma Experimental/inmunología , Ratones , Mutación , Receptores de Antígenos de Linfocitos T/análisis , Receptores de Antígenos de Linfocitos T/genética , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/genética , Retroviridae/genética , Linfocitos T/trasplante , Timo/trasplanteRESUMEN
BACKGROUND & AIMS: Patients who undergo surgery for pancreatic ductal adenocarcinoma (PDAC) frequently receive adjuvant gemcitabine chemotherapy. Key determinants of gemcitabine cytotoxicity include the activities of the human equilibrative nucleoside transporter 1 (hENT1), deoxycytidine kinase (dCK), and ribonucleotide reductase subunit 1 (RRM1). We investigated whether tumor levels of these proteins were associated with efficacy of gemcitabine therapy following surgery. METHODS: Sequential samples of resected PDACs were retrospectively collected from 434 patients at 5 centers; 142 patients did not receive adjuvant treatment (33%), 243 received adjuvant gemcitabine-based regimens (56%), and 49 received nongemcitabine regimens (11%). We measured protein levels of hENT1, dCK, and RRM1 by semiquantitative immunohistochemistry with tissue microarrays and investigated their relationship with patients' overall survival time. RESULTS: The median overall survival time of patients was 32.0 months. Among patients who did not receive adjuvant treatment, levels of hENT1, RRM1, and dCK were not associated with survival time. Among patients who received gemcitabine, high levels of hENT1 and dCK were significantly associated with longer survival time (hazard ratios of 0.34 [P < .0001] and 0.57 [P = .012], respectively). Interaction tests for gemcitabine administration and hENT1 and dCK status were statistically significant (P = .0007 and P = .016, respectively). On multivariate analysis of this population, hENT1 and dCK retained independent predictive values, and those patients with high levels of each protein had the longest survival times following adjuvant therapy with gemcitabine. CONCLUSIONS: High levels of hENT1 and dCK in PDAC predict longer survival times in patients treated with adjuvant gemcitabine.
Asunto(s)
Antimetabolitos Antineoplásicos/uso terapéutico , Carcinoma Ductal Pancreático/tratamiento farmacológico , Desoxicitidina Quinasa/análisis , Desoxicitidina/análogos & derivados , Tranportador Equilibrativo 1 de Nucleósido/análisis , Neoplasias Pancreáticas/tratamiento farmacológico , Proteínas Supresoras de Tumor/análisis , Antimetabolitos Antineoplásicos/metabolismo , Transporte Biológico , Biotransformación , Carcinoma Ductal Pancreático/química , Carcinoma Ductal Pancreático/mortalidad , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/cirugía , Quimioterapia Adyuvante , Distribución de Chi-Cuadrado , Desoxicitidina/metabolismo , Desoxicitidina/uso terapéutico , Femenino , Francia , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Masculino , Análisis Multivariante , Pancreatectomía , Neoplasias Pancreáticas/química , Neoplasias Pancreáticas/mortalidad , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/cirugía , Modelos de Riesgos Proporcionales , Estudios Retrospectivos , Ribonucleósido Difosfato Reductasa , Medición de Riesgo , Factores de Riesgo , Factores de Tiempo , Análisis de Matrices Tisulares/métodos , Resultado del Tratamiento , GemcitabinaRESUMEN
PURPOSE: To determine cross-resistance to anti-tumor treatments in 2',2'difluorodeoxycytidine (dFdC, gemcitabine)-resistant human tumor cells. METHODS AND MATERIALS: Human lung carcinoma cells SW-1573 (SWp) were made resistant to dFdC (SWg). Sensitivity to cisplatin (cDDP), paclitaxel, 5-fluorouracil (5-FU), methotrexate (MTX), cytarabine (ara-C), and dFdC was measured by a proliferation assay. Radiosensitivity and radioenhancement by dFdC of this cell panel and the human ovarian carcinoma cell line A2780 and its dFdC-resistant variant AG6000 were determined by clonogenic assay. Bivariate flowcytometry was performed to study cell cycle changes. RESULTS: In the SWg, a complete deoxycytidine kinase (dCK) deficiency was found on mRNA and protein level. This was accompanied by a 10-fold decrease in dCK activity which resulted in the >1000-fold resistance to dFdC. Sensitivity to other anti-tumor drugs was not altered, except for ara-C (>100-fold resistance). Radiosensitivity was not altered in the dFdC-resistant cell lines SWg and AG6000. High concentrations (50-100 microM dFdC) induced radioenhancement in the dFdC-resistant cell lines similar to the radioenhancement obtained at lower concentrations (10 nM dFdC) in the parental lines. An early S-phase arrest was found in all cell lines after dFdC treatment where radioenhancement was achieved. CONCLUSIONS: In the dFdC-resistant lung tumor cell line SWg, the deficiency in dCK is related to the resistance to dFdC and ara-C. No cross-resistance was observed to other anti-tumor drugs used for the treatment in lung cancer. Sensitivity to ionizing radiation was not altered in two different dFdC-resistant cell lines. Resistance to dFdC does not eliminate the ability of dFdC to sensitize cells to radiation.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/terapia , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Resistencia a Antineoplásicos , Neoplasias Pulmonares/terapia , Tolerancia a Radiación , Carcinoma de Pulmón de Células no Pequeñas/patología , Cisplatino/farmacología , Desoxicitidina Quinasa/análisis , Desoxicitidina Quinasa/genética , Humanos , Neoplasias Pulmonares/patología , Paclitaxel/farmacología , Células Tumorales Cultivadas , GemcitabinaRESUMEN
In vitro studies have demonstrated that deoxycytidine kinase (dCK) plays a crucial role in the mechanism of resistance to cytarabine (AraC). The resistant phenotype in vitro is always a result of mutational inactivation of dCK, leading to defects in the metabolic pathways of AraC. Although inactivation of dCK has shown to be one of the major mechanism of resistance to AraC in vitro, limited in vivo data are available. To improve research concerning the involvement of dCK inactivation in patients with acute myeloid leukemia (AML), we have set up a protocol that allows direct assessment of dCK expression and activity in primary human cells. In this protein activity truncation assay (PAT assay), the complete coding region of dCK is amplified by RT-PCR and a T7 RNA polymerase promoter sequence is introduced upstream of the coding region in a nested PCR reaction. After in vitro transcription-translation dCK proteins are analyzed for their molecular weight and phosphorylating capacities. We show that this relatively quick method can be used in purified, primary human leukemic blasts. In addition, inactivation of dCK by point mutations, deletions or genomic rearrangements can easily be detected in AraC-resistant cell lines. This novel assay may contribute to further elucidate the mechanism of AraC resistance in vivo.
Asunto(s)
Desoxicitidina Quinasa/genética , Leucemia Mieloide/enzimología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Enfermedad Aguda , Animales , Antimetabolitos Antineoplásicos/farmacología , Células de la Médula Ósea/enzimología , Citarabina/farmacología , Desoxicitidina Quinasa/análisis , Desoxicitidina Quinasa/metabolismo , Resistencia a Antineoplásicos , Humanos , Leucemia Mieloide/patología , Fitohemaglutininas/farmacología , Biosíntesis de Proteínas , Ratas , Linfocitos T/enzimología , Transcripción Genética , Células Tumorales CultivadasRESUMEN
The current study was initiated to explore the mechanisms underlying the previously demonstrated association between the proliferative activity of leukaemic blasts and the response to cytosine arabinoside (AraC)-based therapy in de novo acute myeloid leukaemia (AML). The activity of key enzymes of AraC metabolism-deoxycytidine kinase (DCK), cytidine deaminase (DCD) and polymerase alpha (PolyA) were determined in blast cells from 33 patients. In addition, formation and retention of intracellular levels of AraC triphosphate (AraCTP) and DNA incorporation of AraC were measured, as was the proliferative activity of leukaemic blasts by [3H]-TdR incorporation before and after stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF) or granulocyte CSF (G-CSF) for 48 h. AraC incorporation into the DNA (median 0.60 pmol/105 cells) was significantly related to the proliferative activity of AML blasts (r = 0.74, P < 0.001). Similarly, priming with GM-CSF or G-CSF increased both the proliferative activity of AML blasts by a median of 1.84- and 1.64-fold, respectively, and the incorporation of AraC into the DNA (1.29- and 1.40-fold respectively). In contrast, no relationship was found between the endogenous proliferative activity (EPA) and enzyme activities regulating AraC activation (DCK; median 4.70 pmol/min/mg protein), inactivation (DCD; median 2.92 pmol/min/mg protein) or inhibitory effects (PolyA; median 1.50 pmol/min/mg protein), nor the formation or retention of AraCTP (median 306.1 ng/107 cell and 1.6 h respectively). When samples were grouped according to EPA (more than or less than the median), slowly proliferating specimens had a higher response to cytokine priming for proliferative activity and incorporation of AraC into DNA. Clinical data of 15 patients were available. Although all eight patients with a high endogenous proliferative activity reached complete remission, only four out of seven patients with a low proliferative activity responded, whereas the other three patients were non-responders (P = 0.077).
Asunto(s)
Citarabina/farmacología , Inmunosupresores/farmacología , Leucemia Mieloide/tratamiento farmacológico , Enfermedad Aguda , Adulto , Anciano , División Celular/efectos de los fármacos , Citarabina/farmacocinética , Citarabina/uso terapéutico , Citidina Desaminasa/análisis , ADN/metabolismo , Desoxicitidina Quinasa/análisis , Femenino , Semivida , Humanos , Inmunosupresores/farmacocinética , Inmunosupresores/uso terapéutico , Leucemia Mieloide/enzimología , Leucemia Mieloide/inmunología , Modelos Lineales , Linfocitos/metabolismo , Masculino , Persona de Mediana Edad , Estadísticas no Paramétricas , Factores de TiempoRESUMEN
Deoxycytidine kinase (dCyd kinase) is important for the phosphorylation of several different nucleoside antimetabolites. To understand the significance of dCyd kinase levels in chemotherapy, dCyd kinase mRNA levels were evaluated in several cells with a quantitative competitive polymerase chain reaction (PCR) assay. dCyd kinase catalytic activity and intracellular ara-CTP production were also compared with the levels of dCyd kinase mRNA. The assay was able to show: (i) that dCyd kinase catalytic activity and dCyd kinase mRNA levels were correlated in cells; (ii) that dCyd kinase mRNA levels were more sensitive in lower levels of 10 amol/micrograms of total RNA; and (iii) in cytosine arabinoside (ara-C)-resistant cells, both dCyd kinase mRNA levels and intracellular ara-CTP levels were lower compared with levels in sensitive cells. The PCR assay for dCyd kinase mRNA could be useful in the selection and monitoring of patients treated with nucleosides that are activated by this enzyme.
Asunto(s)
Desoxicitidina Quinasa/análisis , Leucemia/enzimología , Enfermedad Aguda , Trifosfato de Arabinofuranosil Citosina/análisis , Desoxicitidina Quinasa/genética , Humanos , Leucemia Mieloide/enzimología , Inhibidores de la Síntesis del Ácido Nucleico/análisis , Reacción en Cadena de la Polimerasa/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimología , ARN Mensajero/análisis , Transcripción GenéticaRESUMEN
2',2'-Difluorodeoxycytidine (gemcitabine, dFdCyd) is a deoxycytidine analogue with promising antitumor activity. In order to be active it must be phosphorylated by deoxycytidine kinase (dCK). We induced resistance to dFCyd in the human ovarian carcinoma cell line A2780 by exposure to increasing concentrations of dFdCyd. The IC50, defined as the concentration of dFdCyd causing 50% growth inhibition, at 72 h exposure increased from 0.6 nM dFdCyd in A2780 to 92 microM in the resistant variant, named AG6000. Although the resistant cell line is routinely cultured in 6 microM dFdCyd, the resistant phenotype can be maintained for at least 10 passages without dFdCyd. AG6000 is cross-resistant to other drug which require activation by dCK, such as 1-beta-D-arabinofuranosylcytosine, 5-aza-2'-deoxycytidine, and 2-chlorodeoxyadenosine. There was no specific dCK activity in extracts from AG600 cells. Western blot analysis using a polyclonal anti-dCK antibody did not reveal any dCK protein in AG6000 cell extracts. Reverse-transcribed and PCR-amplified mRNA, using specific dCK primers, demonstrated that AG6000 expressed a normal length amplicon of 701 base pairs, besides an aberrant amplicon of 500 base pairs. Chromosome spreads from the cell lines showed no major differences between A2780 and AG6000. The latter cell line was also cross-resistant to 2',2'-difluorodeoxyurdine, the deamination product of dFdCyd. Additionally, cross-resistance to the multidrug resistant drugs doxorubicin and vincristine was observed. This was not associated with the induction of P-glycoprotein, as determined by the RNase protection assay. Injection of AG6000 cells s.c. into nude mice demonstrated that the cell line had retained its tumorigenicity; AG6000 xenografts were not sensitive to dFdCyd treatment, in contrast to the parental A2780 tumors. No dFdCyd triphosphate accumulation was found in the resistant tumors, in contrast to the parental A2780 tumors. These results indicate that the dFdCyd resistance phenotype is stable, and mainly due to dCK deficiency.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Desoxicitidina Quinasa/análisis , Desoxicitidina/análogos & derivados , Neoplasias Ováricas/enzimología , Animales , Secuencia de Bases , Desoxicitidina/farmacología , Resistencia a Medicamentos , Femenino , Humanos , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Neoplasias Ováricas/patología , ARN Mensajero/análisis , Células Tumorales Cultivadas , GemcitabinaRESUMEN
The incorporation of pyrimidine nucleotide precursors into Helicobacter pylori and the activities of enzymes involved in their synthetic pathways were investigated by radioactive tracer analysis and 31P nuclear magnetic resonance spectroscopy. The bacterium was found to take up aspartate and bicarbonate and to incorporate carbon atoms from these precursors into its genomic DNA. Orotate, an intermediate of de novo pyrimidine biosynthesis, and uracil and uridine, precursors for pyrimidine pathways, were also incorporated by the micro-organism. Radiolabelled substrates were used to assess the activities of aspartate transcarbamoylase, orotate phosphoribosyltransferase, orotidylate decarboxylase, CTP synthetase, uracil phosphoribosyltransferase, thymidine kinase and deoxycytidine kinase in bacterial lysates. The study provided evidence for the presence in H. pylori of an operational de novo pathway, and a less active salvage pathway for the biosynthesis of pyrimidine nucleotides.
Asunto(s)
Ligasas de Carbono-Nitrógeno , Helicobacter pylori/metabolismo , Nucleótidos de Pirimidina/biosíntesis , Aspartato Carbamoiltransferasa/análisis , Ácido Aspártico/metabolismo , Bicarbonatos/metabolismo , Desoxicitidina Quinasa/análisis , Helicobacter pylori/enzimología , Marcaje Isotópico , Ligasas/análisis , Espectroscopía de Resonancia Magnética , Orotato Fosforribosiltransferasa/análisis , Pentosiltransferasa/análisis , Isótopos de FósforoRESUMEN
Deoxycytidine (CdR) kinase (E.C. 2.7.1.74) acts as a salvage enzyme in DNA biosynthesis, but little is known about this important nucleoside kinase in the brain. We report that CdR kinase activity in the 100,000 x g cytosolic fraction of normal adult rat brain cortex was 0.89 +/- 0.04 nmol/hr/mg protein which is twice that of the liver enzyme. For brain CdR kinase the apparent Km for CdR, ATP and Mg++ were 0.22, 1.1 and 0.63 mM, respectively. When the cytosolic preparation was incubated at 37 degrees C, CdR kinase activity rapidly decreased (t1/2 = 15 min); CdR (400 microM) protected the enzyme. Addition of DFDC (0 to 100 microM) competitively inhibited brain CdR kinase activity with Ki = 17 microM. DFDC elevated the apparent Km for CdR of brain CdR kinase 3.5-fold, from 0.22 to 0.8 mM. DFDC did not inhibit brain TdR kinase. AZT, which competitively inhibited TdR kinase (Ki = 0.6 microM), did not affect brain CdR kinase activity. These results indicate that the cytosol of rat brain contains CdR kinase which is inhibited by the deoxycytidine analog, DFDC. The enzyme is protected from thermal denaturation by CdR but not by TdR.
Asunto(s)
Corteza Cerebral/enzimología , Desoxicitidina Quinasa/análisis , Timidina Quinasa/análisis , Animales , Citosol/enzimología , Desoxicitidina/análogos & derivados , Masculino , Ratas , Ratas Wistar , Especificidad por Sustrato , Zidovudina/farmacologíaRESUMEN
Deoxycytidine kinase (dCK) is necessary for the activity of several nucleosides used for the chemotherapy of cancer and AIDS. However, the measurement of dCK catalytic activity in crude cell extracts may be imprecise, due to the presence of phosphatases and nucleotidases that degrade the enzyme products. We describe a simple immunoassay for dCK that can measure accurately as little as 5 ng enzyme protein in crude tissue extracts. The assay enabled us to show (i) that mutant cells deficient in dCK activity lack immunoreactive dCK protein, (ii) that dCK catalytic activity and immunoreactivity correlate closely in human tumors, and (iii) that immunoreactive dCK is particularly high in lymphocytes and lymphoid malignancies, although certain solid tumors may also contain the enzyme. The immunoassay of dCK could prove useful in the selection and monitoring of patients who are being treated with nucleosides that are activated by this enzyme.
Asunto(s)
Desoxicitidina Quinasa/análisis , Neoplasias/enzimología , Animales , Astrocitoma/enzimología , Secuencia de Bases , Desoxicitidina Quinasa/inmunología , Desoxicitidina Quinasa/metabolismo , Humanos , Técnicas para Inmunoenzimas , Leucemia de Células Pilosas/enzimología , Leucemia Linfocítica Crónica de Células B/enzimología , Melanoma/enzimología , Datos de Secuencia Molecular , Conejos , Estándares de Referencia , Células Tumorales CultivadasRESUMEN
Human cells salvage pyrimidine deoxyribonucleosides via 5'-phosphorylation which is also the route of activation of many chemotherapeutically used nucleoside analogs. Key enzymes in this metabolism are the cytosolic thymidine kinase (TK1), the mitochondrial thymidine kinase (TK2) and the cytosolic deoxycytidine kinase (dCK). These enzymes are expressed differently in different tissues and cell cycle phases, and they display overlapping substrate specificities. Thymidine is phosphorylated by both thymidine kinases, and deoxycytidine is phosphorylated by both dCK and TK2. The enzymes also phosphorylate nucleoside analogs with very different efficiencies. Here we present specific radiochemical assays for the three kinase activities utilizing analogs as substrates that are by more than 90 percent phosphorylated solely by one of the kinases; i.e. 3'-azido-2',3'-dideoxythymidine (AZT) as substrate for TK1, 1-beta-D-arabinofuranosylthymidine (AraT) for TK2 and 2-chlorodeoxyadenosine (CdA) for dCK. We determined the fraction of the total deoxycytidine and thymidine phosphorylating activity that was provided by each of the three enzymes in different human cells and tissues, such as resting and proliferating lymphocytes, lymphocytic cells of leukemia patients (chronic lymphocytic, chronic myeloic and hairy cell leukemia), muscle, brain and gastrointestinal tissue. The detailed knowledge of the pyrimidine deoxyribonucleoside kinase activities and substrate specificities are of importance for studies on chemotherapeutically active nucleoside analogs, and the assays and data presented here should be valuable tools in that research.
Asunto(s)
Desoxicitidina Quinasa/análisis , Isoenzimas/análisis , Leucocitos/enzimología , Timidina Quinasa/análisis , Arabinonucleósidos/metabolismo , Cladribina/metabolismo , Desoxicitidina/metabolismo , Desoxicitidina Quinasa/metabolismo , Humanos , Isoenzimas/metabolismo , Cinética , Leucemia , Mitocondrias/enzimología , Especificidad de Órganos , Fosforilación , Técnica de Dilución de Radioisótopos , Valores de Referencia , Especificidad por Sustrato , Timidina/análogos & derivados , Timidina/metabolismo , Timidina Quinasa/metabolismo , Tritio , Células Tumorales Cultivadas , Zidovudina/metabolismoRESUMEN
Mononuclear phagocytes play a central role in the pathogenesis of chronic inflammatory diseases. It is therefore important to define chemotherapeutically exploitable metabolic pathways that distinguish monocytes from other cell types. Blood monocytes do not synthesize deoxynucleotides de novo, and their transformation to macrophages occurs without cell division. Whether or not monocytes can repair DNA damage, and whether or not DNA repair is necessary for their survival, is unknown. The present experiments demonstrate that normal human monocytes, unlike neutrophils, rapidly repair DNA strand breaks induced by gamma-irradiation. Monocyte extracts contain functional immunoreactive DNA polymerase-alpha. DNA repair synthesis in normal monocytes is blocked by aphidicolin, an inhibitor of DNA polymerase-alpha with respect to dCTP. Aphidicolin is also directly toxic to normal monocytes, but has no effect on nondividing lymphocytes or fibroblasts. Compared to most other cell types, monocytes and macrophages have very low dCTP pools, but abundant deoxycytidine kinase activity. This suggests that dCTP derived from salvage pathways is important for DNA repair in these cells. Consistent with this notion, exogenous deoxycytidine could partially protect monocytes from aphidicolin killing. The unexpected toxicity of aphidicolin toward normal human monocytes may be attributable to their high rate of spontaneous DNA strand break formation, to the importance of DNA polymerase-alpha for DNA repair in these cells, and to their minute dCTP pools.
Asunto(s)
Supervivencia Celular/fisiología , Reparación del ADN/fisiología , Monocitos/fisiología , 2-Cloroadenosina/toxicidad , Afidicolina/toxicidad , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , ADN Polimerasa II/antagonistas & inhibidores , Reparación del ADN/efectos de los fármacos , Desoxicitidina Quinasa/análisis , Humanos , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Timidina Quinasa/análisisRESUMEN
In this study we describe the establishment of a leukemic cell line (BNML-CL/ara-C), originating from the 1-beta-D-arabinofuranosylcytosine (ara-C)-resistant brown Norway rat myelocytic leukemia model (BNML/ara-C), that retains the in vivo generated ara-C resistance. Its biological and biochemical characteristics have been compared with a cell line, derived from the ara-C-sensitive BNML model (BNML-CL/O). Resistance to ara-C was attributed to a decrease in phosphorylation of ara-C. Deoxycytidine (dCyd) kinase activity in crude cell extracts with dCyd as substrate showed similar enzyme activities in both cell lines, whereas with ara-C as substrate no dCyd kinase activity was detectable in the ara-C-resistant cell line. Two isoenzymes of dCyd kinase with different substrate specificities have been described (Cheng, Y.C., Domin, B., and Lee, L.S. Biochim. Biophys. Acta, 481: 481-492, 1977), cytoplasmic (dCyd kinase I, substrates: dCyd and ara-C) and mitochondrial (dCyd kinase II, substrates: dCyd and thymidine). In the ara-C-sensitive BNML model, thymidine induced a reduction of dCyd kinase activity when dCyd was used as substrate. However, thymidine did not affect kinase activity with ara-C was used as substrate. In the BNML-CL/ara-C, thymidine even induces a dCyd kinase inhibition of 85% with dCyd as substrate. It is likely that the ara-C-specific dCyd kinase deficiency in BNML-CL/ara-C cells was due to a selective loss of dCyd kinase I, whereas dCyd kinase II activity remained intact.
Asunto(s)
Citarabina/metabolismo , Desoxicitidina Quinasa/deficiencia , Leucemia Mieloide/patología , Animales , Supervivencia Celular/efectos de los fármacos , Aberraciones Cromosómicas , Citarabina/farmacología , Desoxicitidina Quinasa/análisis , Desoxirribonucleótidos/análisis , Resistencia a Medicamentos , Isoenzimas/análisis , Leucemia Mieloide/genética , Ratas , Ratas Endogámicas BN , Especificidad por Sustrato , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Deficiency of the purine metabolic enzyme adenosine deaminase causes severe immunodeficiency. Retroviruses have been reported to decrease the activity of adenosine deaminase, and many retroviruses, including feline leukemia virus, cause immunodeficiency. Levels of purine metabolic enzymes including adenosine deaminase and consequences of adenosine deaminase inhibition were investigated in feline leukemia virus-infected fresh tumor cells and infected and uninfected cell lines. No evidence of virus effect on levels of adenosine deaminase or other purine metabolic enzymes was detected. Neoplastic cells demonstrated considerable heterogeneity of activity levels of purine metabolic enzymes.
Asunto(s)
Linfoma/veterinaria , Fosfotransferasas (Aceptor de Grupo Alcohol) , Purinas/metabolismo , 5'-Nucleotidasa/análisis , Adenosina Desaminasa/análisis , Animales , Gatos , Línea Celular , Desoxicitidina Quinasa/análisis , Virus de la Leucemia Felina , Linfoma/enzimología , Linfoma/metabolismo , Fosfotransferasas/análisis , Células Tumorales CultivadasRESUMEN
In this study we investigated the Ara-CTP-forming capacity of leukemic cells in different phases of the cell cycle. Cells from two leukemic cell lines and leukemic bone marrow cells from patients and rats (BNML model) with acute myelocytic leukemia were separated according to cell cycle phase by means of an albumin density gradient in a specially designed sedimentation chamber. We found that the activity of CdR kinase and Cyt deaminase is much less influenced by cell-cycle phase progression than TdR kinase activity. For the leukemic cell lines HL-60 and BNML-CL/O CdR kinase activity is even independent of cell-cycle phase. In addition, Ara-CTP formation is not restricted to cells in S-phase. Cell cycle phase-independent Ara-CTP formation creates a situation in which cells which are not in S-phase during exposure to Ara-C might undergo the cytotoxic effects of Ara-C as soon as they enter S-phase.
Asunto(s)
Trifosfato de Arabinofuranosil Citosina/metabolismo , Arabinonucleotidos/metabolismo , Desoxicitidina Quinasa/análisis , Leucemia/metabolismo , Fosfotransferasas/análisis , Timidina Quinasa/análisis , Uridina Quinasa/análisis , Animales , Ciclo Celular , ADN de Neoplasias/análisis , Humanos , Ratas , Células Tumorales CultivadasRESUMEN
In an attempt to maximize the therapeutic index and to overcome the large variations in 1-beta-D-arabinofuranosylcytosine (ara-C) plasma levels and host toxicities that have been documented with standard HDara-C regimens (3 g/m2 over 3 h every 12 h x 8 or x12 doses), pediatric patients with acute lymphocytic leukemia or lymphoma in relapse were treated with a regimen of loading bolus followed immediately by continuous infusion of ara-C. In addition, patients received a single dose of etoposide (VP-16, 1 g/m2) prior to the ara-C administration. In four patients, total body irradiation was administered as part of a bone marrow transplantation preparative regimen after the ara-C administration. The regimen was designed to attain and maintain plasma steady-state concentrations (Css) of ara-C three to four times the Km2 value of ara-C, which was determined with purified deoxycytidine kinase from the patients' tumor cells prior to treatment. Eight patients age 0.75 to 16 years with relapsed acute lymphocytic leukemia (three patients) or lymphoma (five patients, one with bone marrow involvement), received a test dose of 3 g/m2 ara-C injected over 1 h, and the plasma kinetics were determined. The peak plasma ara-C concentration of ara-C ranged from 57 to 199 microM with an average concentration of 103 +/- 49 microM; the half-lives of distribution (t1/2, alpha) and elimination (t1/2, beta) averaged 17 +/- 7 min and 4.04 +/- 3.1 h, respectively. The mean area under the plasma concentration time curve from 0 to 12 h (AUC0----12 h) of ara-C averaged 386.8 +/- 328.0 microMh (mean, +/- SD, n = 8). The peak concentration of uracil arabinoside averaged 501 +/- 123 microM, and it was eliminated with a t1/2, el of 2.3 +/- 0.6 h. The patients then received an individualized loading bolus (mean = 0.5 g/m2) followed by a continuous infusion regimen of ara-C (mean = 130 mg/m2/h), to achieve a Css in the range of 20 to 35 microM. The obtained plasma Css were similar to the desired ones, averaging in variation 10.7% +/- 8.2%. The percentage of variation of correlation of the AUC following the loading bolus plus the continuous infusion from 12 to 72 h was only 12.4% (mean = 2158 microMh, n = 8), whereas the percentage of variation of correlation of the AUC after the test dose of ara-C in the same patients was 84.8%.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Citarabina/farmacocinética , Leucemia/tratamiento farmacológico , Linfoma/tratamiento farmacológico , Adolescente , Niño , Preescolar , Citarabina/administración & dosificación , Citarabina/efectos adversos , Desoxicitidina Quinasa/análisis , Humanos , LactanteRESUMEN
Deoxycytidine kinase from human leukemic spleen has been purified 6000-fold to apparent homogeneity with an overall yield of 10%. The purification was achieved by using DEAE chromatography, hydroxylapatite chromatography, and affinity chromatography on dTTP-Sepharose. Only one form of deoxycytidine kinase activity was found during all the chromatographic procedures. The subunit molecular mass, as judged by sodium dodecyl sulfate--polyacrylamide gel electrophoresis, was 30 kilodaltons. The pure enzyme phosphorylates deoxycytidine, deoxyadenosine, and deoxyguanosine, demonstrating for the first time that the same enzyme molecule has the capacity to use these three nucleosides as substrates. The apparent molecular weight of the active enzyme, determined by gel filtration and glycerol gradient centrifugation, was 60,000. Thus, the active form of human deoxycytidine kinase is a dimer. The kinetic behavior of pure human deoxycytidine kinase was studied in detail with regard to four different phosphate acceptors and two different phosphate donors. The apparent Km values were 1, 20, 150, and 120 microM for deoxycytidine, arabinosylcytosine, deoxyguanosine, and deoxyadenosine, respectively. The Vmax values were 5-fold higher for the purine nucleosides as compared to the pyrimidine substrates. We observe competitive inhibition of the phosphorylation of one substrate by the presence of either of the three other substrates, but the apparent Ki values differed greatly from the corresponding Km values, suggesting the existence of allosteric effects. The double-reciprocal plots for ATP-MgCl2 as phosphate donor were convex, indicating negative cooperative effects. In contrast, plots with varying dTTP-MgCl2 concentration as phosphate donor were linear with an apparent Km of 2 microM. The enzyme activity was strongly inhibited by dCTP, in a noncompetitive way with deoxycytidine and in a competitive way with ATP-MgCl2.
Asunto(s)
Desoxicitidina Quinasa/aislamiento & purificación , Leucemia/enzimología , Fosfotransferasas/aislamiento & purificación , Bazo/enzimología , Unión Competitiva , Cromatografía por Intercambio Iónico , Desoxicitidina Quinasa/análisis , Estabilidad de Enzimas , Humanos , CinéticaRESUMEN
Cadeguomycin markedly stimulated the uptake of thymidine, deoxycytidine and uridine into the acid-insoluble fraction of K562 human leukemic cells, but did not significantly affect adenosine incorporation. The enhancement of pyrimidine nucleoside uptake was 6 approximately 17 fold over the control. Aspartate incorporation into nucleic acid was not significantly blocked by the antibiotic, suggesting that the stimulation of pyrimidine nucleoside incorporation is not due to the inhibition of de novo pyrimidine nucleotide synthesis. Net DNA and RNA syntheses, observed by [32P]phosphate uptake, were not significantly affected by cadeguomycin. The enzymatic activity of thymidine, deoxycytidine and uridine kinases was higher in cadeguomycin-treated cells than in untreated cells, suggesting that the enhancement of pyrimidine nucleoside uptake occurs in the phosphorylation process. The stimulatory activity of cadeguomycin of thymidine uptake was reversed by guanosine and deoxyguanosine, but not by adenosine and deoxyadenosine, suggesting that intracellular metabolism and/or action of cadeguomycin is related to that of guanosine and deoxyguanosine. The stimulation of pyrimidine nucleoside incorporation by cadeguomycin was also found with YAC-1 cells, but not with the other cell lines. The enhancement effect of the antibiotic seems to be not directly related to its cytotoxicity.