RESUMEN
Although recurrent chromosomal alterations occur in chronic lymphocytic leukemia (CLL), relatively few affected tumor suppressors and oncogenes have been implicated. To improve genetic characterization of CLL, we performed high-resolution gene copy number analysis of 20 CLL patients using oligonucleotide array comparative genomic hybridization (aCGH). The most recurrent losses were observed in 13q and 11q with variable sizes. The 11q losses varied between 7.44 Mb and 41.72 Mb in size and targeted ATM among others. Lost regions in 13q were generally smaller, spanning from 0.79 Mb to 29.33 Mb. The minimal common region (158 kb) in 13q14.3, which was also homozygously deleted in some cases, harbored five genes: TRIM13, KCNRG, DLEU2, DLEU1, and FAM10A4. Additionally, two micro-RNA genes (MIRN15A and MIRN16-1) locate to the region. New cryptic losses were detected in 1q23.2-->q23.3, 3p21.31, 16pter-->p13.3, 17p13.3-->p13.2, 17q25.3-->qter, and 22q11.22. In conclusion, our oligonucleotide aCGH study revealed novel aberrations and provided detailed genomic profiles of the altered regions.
Asunto(s)
Eliminación de Gen , Leucemia Linfocítica Crónica de Células B/genética , Hibridación de Ácido Nucleico , Aberraciones Cromosómicas , Estudios de Cohortes , Humanos , CariotipificaciónRESUMEN
One of the most widely used antitumor drugs is cis-diamminedichloroplatinum(II) (cisplatin), and mechanisms of cisplatin resistance have been investigated in numerous model systems. Many studies have used mouse leukemia L1210/0 as a reference wild-type cell line, and cisplatin-resistant subclones have been derived from it. Increased DNA excision repair capacity is thought to play a key role in the acquired cisplatin resistance, and this has influenced development of drugs for clinical trials. We report here that the L1210/0 line is in fact severely deficient in nucleotide excision repair of damaged DNA in vivo and in vitro. L1210/0 cell extracts could be complemented by extracts from repair-defective human xeroderma pigmentosum (XP) or rodent excision repair cross-complementing (ERCC) mutant cells, except for XPG/ERCC5 mutants. Purified XPG protein could restore repair proficiency to L1210/0 extracts. Expression of mouse XPG mRNA was similar in all L1210 lines studied, suggesting a point mutation or small alteration of XPG in L1210/0 cells. The DNA repair capacity of a cisplatin-resistant subline, L1210/DDP10, is similar to that of type culture collection L1210 cells and to those of other normal mammalian cell lines. Nucleotide excision repair of DNA is thus clearly important in the intrinsic cellular defense against cisplatin. However, in contrast to what is generally believed, enhancement of DNA repair above the normal level in these rodent cells does not appear to be a mechanism of acquired resistance to the drug.
Asunto(s)
Cisplatino/toxicidad , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/genética , Animales , Secuencia de Bases , ADN/efectos de los fármacos , ADN/efectos de la radiación , Cartilla de ADN/química , Resistencia a Medicamentos , Endonucleasas , Expresión Génica , Leucemia L1210 , Ratones , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares , ARN Mensajero/genética , Factores de Transcripción , Rayos UltravioletaRESUMEN
The G(-248)A polymorphism in the promoter region of the Bax gene was recently associated with low Bax expression, more advanced stage, treatment resistance and short overall survival in B-cell chronic lymphocytic leukemia (CLL), the latter particularly in treated patients. To investigate this further, we analyzed 463 CLL patients regarding the presence or absence of the G(-248)A polymorphism and correlated with overall survival, treatment status and known prognostic factors, for example, Binet stage, VH mutation status and genomic aberrations. In this material, similar allele and genotype frequencies of the Bax polymorphism were demonstrated in CLL patients and controls (n=207), where 19 and 21% carried this polymorphism, respectively, and no skewed distribution of the polymorphism was evident between different Binet stages and VH mutated and unmutated CLLs. Furthermore, no difference in overall survival was shown between patients displaying the G(-248)A polymorphism or not (median survival 85 and 102 months, respectively, P=0.21), and the polymorphism did not influence outcome specifically in treated CLL. Neither did the polymorphism affect outcome in prognostic subsets defined by VH mutation status or genomic aberrations. In conclusion, the pathogenic role and clinical impact of the Bax polymorphism is limited in CLL.
Asunto(s)
Biomarcadores de Tumor/genética , Leucemia Linfocítica Crónica de Células B/genética , Polimorfismo Genético/genética , Regiones Promotoras Genéticas , Proteína X Asociada a bcl-2/genética , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/biosíntesis , Estudios de Cohortes , Análisis Citogenético , Femenino , Humanos , Leucemia Linfocítica Crónica de Células B/diagnóstico , Masculino , Persona de Mediana Edad , Pronóstico , Estudios Retrospectivos , Tasa de Supervivencia , Proteína X Asociada a bcl-2/biosíntesisRESUMEN
The present experiments were conducted to test the effects of the potent cytidine deaminase inhibitor tetrahydrouridine (THU) on the metabolism and cytotoxicity of 5-methyl-2'-deoxycytidine (5-Med-Cyd) in several human leukemia cell lines in vitro. It was observed that 5-Med-Cyd exerts its effects via deamination to thymidine, which is particularly toxic to human promyelocytic (HL-60) and T-cell (JM) leukemia cell lines in vitro. The deamination and the cytotoxicity of 5-Med-Cyd were effectively hindered by 10(-3) M THU in 3-day cultures of HL-60 cells. Although the catabolism of [14C]5-Med-Cyd in the HL-60 cell cultures was blocked by THU, no radioactive 5-Med-Cyd was incorporated into DNA. The cytotoxicity and DNA incorporation of fluorodeoxycytidine are enhanced by THU. Unlike that compound 5-Med-Cyd resembled more bromodeoxycytidine and iododeoxycytidine; THU decreases the toxicity of both of these deoxycytidine analogues.
Asunto(s)
Desoxicitidina/análogos & derivados , Neoplasias/patología , Tetrahidrouridina/farmacología , Uridina/análogos & derivados , Biotransformación , Línea Celular , Supervivencia Celular/efectos de los fármacos , Desoxicitidina/metabolismo , Desoxicitidina/toxicidad , Humanos , Cinética , Neoplasias/metabolismoRESUMEN
5-Hydroxymethyl-2'-deoxyuridine (5HmdUrd) and 1-beta-D-arabinofuranosylcytosine (Ara-C) had a dose-dependent synergistic or antagonistic action on growth of human promyelocytic leukemic (HL-60) cells in suspension culture. For instance, in 3-day cultures, the cell number was reduced from 100% (with either 100 nM Ara-C or 10 microM 5HmdUrd alone) to 65% (with 100 nM Ara-C plus 10 microM 5HmdUrd), or from 35% (with 1.0 microM Ara-C alone) to 10% (with 1.0 microM Ara-C plus 10 microM 5HmdUrd), compared to the control cultures without drugs. 1.0 and 10 microM 5HmdUrd potentiated the incorporation of radioactive Ara-C (1.0 microM) into HL-60 cell nucleic acids in 2-day cultures by 56 and 64%, respectively. 5HmdUrd-induced enhancement of Ara-C incorporation is one explanation for the synergism of these two drugs. On the other hand, 10 nM Ara-C partially inhibited the toxicity of 100 microM 5HmdUrd. Radioactive 5HmdUrd was incorporated into DNA, but not RNA, the rate being 5% of that observed with thymidine. [3H]5HmdUrd-derived radioactivity remained stable in DNA for at least 24 h, indicating that the compound was not excised to a significant extent from DNA in these conditions. The incorporation of Ara-C and 5HmdUrd into DNA appeared to take place via different pathways, which is a second explanation for their synergism. Ara-C is the most important drug in the clinical chemotherapy of acute nonlymphoblastic leukemia. Experience with 5HmdUrd in experimental antileukemia chemotherapy has been promising. This novel combination of antileukemic agents merits further evaluation.
Asunto(s)
Citarabina/farmacología , ADN de Neoplasias/biosíntesis , Timidina/análogos & derivados , Línea Celular , Supervivencia Celular/efectos de los fármacos , Citarabina/antagonistas & inhibidores , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Humanos , Cinética , Leucemia Mieloide Aguda , Timidina/metabolismo , Timidina/farmacologíaRESUMEN
The factors determining the growth and survival of cells in B chronic lymphocytic leukemia (CLL) have remained poorly understood. We investigated the effects of optimal mitogen combinations (OMCs) on the expression of 26 surface membrane antigens among 33 CLL patients. The seven OMCs used were selected after pre-testing 14 combinations of (1) S. aureus Cowan I (SAC), (2) interleukin-2 (IL-2), (3) tumor necrosis factor alpha (TNF-alpha) and (4) 12-O-tetradecanoylphorbol 13-acetate (TPA; also known as phorbol 12-myristate 13-acetate or PMA). In flow cytometry we revealed that OMCs induced statistically highly significant upregulation of the expression of CD5, CD11c, CD19, CD22, CD23, CD25, CD38, CD40, CD45, CD45RO, CD95, CD126, CD130 and FMC7, and downregulation of CD20 and CD124 expression. Interestingly, the expression of CD27, CD45RA, CD79b, CD80, CD122 and that of the immunoglobulin gene superfamily members CD21, Ig-kappa, Ig-lambda, Ig-delta and Ig-micro were not significantly affected under similar conditions. The expression of several antigens was co-regulated, suggesting common regulatory pathways. These antigens include CD11c/CD5, CD11c/CD22, CD11c/CD126, CD11c/FMC7 as well as CD27/CD45, CD27/CD45RA and CD27/CD79b. Upregulation of surface antigen expression, induced by OMCs, should be applicable in antibody therapy in vitro and in vivo, and in negative stem cell selection for autotransplantation. Furthermore, the current strategy to enhance cell surface antigen expression may be a versatile tool to raise humoral and cell-mediated host defense against CLL cells. Upregulation of proteins mediating positive growth signals (eg CD25, CD40) and negative signals or apoptosis (eg CD95) may be used to sensitize cells to chemotherapy and programmed cell death.
Asunto(s)
Antígenos de Diferenciación/metabolismo , Antígenos de Neoplasias/metabolismo , Linfocitos B/efectos de los fármacos , Sustancias de Crecimiento/farmacología , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Anciano , Antígenos CD/metabolismo , Linfocitos B/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Femenino , Citometría de Flujo , Humanos , Inmunofenotipificación , Técnicas In Vitro , Leucemia Linfocítica Crónica de Células B/metabolismo , Masculino , Persona de Mediana Edad , Regulación hacia ArribaRESUMEN
Chronic lymphocytic leukemia (CLL) is a phenotypically distinguishable form of B-lymphoid leukemias. The regularity of surface membrane antigen expression patterns, their interrelationships as well as the effects of the three frequent chromosomal aberrations, ie 11q deletion, 13q deletion and trisomy 12, were investigated in 35 classic CLL cases by flow cytometry. The two-way cluster analysis of 31 individual antigens revealed three expression patterns: (1) most cells in most cases positive (CD5, CD19, CD20, CD23, CD27, CD40, CD45, CD45RA); (2) most cells in most cases negative (CD10, CD14, CD34, CD122, CD154, mIgG); and (3) a mixed pattern with a variable number of positive cases and a variable percentage of positive cells in individual cases (CD11c, CD21, CD22, CD25, CD38, CD45RO, CD79b, CD80, CD95, CD124, CD126, CD130, FMC7, mIgD, mIgkappa, mIglambda, mIgM). The expressions of several antigens were strongly interdependent, even when antigens belonged to entirely different gene families. Such antigen pairs were: CD11c/CD21; CD19/CD45; CD19/CD79b; CD22/CD45RA; CD23/Igkappa; CD25/mIgM; CD27/CD45; CD45/CD79b; CD45RA/Igkappa. In contrast, the expression of some antigens was mutually exclusive, the best examples being CD45RA/CD45RO, CD38/CD80 and CD45RA/CD80. Deletion of chromosome arm 11q attenuated expression of splicing variant CD45RA, but enhanced CD45RO expression. In contrast, cases of trisomy 12 were associated with enhanced CD45RA and attenuated CD45RO expression. Similarly, trisomy 12 was associated with enhanced CD27 and mIgkappa expression. The variable levels of signaling surface membrane antigens, their interactions and interference by genetic aberrations are likely to affect the clinical progression and drug response of CLL.
Asunto(s)
Antígenos CD/análisis , Antígenos de Neoplasias/análisis , Antígenos de Superficie/análisis , Subgrupos de Linfocitos B/inmunología , Aberraciones Cromosómicas , Leucemia Linfocítica Crónica de Células B/inmunología , Células Madre Neoplásicas/inmunología , Receptores de Antígenos de Linfocitos B/análisis , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Adulto , Anciano , Antígenos CD/genética , Antígenos de Diferenciación/análisis , Antígenos de Neoplasias/genética , Antígenos de Superficie/genética , Deleción Cromosómica , Cromosomas Humanos Par 11/ultraestructura , Cromosomas Humanos Par 12 , Cromosomas Humanos Par 13/ultraestructura , Análisis por Conglomerados , Femenino , Humanos , Inmunofenotipificación , Leucemia Linfocítica Crónica de Células B/genética , Antígenos Comunes de Leucocito/análisis , Antígenos Comunes de Leucocito/genética , Masculino , Glicoproteínas de Membrana , Persona de Mediana Edad , NAD+ Nucleosidasa/análisis , Receptores de Antígenos de Linfocitos B/genética , Eliminación de Secuencia , TrisomíaRESUMEN
Loss of genomic material in 11q is one of the most common structural chromosome aberrations in B cell chronic lymphocytic leukemia (B-CLL). In order to characterize the deletions of 11q23 in B-CLL, we performed fluorescence in situ hybridization (FISH) with eight YAC (yeast artificial chromosome) probes on peripheral leukocytes of 30 patients. These YACs form a contig spanning 7.8 Mb at 11q23.1-q23.3. We found deletions in nine out of 30 cases (30%) and five of them had discontinuous deletions in this region. The region represented by YAC 755b11 (1.6 Mb in size) was involved in all cases with deletions, supporting the hypothesis that this region might contain a novel gene of pathogenic importance to B-CLL. A more distal region represented by YAC 785e12 (760 kb in size) was deleted frequently and specifically. Whether there is another novel gene of pathogenic importance to B-CLL and what is its potential relationship to the deletions in the region represented by YAC 755b11, are issues that require further studies.
Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 11 , Leucemia Linfocítica Crónica de Células B/genética , Anciano , Femenino , Humanos , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana EdadRESUMEN
Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with regard to its clinical course. The limitations of the methods currently available for prognostic assessment in CLL do not allow accurate prediction of the risk of disease progression in individual patients. The recently developed cDNA array technique provides a unique opportunity to study gene expression in various malignancies. To identify new molecular markers for prognostication of CLL patients, we analyzed cDNA arrays by using hierarchical clustering and standard statistic t-test on 34 CLL patients. We found significant expression differences in 78 genes compared to the reference tonsillar B lymphocytes. A cluster of genes, LCP1, PARP, BLR1, DEK, NPM, MCL1, SLP76, STAM, HIVEP1, EVI2B, CD25, HTLF, HIVEP2, BCL2, MNDA, PBX3, EB12, TCF1, CGRP, CD14, ILB, GZMK, GPR17 and CD79B, was associated (P < 0.05) with the unfavorable 11q deletion and also with the unfavorable Binet stages B and C. We present here gene expression profiling that is associated with CLL patients with the 11q23 deletion. Many of the genes in the cluster have not previously been shown to be related to the initiation or progression of CLL. These novel findings provide fundamental information for further attempts to understand the interaction of the clustered genes in the leukomogenesis of CLL in order to better design treatments aimed at specific molecular target(s).
Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 11 , Leucemia Linfocítica Crónica de Células B/genética , Anciano , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Marcadores Genéticos , Humanos , Leucemia Linfocítica Crónica de Células B/diagnóstico , Leucemia Linfocítica Crónica de Células B/metabolismo , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , ARN Neoplásico/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
In acute myelogenous leukemia (AML) intensive postremission treatment is needed for an optimal result. However, it is not known how long the treatment should last and how many courses are necessary. The object of this prospective study was to compare four and eight intensive chemotherapy cycles in the treatment of adult de novo AML. In a multicenter study, 248 consecutive patients, aged from 16 to 65 years, were treated with intensive induction treatment. The patients in remission after two courses were randomized to receive either two (short arm) or six (long arm) additional intensive cycles of chemotherapy. The median follow-up time of the living patients is 68 months. Of the patients, 77% achieved complete remission, and 36% of all patients survived for 5 years. Seventy-three patients were randomized to the short arm and 66 to the long arm. There was no significant difference in the relapse-free survival (median 21 months vs 17 months) or overall survival (43 months vs 39 months) between the short and long arms, respectively. Treatment-related deaths occurred in 31 patients (13%), 11 of them in first remission. More than one-third of the patients survived for 5 years. It seems probable that the first few months after diagnosis are decisive for the prognosis if the chemotherapy is intensive, and further treatment cannot markedly influence the outcome.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Aclarubicina/administración & dosificación , Adolescente , Adulto , Anciano , Amsacrina/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Citarabina/administración & dosificación , Daunorrubicina/administración & dosificación , Supervivencia sin Enfermedad , Esquema de Medicación , Etopósido/administración & dosificación , Femenino , Humanos , Masculino , Persona de Mediana Edad , Prednisolona/administración & dosificación , Estudios Prospectivos , Inducción de Remisión , Vincristina/administración & dosificaciónRESUMEN
5-Methyl-2'-deoxycytidine (5MedCyd) is a minor constituent of mammalian cell DNA. We report the production and characterization of highly specific rabbit anti-5MedCyd antiserum. The antiserum was suitable for the radioimmunological measurement of 5MedCyd. This simple radioimmunoassay was capable of quantitating calf thymus DNA methylation at nanomolar levels of total DNA.
Asunto(s)
ADN/análisis , Desoxicitidina/análogos & derivados , Animales , Radioisótopos de Carbono , Bovinos , Desoxicitidina/análisis , Desoxirribonucleasa I , Hidrólisis , Sueros Inmunes , Cinética , Metilación , Radioinmunoensayo/métodos , TimoRESUMEN
5-Hydroxymethyl-2'-deoxyuridine is an antileukemic thymidine analogue. It is also a well known thymidine-derivative in DNA exposed to ionizing irradiation. We report the production and characterization of specific rabbit anti-5HmdUrd antisera. The antisera were used for the radioimmunological measurement of 5HmdUrd. The radioimmunoassay was capable of quantitating 2 pmol of 5 HmdUrd per tube corresponding to 0.2 mumol/l in a 10 microliter plasma sample. A good correlation between the results obtained with the radioimmunoassay and HPLC was demonstrated when the methods were applied to the measurement of plasma levels of 5HmdUrd in mice receiving experimental chemotherapy.
Asunto(s)
Radioinmunoensayo , Timidina/análogos & derivados , Animales , Anticuerpos/inmunología , Anticuerpos/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Reacciones Cruzadas , Ratones , Ratones Endogámicos DBA , Conejos , Timidina/sangre , Timidina/inmunologíaRESUMEN
A novel radioimmunoassay of 5MedCyd is described. The assay, employing a highly specific antiserum raised in rabbits against BSA-conjugated 5MeCyd, used 5-125iodo-2'-deoxycytidine as the tracer. The measuring range for the assay was found to be 1-1000 pmol per assay of 5MedCyd. When the methods were applied to the measurement of methylation in DNA samples a good correlation between the results obtained with the radioimmunoassay and HPLC was demonstrated. The method has several advantages over the more laborious and sophisticated techniques previously available: high sensitivity, large assay range, rapidity and potential for large number of simultaneous assays, simplicity, and low cost provided that the laboratory has equipment for gamma counting.
Asunto(s)
Citosina/análogos & derivados , ADN/análisis , Radioinmunoensayo , 5-Metilcitosina , Animales , Especificidad de Anticuerpos , Bromodesoxicitidina/análogos & derivados , Bovinos , Cromatografía Líquida de Alta Presión , Reacciones Cruzadas , Citosina/análisis , Desoxicitidina/análogos & derivados , Peces , Metilación , ConejosRESUMEN
We describe a solid-phase fluoroimmunoassay, based on competition between europium-labeled 5 MeCyd (5-methylcytidine) and sample 5MedCyd (5-methyl-2'-deoxycytidine) for polyclonal anti-5MedCyd antibodies (rabbit). Europium labeling of antigen was performed using a novel polylysine-5MeCyd conjugate. Standard and sample preparations containing 5MedCyd inhibited the binding of the europium-labeled 5MeCyd to the antibody molecules. A second antibody, directed against rabbit IgG, was coated on the solid phase, and bound the IgG-5MeCyd-polylysine-europium complex, giving rapid and complete separation of antibody-bound and free antigen. The measuring range was from 3.7 to 2500 pmol of 5MedCyd per assay. A good correlation between the results obtained with TR-FIA and HPLC was demonstrated when the methods were applied to the measurement of methylation in various DNA samples, enzymatically hydrolyzed to their constituent deoxyribonucleosides. This new TR-FIA possesses the same advantages (high sensitivity, wide assay range, rapidity, simplicity, and low cost) as the previous assay developed in our laboratories. The superiority of the new system is based on (i) its low inter- and intra-assay variation, (ii) low antiserum consumption, and (iii) a protocol, which permits the use of second-antibody-coated microtitration strips common to other assays.
Asunto(s)
Antígenos , Desoxicitidina/análogos & derivados , Europio , Fluoroinmunoensayo , Animales , Cromatografía Líquida de Alta Presión , ADN , Desoxicitidina/análisis , Desoxicitidina/inmunología , Fluoroinmunoensayo/métodos , Fluoroinmunoensayo/normas , Humanos , Hidrólisis , Polilisina/inmunología , ConejosRESUMEN
Several 2-substituted deoxyadenosine derivatives were synthesized and screened for cytotoxicity toward hematopoietic cells in culture. To prepare intermediates for these syntheses, the sodium salts of 2,6-dibromopurine and 2,6-bis(methylthio)purine were reacted with 1-chloro-3,5-di-p-toluyl-alpha-D-erythro-pentofuranose in acetonitrile. Similar reactions using 6-chloropurines have been reported to give only 9-beta and 7-beta nucleosides as major and minor products, respectively. 2,6-Dibromopurine, however, gave 9-beta and 9-alpha isomers as major and minor products, along with a lesser amount of the 7-beta isomer. 2,6-Bis(methylthio)purine, in contrast, produced 9-beta and 7-beta isomers as major and minor products. These results are discussed in terms of sugar anomerization and possible steric and kinetic effects of base substituents in the sodium salt glycosylation reaction. Reactions of the 9-beta nucleoside isomers with ammonia and alkylamines produced several 2-bromo, 2-methylthio, and 2-amino deoxyadenosines. All of the compounds showed weaker cytotoxic activity than 2-bromodeoxyadenosine against hematopoietic cells in culture, when [14C]leucine incorporation into cellular proteins was measured.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Desoxiadenosinas/síntesis química , Animales , Desoxiadenosinas/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Isomerismo , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Stereoselective procedures are described for the synthesis of 6-alkyluridines by Lewis acid-catalyzed condensation of (a) trimethylsilylated 6-alkyl-4-alkylthiouracils with 1-O-acetyl-2,3,5-tri-O-benzoyl-beta-D-ribofuranose (ABR) and (b) trimethylsilylated 6-alkyl-3-benzyluracils with ABR. The 4-methylthio group was subsequently removed with the use of 1 N trifluoroacetic acid and the 3-benzyl group by a new modified procedure with the use of the complex BBr3-THF. Furthermore, 6-(hydroxymethyl)uridine (39) and 5-fluoro-6-(hydroxymethyl)uridine (40) were obtained by sequential oxidation with SeO2 and reduction with tetrabutylammonium borohydride of the 6-methyl group of 6-methyluridine (5) and 5-fluoro-6-methyluridine (35), and their corresponding 6-fluoromethyl congeners 41 and 42 were obtained by DAST treatment of 39 and 40, respectively. For all the foregoing nucleosides in the fixed syn conformation about the glycosyl bond, 1H NMR spectroscopy further demonstrated that the pentose rings exist predominantly in the conformation N (3'-endo). Most of the nucleosides were weak substrates of Escherichia coli pyrimidine nucleoside phosphorylase. Enhanced susceptibility to phosphorolysis was exhibited by two of them, 39 and 41, with 6-CH2OH and 6-CH2F substituents capable of formation of an additional hydrogen bond with the enzyme. The 5-fluoro-6-substituted uridines were the poorest substrates. Cytotoxicities of the nucleosides were examined vs the human tumor cell lines MOLT-3, U-937, K-562, and IM-9, as well as PHA-stimulated human lymphocytes. Two of the analogues, 5-fluoro-6-(fluoromethyl)uridine (42) and 5-fluoro-6-(hydroxymethyl)uridine (40), exhibited cytotoxicities comparable to that of 5-fluorouracil.
Asunto(s)
Antineoplásicos/síntesis química , Uridina Fosforilasa/metabolismo , Uridina/síntesis química , Antineoplásicos/farmacología , Humanos , Conformación Molecular , Relación Estructura-Actividad , Células Tumorales Cultivadas , Uridina/química , Uridina/farmacologíaRESUMEN
Modification of DNA-cytosine by a 5-methyl group is thought to be an important mechanism which regulates the expression of eukaryotic genes. This modification takes place after semiconservative replication. There is very little evidence, if any, that 5MeCyt could be naturally incorporated into mammalian DNA in semiconservative replication. We have clarified the possibility of incorporating 5MedCyd pharmacologically into human leukemic cells in vitro. To this end, we developed a novel small-scale synthesis method for 14C-labeled 5MedCyd starting from commercially available [14C]dThd derivatives. Particular attention was focused upon possible incorporation of radioactive 5MedCyd derivatives into the acid-soluble cellular fraction as well as into nucleic acids and protein in human cells. The results showed that [2(-14)C]- and [methyl-14C]5MedCyd were incorporated into human leukemic cells to a similar extent. The radioactivity originating from these compounds was incorporated mainly into the acid-soluble pool and nucleic acids. The exact nature of the intracellular radioactive molecules in RNA is not known, but the radioactive label in DNA hydrolyzate co-chromatographed exclusively with thymine. Hence, 5MedCyd is deaminated to thymidine before incorporating into DNA. This deamination had taken place already (partially) in the culture medium. Human leukemic cells do effectively protect their DNA from incorporation of exogenous 5MedCyd.
Asunto(s)
ADN/metabolismo , Desoxicitidina/análogos & derivados , Leucemia Linfoide/metabolismo , 5-Metilcitosina , Línea Celular , Citosina/análogos & derivados , Citosina/metabolismo , Desoxicitidina/metabolismo , Humanos , Leucina/metabolismo , ARN/metabolismo , Timidina/metabolismo , Uridina/metabolismoRESUMEN
The toxicity and metabolism of a thymidine analogue, 5-hydroxymethyl-2'-deoxyuridine (5HmdUrd) were studied with human leukemia cells (HL-60) and with human platelets. 3 X 10(-5) M 5HmdUrd caused a 50% inhibition in the proliferation of HL-60 cells. The compound was hydrolyzed to 5-hydroxymethyluracil (5HmUra) by the enzyme thymidine phosphorylase (EC 2.4.2.4) present in leukemia cells; this catabolic product was non-toxic. The catabolism of 5HmdUrd by human platelet thymidine phosphorylase could be inhibited by 6-aminothymine. The toxicity of 5HmdUrd was effectively reversed by deoxycytidine and 5HmdUrd increased the incorporation of deoxycytidine into dCTP and DNA several fold. The two latter phenomena are explicable in terms of a feedback action to ribonucleotide reductase, resulting in deoxycytidylate starvation, which is a known effect of excess thymidine. We report here also our preliminary observations that 5HmdUrd is active against mouse leukemia in vivo.
Asunto(s)
Antineoplásicos/farmacología , Leucemia/tratamiento farmacológico , Timidina/análogos & derivados , Animales , Plaquetas/metabolismo , Línea Celular , Desoxicitidina/metabolismo , Humanos , Leucemia L1210/tratamiento farmacológico , Timidina/metabolismo , Timidina/farmacologíaRESUMEN
The chemosensitivity of leukemia cells, obtained from the peripheral blood of 35 patients with B-cell chronic lymphocytic leukemia, was determined by a leucine-incorporation assay in vitro. There was good correlation between the sensitivities to two purine analogs, 2-chlorodeoxyadenosine and 9-beta-D-arabinofuranosyl-2-fluoroadenine among previously untreated patients when tested at the 80% inhibition level. Previous exposure to chlorambucil did not affect the sensitivity to these compounds.
Asunto(s)
Antineoplásicos/farmacología , Cladribina/farmacología , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Vidarabina/análogos & derivados , Células Cultivadas , Humanos , Vidarabina/farmacologíaRESUMEN
The expression of the DNA excision repair enzyme uracil-DNA glycosylase was investigated in bone marrow and peripheral samples from seven patients with acute lymphoblastic leukemia (ALL), from 17 patients with acute non-lymphocytic leukemia (ANLL), and from one patient with chronic granulocytic leukemia (CGL) in blast crisis. In addition, uracil-DNA glycosylase activities were determined in nine human leukemia/lymphoma cell lines. There was a clear correlation between the percentage of blast cells and the enzyme activity when mononuclear cell fractions from patient samples were analysed. The following uracil-DNA glycosylase activities were recorded (mean +/- S.D., number of samples): ALL = 45.6 +/- 14.8 U/mg of protein, N = 10; ANLL = 41.1 +/- 13.8 U/mg of protein, N = 22; CGL (blast crisis) = 44.7 U/mg of protein. The uracil-DNA glycosylase activity in nine human leukemia/lymphoma cell lines ranged from 35.2 to 66.0 U/mg of protein, and no striking differences were observed between the T-ALL, B-ALL, null cell ALL or myeloid lines. Similarly, the various biological features, such as the common ALL surface antigen, the terminal deoxynucleotidyl transferase enzyme, the sub-type of leukemia, chromosomal aberrations, or previous chemotherapy, did not apparently affect the expression of uracil-DNA glycosylase. We propose that the integrity of the genetic information is well protected by uracil-DNA glycosylase in different forms of leukemia, including cases with a low proportion of S-phase blasts, as assessed by flow cytometry in the present work. When compared to the activities in benign hematopoietic progenitor cells, studied previously in this laboratory, no big differences between the benign and malignant hematopoiesis were demonstrated. Hence, it is unlikely that selectivity of chemotherapy towards malignant vs benign hematopoietic growth could be based on the enzyme uracil-DNA glycosylase.