Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Lactente , Criança , Prognóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteína de Leucina Linfoide-Mieloide/genética , Contagem de Leucócitos , Sistema Nervoso CentralRESUMO
The poor outcomes in infant acute lymphoblastic leukemia (ALL) necessitate new treatments. Here we discover that EIF4E protein is elevated in most cases of infant ALL and test EIF4E targeting by the repurposed antiviral agent ribavirin, which has anticancer properties through EIF4E inhibition, as a potential treatment. We find that ribavirin treatment of actively dividing infant ALL cells on bone marrow stromal cells (BMSCs) at clinically achievable concentrations causes robust proliferation inhibition in proportion with EIF4E expression. Further, we find that ribavirin treatment of KMT2A-rearranged (KMT2A-R) infant ALL cells and the KMT2A-AFF1 cell line RS4:11 inhibits EIF4E, leading to decreases in oncogenic EIF4E-regulated cell growth and survival proteins. In ribavirin-sensitive KMT2A-R infant ALL cells and RS4:11 cells, EIF4E-regulated proteins with reduced levels of expression following ribavirin treatment include MYC, MCL1, NBN, BCL2 and BIRC5. Ribavirin-treated RS4:11 cells exhibit impaired EIF4E-dependent nuclear to cytoplasmic export and/or translation of the corresponding mRNAs, as well as reduced phosphorylation of the p-AKT1, p-EIF4EBP1, p-RPS6 and p-EIF4E signaling proteins. This leads to an S-phase cell cycle arrest in RS4:11 cells corresponding to the decreased proliferation. Ribavirin causes nuclear EIF4E to re-localize to the cytoplasm in KMT2A-AFF1 infant ALL and RS4:11 cells, providing further evidence for EIF4E inhibition. Ribavirin slows increases in peripheral blasts in KMT2A-R infant ALL xenograft-bearing mice. Ribavirin cooperates with chemotherapy, particularly L-asparaginase, in reducing live KMT2A-AFF1 infant ALL cells in BMSC co-cultures. This work establishes that EIF4E is broadly elevated across infant ALL and that clinically relevant ribavirin exposures have preclinical activity and effectively inhibit EIF4E in KMT2A-R cases, suggesting promise in EIF4E targeting using ribavirin as a means of treatment.
Assuntos
Fator de Iniciação 4E em Eucariotos/genética , Terapia de Alvo Molecular/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Ribavirina/uso terapêutico , Linhagem Celular Tumoral , Pré-Escolar , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Fator de Iniciação 4E em Eucariotos/fisiologia , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Indóis , Lactente , Análise em Microsséries , Família Multigênica/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Biossíntese de Proteínas/efeitos dos fármacos , Pirróis/uso terapêutico , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Infant acute lymphoblastic leukemia (ALL) has never occurred in families except for the â¼100% concordant cases in monozygous twins attributed to twin-to-twin metastases. We report the first kindred with infant ALL in non-twin siblings. The siblings were diagnosed with MLL-rearranged (MLL-R) ALL 26 months apart. The second affected sibling had an unaffected dichorionic monozygous co-twin. Both had fatal outcomes. PROCEDURES: Translocations were characterized by karyotype, FISH, multiplex FISH, and MLL breakpoint cluster region (bcr) Southern blot analysis. Breakpoint junctions and fusion transcripts were cloned by PCR. TP53 mutation and NADPH quinone oxidorecuctase 1 (NQO1) C609T analyses were performed, and pedigree history and parental occupations were ascertained. The likelihood of chance occurrence of infant ALL in non-twin siblings was computed based on a binomial distribution. Zygosity was determined by single nucleotide polymorphism (SNP) array. RESULTS: The translocations were not related or vertically transmitted. The complex karyotype of the proband's ALL had chromosome 2, 3, 4, and 11 abnormalities causing a 5'-MLL-AFF1-3' fusion and a non-productive rearrangement of 3'MLL with a chromosome 3q intergenic region. The affected twin's ALL exhibited a simple t(4;11). The complex karyotype of the proband's ALL suggested a genotoxic insult, but no exposure was identified. There was no germline TP53 mutation. The NQO1 C609T risk allele was absent. The likelihood of infant ALL occurring in non-twin siblings by chance alone is one in 1.198 × 10(9) families. CONCLUSIONS: Whether because of a deleterious transplacental exposure, novel predisposition syndrome, or exceedingly rare chance occurrence, MLL-R infant ALL can occur in non-twin siblings. The discordant occurrence of infant ALL in the monozygous twins was likely because they were dichorionic.
Assuntos
Cromossomos Humanos/genética , Rearranjo Gênico , Histona-Lisina N-Metiltransferase/genética , Proteína de Leucina Linfoide-Mieloide/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Irmãos , Translocação Genética , Gêmeos Dizigóticos , Humanos , Lactente , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologiaRESUMO
Survival in infants younger than 1 year who have acute lymphoblastic leukemia (ALL) is inferior whether MLL is rearranged (R) or germline (G). MLL translocations confer chemotherapy resistance, and infants experience excess complications. We characterized in vitro sensitivity to the pan-antiapoptotic BCL-2 family inhibitor obatoclax mesylate in diagnostic leukemia cells from 54 infants with ALL/bilineal acute leukemia because of the role of prosurvival BCL-2 proteins in resistance, their imbalanced expression in infant ALL, and evidence of obatoclax activity with a favorable toxicity profile in early adult leukemia trials. Overall, half maximal effective concentrations (EC50s) were lower than 176 nM (the maximal plasma concentration [Cmax] with recommended adult dose) in 76% of samples, whether in MLL-AF4, MLL-ENL, or other MLL-R or MLL-G subsets, and regardless of patients' poor prognostic features. However, MLL status and partner genes correlated with EC50. Combined approaches including flow cytometry, Western blot, obatoclax treatment with death pathway inhibition, microarray analyses, and/or electron microscopy indicated a unique killing mechanism involving apoptosis, necroptosis, and autophagy in MLL-AF4 ALL cell lines and primary MLL-R and MLL-G infant ALL cells. This in vitro obatoclax activity and its multiple killing mechanisms across molecular cytogenetic subsets provide a rationale to incorporate a similarly acting compound into combination strategies to combat infant ALL.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Pirróis/uso terapêutico , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Histona-Lisina N-Metiltransferase , Humanos , Indóis , Lactente , Recém-Nascido , Proteína de Leucina Linfoide-Mieloide/genética , Necrose/tratamento farmacológico , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidoresRESUMO
Gene expression profiling was performed on 97 cases of infant ALL from Children's Oncology Group Trial P9407. Statistical modeling of an outcome predictor revealed 3 genes highly predictive of event-free survival (EFS), beyond age and MLL status: FLT3, IRX2, and TACC2. Low FLT3 expression was found in a group of infants with excellent outcome (n = 11; 5-year EFS of 100%), whereas differential expression of IRX2 and TACC2 partitioned the remaining infants into 2 groups with significantly different survivals (5-year EFS of 16% vs 64%; P < .001). When infants with MLL-AFF1 were analyzed separately, a 7-gene classifier was developed that split them into 2 distinct groups with significantly different outcomes (5-year EFS of 20% vs 65%; P < .001). In this classifier, elevated expression of NEGR1 was associated with better EFS, whereas IRX2, EPS8, and TPD52 expression were correlated with worse outcome. This classifier also predicted EFS in an independent infant ALL cohort from the Interfant-99 trial. When evaluating expression profiles as a continuous variable relative to patient age, we further identified striking differences in profiles in infants less than or equal to 90 days of age and those more than 90 days of age. These age-related patterns suggest different mechanisms of leukemogenesis and may underlie the differential outcomes historically seen in these age groups.
Assuntos
Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Fatores Etários , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteínas de Transporte/genética , Análise por Conglomerados , Estudos de Coortes , Proteínas de Ligação a DNA/genética , Feminino , Redes Reguladoras de Genes , Proteínas de Homeodomínio/genética , Humanos , Lactente , Estimativa de Kaplan-Meier , Masculino , Modelos Genéticos , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas Nucleares/genética , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Prognóstico , Fatores de Transcrição/genética , Fatores de Elongação da Transcrição , Resultado do Tratamento , Proteínas Supressoras de Tumor/genética , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
Infant leukaemia is an embryonal disease in which the underlying MLL translocations initiate in utero. Zebrafish offer unique potential to understand how MLL impacts haematopoiesis from the earliest embryonic timepoints and how translocations cause leukaemia as an embryonal process. In this study, a zebrafish mll cDNA syntenic to human MLL spanning the 5' to 3' UTRs, was cloned from embryos, and mll expression was characterized over the zebrafish lifespan. The protein encoded by the 35-exon ORF exhibited 46·4% overall identity to human MLL and 68-100% conservation in functional domains (AT-hooks, SNL, CXXC, PHD, bromodomain, FYRN, taspase1 sites, FYRC, SET). Maternally supplied transcripts were detected at 0-2 hpf. Strong ubiquitous early zygotic expression progressed to a cephalo-caudal gradient during later embryogenesis. mll was expressed in the intermediate cell mass (ICM) where primitive erythrocytes are produced and in the kidney where definitive haematopoiesis occurs in adults. mll exhibits high cross species conservation, is developmentally regulated in haematopoietic and other tissues and is expressed from the earliest embryonic timepoints throughout the zebrafish lifespan. Haematopoietic tissue expression validates using zebrafish for MLL haematopoiesis and leukaemia models.
Assuntos
Sistema Hematopoético/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Peixe-Zebra/metabolismo , Envelhecimento/genética , Envelhecimento/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Biologia Computacional , DNA Complementar/genética , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese/fisiologia , Humanos , Dados de Sequência Molecular , Proteína de Leucina Linfoide-Mieloide/genética , Fases de Leitura Aberta , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Especificidade da Espécie , Peixe-Zebra/genéticaRESUMO
Translocations and other rearrangements of the MLL gene at chromosome band 11q23 are biologically and clinically important molecular abnormalities in infant acute leukemias, leukemias associated with chemotherapeutic topoisomerase II poisons and, less often, acute leukemias in adults or myelodysplastic syndrome. Depending on the disease and the regimen, MLL-rearranged leukemias may be associated with inferior prognosis, and MLL rearrangements with some of the more than 60 known MLL-partner genes confer especially adverse effects as response to treatment (Blood 108:441-451, 2006). MLL rearrangements are usually evident as overt balanced chromosomal translocations by conventional cytogenetic analysis but up to one-third are cryptic rearrangements and occur in leukemias with del(11)(q23), a normal karyotype, or trisomy 11, the latter two of which sometimes are associated with partial tandem duplications of MLL itself (Proc Natl Acad Sci USA 97:2814-2819, 2000; Proc Natl Acad Sci USA 94:3899-3902, 1997). In addition, subsets of MLL rearrangements are complex at a cytogenetic level and/or molecular level, and fuse MLL with two different partner genes. Rapid and accurate methods to identify and characterize genomic breakpoint junctions and fusion transcripts resulting from the many types of MLL rearrangements are essential for risk group stratification, treatment protocol assignments, new partner gene discovery, understanding leukemia etiology and pathogenesis, and elucidating the impact of less common MLL-partner genes on biology and prognosis. Due to the vast heterogeneity in partner genes, typical gene-specific PCR based methods are not practical, especially when cytogenetics are normal or do not suggest involvement of a known partner gene of MLL. We have advanced seven different panhandle PCR based methods for cloning 5'-MLL-partner gene-3' and 5'-partner gene-MLL-3' genomic breakpoint junctions and identifying 5'-MLL-partner gene-3' fusion transcripts, all of which employ a stem-loop template shaped schematically like a pan with a handle and amplify the template without knowledge of the unknown partner sequence using primers all derived from MLL alone.
Assuntos
Quebra Cromossômica , Clonagem Molecular/métodos , Leucemia/genética , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Fusão Oncogênica/genética , Reação em Cadeia da Polimerase/métodos , Processamento Alternativo/genética , Cromossomos Humanos Par 11/genética , Éxons/genética , Histona-Lisina N-Metiltransferase , HumanosRESUMO
Chemotherapy resistance from imbalanced apoptosis regulation may contribute to poor outcome in leukaemias with t(4;11). Anti-apoptotic BCL-2 expression and target modulation were characterized in cell lines with t(4;11) and BCL-2 expression was examined in MLL and non-MLL infant/paediatric leukaemia cases by Western blot analysis and/or real-time polymerase chain reaction. Cytotoxicity of Genasensetrade mark (Oblimersen Sodium, G3139) alone or combined with cytotoxic drugs was assessed by MTT [(3-4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assays of the cell lines, applying pharmacostatistical response surface modelling of drug interactions. Apoptosis and cell cycle were evaluated by flow cytometry in RS4:11 cells. Primary leukaemias and cell lines with t(4;11) expressed abundant BCL2 mRNA and protein. Variable, sometimes substantial BCL2 mRNA was detected in other leukaemia subtypes. G3139 reduced BCL2 mRNA and protein in RS4:11 cells. The most sensitive cell line to single-agent G3139 was RS4:11. Low G3139 concentrations sensitized RS4:11 and MV4-11 cells to select anti-leukaemia cytotoxic drugs. In RS4:11 cells, combining G3139 with doxorubicin (ADR) increased active caspase 3 and TUNEL staining compared to ADR alone, indicating greater apoptosis, and G3139 increased S-phase progression. The abundant BCL-2 affords a molecular target in leukaemias with t(4;11). G3139 exhibits preclinical activity and synergy with select cytotoxic agents in RS4:11 and MV4-11 cells, and these effects occur through apoptosis.
Assuntos
Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 4/genética , Leucemia Mieloide Aguda/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Translocação Genética , Antibióticos Antineoplásicos/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , Tionucleotídeos/farmacologia , Células Tumorais CultivadasRESUMO
We prospectively observed a child exposed to intensive multimodality therapy for metastatic neuroblastoma from emergence of a MLL translocation to disease diagnosis. The t(4;11)(p12;q23) was detected in the marrow 17 months after starting treatment following topoisomerase II poisons, alkylating agents, local radiation, hematopoietic stem cell transplantation, anti-GD2 monoclonal antibody with granulocyte macrophage-colony-stimulating factor, and a high cumulative dose of oral etoposide. Reciprocal genomic breakpoint junctions and fusion transcripts joined MLL with FRYL, the Drosophila melanogaster protein homologue of which regulates cell fate. Etoposide metabolites induced topoisomerase II cleavage complexes that could form both breakpoint junctions. Cells harboring the translocation replaced the marrow without clinical evidence of leukemia and differentiation appeared unaffected for 37 months. Subsequent bilineage dysplasia and increased blasts in addition to the translocation fulfilled criteria for MDS. The MEIS1 target gene of typical MLL fusion oncoproteins was underexpressed before and at MDS diagnosis. These results are consistent with repair of topoisomerase II cleavage from etoposide metabolites as the translocation mechanism, whereas other agents in the regimen may have contributed to progression of the clone with the translocation to MDS. MLL-FRYL did not increase MEIS1 expression, conferred a proliferative advantage without altering differentiation, and had protracted latency to disease.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/biossíntese , Síndromes Mielodisplásicas/metabolismo , Proteína de Leucina Linfoide-Mieloide/biossíntese , Proteínas de Neoplasias/biossíntese , Neuroblastoma/metabolismo , Proteínas de Fusão Oncogênica/biossíntese , Alquilantes/administração & dosagem , Alquilantes/efeitos adversos , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Medula Óssea/metabolismo , Medula Óssea/patologia , Pré-Escolar , Cromossomos Humanos Par 12/genética , Cromossomos Humanos Par 12/metabolismo , Cromossomos Humanos Par 4/genética , Cromossomos Humanos Par 4/metabolismo , Terapia Combinada/efeitos adversos , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Etoposídeo/administração & dosagem , Etoposídeo/efeitos adversos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Fator Estimulador de Colônias de Granulócitos e Macrófagos/efeitos adversos , Histona-Lisina N-Metiltransferase , Proteínas de Homeodomínio/genética , Humanos , Masculino , Síndromes Mielodisplásicas/diagnóstico , Síndromes Mielodisplásicas/etiologia , Síndromes Mielodisplásicas/patologia , Proteína Meis1 , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Neoplasias/genética , Neuroblastoma/complicações , Neuroblastoma/diagnóstico , Neuroblastoma/patologia , Neuroblastoma/terapia , Proteínas de Fusão Oncogênica/genética , Transplante de Células-Tronco de Sangue Periférico , Estudos Prospectivos , Homologia de Sequência de Aminoácidos , Inibidores da Topoisomerase II , Translocação Genética/genética , Transplante AutólogoRESUMO
Radiation sensitization by 2',2'-difluoro-2'-deoxycytidine (dFdCyd) has correlated with dATP depletion [dFdCDP-mediated inhibition of ribonucleotide reductase (RR)] and S-phase accumulation. We hypothesized that radiosensitization by dFdCyd is due to nucleotide misincorporations in the presence of deoxynucleotide triphosphate pool imbalances, which, if not repaired, augments cell death following irradiation. The ability of dFdCyd to produce misincorporations was measured as pSP189 plasmid mutations in hMLH1-deficient [mismatch repair (MMR) deficient] and hMLH1-expressing (MMR proficient) HCT116 cells. Only MMR-deficient cells showed a significant increase in nucleotide misincorporations (2- to 3-fold increase; P
Assuntos
Pareamento Incorreto de Bases , Desoxicitidina/análogos & derivados , Radiossensibilizantes/farmacologia , Linhagem Celular Tumoral , Desoxicitidina/farmacologia , Humanos , Mutação , RNA Interferente Pequeno , GencitabinaRESUMO
Panhandle PCR techniques to amplify known sequence flanked by unknown sequence have been useful for MLL genomic breakpoint junctions and fusion transcripts because MLL has a large number of partner genes. However, genomic panhandle PCR approaches are impeded when the restriction fragment that contains the breakpoint junction is too large to amplify. We devised new panhandle PCR approaches for MLL genomic breakpoint junctions that create the template from BglII restriction fragments by attaching MLL sequence to a BglII site in the partner gene. This leads to the annealing of MLL and its complement in the handle and creates an intrastrand loop containing the breakpoint junction sequence for amplification with primers all from MLL. BglII panhandle PCR for der(11) breakpoint junctions was accomplished by ligating a phosphorylated oligonucleotide containing a BglII overhang and sequence complementary to MLL exon 7 to the 3' ends of BglII digested DNA, and forming the template from the sense strand of DNA. In BglII reverse panhandle PCR for der(other) breakpoint junctions, a phosphorylated oligonucleotide containing a BglII overhang and the complement of antisense sequence in MLL exon 10 was ligated to the 3' ends of BglII digested DNA, and the template was formed from the antisense strand of DNA. These approaches amplified 5'-MLL-MLLT4-3' and 5'-AFF1-MLL-3' breakpoint junctions. The former is significant because few t(6;11) genomic breakpoint junctions have been sequenced. BglII panhandle PCR approaches increase the possibilities for cloning MLL genomic breakpoint junctions where there is heterogeneity in partner genes and breakpoint locations.
Assuntos
Proteínas de Bactérias , Quebra Cromossômica , Cromossomos Humanos Par 11 , Clonagem Molecular/métodos , Desoxirribonucleases de Sítio Específico do Tipo II , Proteína de Leucina Linfoide-Mieloide/genética , Reação em Cadeia da Polimerase/métodos , Translocação Genética , Adolescente , Sequência de Bases , Cromossomos Humanos Par 4 , Cromossomos Humanos Par 6 , Humanos , Modelos Biológicos , Modelos Genéticos , Dados de Sequência Molecular , Proteína de Leucina Linfoide-Mieloide/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismoRESUMO
Because treatment regimens for breast cancer commonly include gemcitabine, we evaluated two promising combinations in preclinical studies: gemcitabine (Gemzar; Eli Lilly and Company, Indianapolis, IN) with either ionizing radiation or docetaxel (Taxotere; Aventis Pharmaceuticals, Inc, Parsippany, NJ). In breast cancer cell lines that expressed either wild-type p53 (MCF-7) or mutant p53 (MCF-7/Adr), sensitivity to the cytotoxic effects of gemcitabine during a 24-hour incubation was similar (IC(50) values 80 and 60 nmol/L in MCF-7 and MCF-7/Adr, respectively). Both cell lines were well radiosensitized by gemcitabine at the corresponding IC(50), with radiation enhancement ratios of 1.6 to 1.7. Although the MCF-7 cells accumulated nearly twice as much gemcitabine triphosphate compared with the MCF-7/Adr cells, a similar reduction in 2'-deoxyadenosine 5'-triphosphate pools was observed. While the number of dying cells, as measured by sub-G1 DNA content or S-phase cells unable to replicate DNA, differed between the wild-type p53 or mutant p53-expressing cell lines, neither parameter correlated with radiosensitization. Docetaxel was a more potent cytotoxic agent than gemcitabine in MCF-7 cells (IC(50) = 1 nmol/L). Strong synergistic cytotoxicity was observed in cells treated with gemcitabine (24 hours) followed by docetaxel (24 hours) or the reverse sequence. However, simultaneous addition of the two drugs was antagonistic. To determine whether synergy with radiation or docetaxel was mediated by increased DNA damage, DNA double-strand breaks (double-strand breaks) were measured by immunostaining for phosphorylated H2AX. Ionizing radiation produced more double-strand breaks than gemcitabine alone, while no significant double-strand breaks formed with docetaxel alone. The addition of docetaxel or ionizing radiation to gemcitabine-treated cells did not increase H2AX foci formation. These results show that the combination of gemcitabine with ionizing radiation or docetaxel produces strong, schedule-dependent synergy in breast cancer cells that is not mediated through increasing DNA double-strand breaks.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/tratamento farmacológico , Desoxicitidina/análogos & derivados , Desoxicitidina/administração & dosagem , Neoplasias da Mama/radioterapia , Linhagem Celular Tumoral , DNA de Neoplasias/efeitos dos fármacos , DNA de Neoplasias/efeitos da radiação , Docetaxel , Sinergismo Farmacológico , Histonas , Humanos , Radiossensibilizantes/administração & dosagem , Radioterapia Adjuvante , Taxoides/administração & dosagem , Ensaio Tumoral de Célula-Tronco , GencitabinaRESUMO
Gemcitabine [2',2'-difluoro-2'-deoxycytidine (dFdCyd)] is a potent ionizing radiation sensitizer in solid tumor cells in vitro and in vivo. Previously, we have demonstrated (Shewach et al., Cancer Res., 54: 3218-3223, 1994) a strong correlation between depletion of dATP (caused by dFdCyd diphosphate-mediated inhibition of ribonucleotide reductase) and radiosensitization. In addition, we and others (Latz et al., Int. J. Radiat. Oncol. Biol. Phys., 41: 875-882, 1998; Ostruszka and Shewach, Cancer Res., 60: 6080-6088, 2000) have shown that the accumulation of cells in S phase prior to irradiation is also important for radiosensitization with dFdCyd. This led us to hypothesize that the incorporation of incorrect nucleotides because of the dATP pool imbalance was important for radiosensitization with dFdCyd, and, therefore, cells deficient in mismatch repair (MMR) would exhibit greater radiosensitization. We tested this hypothesis by evaluating the ability of HCT116 colon carcinoma cell lines, which differ in MMR proficiency, to be radiosensitized by dFdCyd. The MMR-proficient cell line (HCT116 + ch3) was more sensitive to dFdCyd alone than were the MMR-deficient cell lines (HCT116, HCT116 + ch2, and HCT116 p53(-/-)). Interestingly, the MMR-proficient cells could not be radiosensitized at concentrations of dFdCyd
Assuntos
Pareamento Incorreto de Bases/fisiologia , Reparo do DNA/fisiologia , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/farmacologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , DNA de Neoplasias/metabolismo , Nucleotídeos de Desoxiadenina/metabolismo , Células HCT116 , Humanos , Nucleotídeos/metabolismo , Tolerância a Radiação/genética , GencitabinaRESUMO
Ganciclovir (GCV) is widely used as a prodrug for selective activation in tumor cells expressing herpes simplex virus thymidine kinase (HSV-TK) because of its ability to induce multi-log cytotoxicity to HSV-TK-expressing as well as nonexpressing bystander cells. We now report that another substrate for HSV-TK, D-carbocyclic 2'-deoxyguanosine (CdG), induces multi-log cytotoxicity in HSV-TK-expressing and bystander cells at concentrations Assuntos
Antivirais/farmacologia
, Desoxiguanosina/análogos & derivados
, Desoxiguanosina/farmacologia
, Ganciclovir/farmacologia
, Pró-Fármacos/farmacologia
, Timidina Quinase/biossíntese
, Proteínas Virais/biossíntese
, Antivirais/metabolismo
, Antivirais/uso terapêutico
, Morte Celular/efeitos dos fármacos
, Morte Celular/genética
, Neoplasias do Colo/tratamento farmacológico
, Neoplasias do Colo/genética
, Neoplasias do Colo/virologia
, Desoxiguanosina/metabolismo
, Desoxiguanosina/uso terapêutico
, Ativação Enzimática/efeitos dos fármacos
, Ganciclovir/metabolismo
, Ganciclovir/uso terapêutico
, Técnicas de Transferência de Genes
, Glioblastoma/tratamento farmacológico
, Glioblastoma/genética
, Glioblastoma/virologia
, Pró-Fármacos/metabolismo
, Pró-Fármacos/uso terapêutico
, Simplexvirus/enzimologia
, Timidina Quinase/genética
, Células Tumorais Cultivadas