RESUMO
Human leukemia cell lines are of great value in investigating basic and applied aspects of cell biology and clinical medicine. There have been 37 leukemia cell lines carrying 11q23 translocation and MLL rearrangements; however, cell lines harboring with t(1;11)(p32;q23) have not been established. We report here for the first time a new acute monocytic leukemia (AMoL) cell line with t(1;11)(p32;q23), designated TZ-1, and herein describe its biological characteristics. Mononuclear cells isolated from the ascites from a patient with AMoL (French-American-British classification; acute myeloid leukemia M5a) were isolated and passaged by liquid culture medium for a year. TZ-1 cells revealed typical monocytic features in morphology and had a t(1;11)(p32;q23) translocation. The immunoprofiling as determined by flow cytometry showed that TZ-1 cells are positive for myeloid and monocytic markers with lymphoid-associated markers. Fluorescence in situ hybridization and reverse transcription-polymerase chain reaction analyses revealed MLL-EPS15 fusion transcript and protein. Taken together, these results suggest that TZ-1 is a new monocytic leukemia cell line with t(1;11) translocation and fusion gene MLL-EPS15. The established cell line, TZ-1, could provide a valuable model in the analysis of the pathogenesis of MLL-EPS15-positive leukemia and in the development of new agents for this type of leukemia.
Assuntos
Cromossomos Humanos Par 11 , Cromossomos Humanos Par 1 , Leucemia Monocítica Aguda/patologia , Translocação Genética , Idoso , Linhagem Celular Tumoral , Humanos , Imunofenotipagem , Hibridização in Situ Fluorescente , Cariotipagem , Leucemia Monocítica Aguda/genética , Leucemia Monocítica Aguda/imunologia , Masculino , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Human cervical cancer is caused by high-risk types of human papillomavirus (HPV) such as HPV16 and HPV18, which possess the E6 and E7 oncogenes, whose concurrent expression is a prerequisite for cancer development and maintaining malignant phenotypes. Silencing these oncogenes is considered to be applicable in molecular therapies of human cervical cancer. However, it remains to be determined whether E6, E7, or both should be silenced to obtain most efficient antitumor activity by an HPV small-interfering RNA (siRNA). Herein, we report two types of siRNAs targeting HPV18 E6, that exerted a negative growth effect on HPV18-positive cervical cancer cells (HeLa and SW756), in part, inducing cell death. One siRNA (Ex-18E6), designed to target both E6-E7 mRNA and its splicing variant, E6*I-E7 mRNA, efficiently knocked down both E6 and E7 expression. The other (Sp-18E6), designed to specifically target E6-E7 mRNA but not E6*I-E7 mRNA, suppressed E6 to a similar level as Ex-18E6; however, it less efficiently inhibited E7 as compared to Ex-18E6. Although both siRNAs induced cell death, Sp-18E6 siRNA induced more prominent cell death than Ex-18E6. Our results suggest that E6-specific suppression may induce more potent anticancer activity than simultaneous E6 and E7 suppression, and that E6-specific targeting is a promising strategy for siRNA-based therapy for HPV-positive cervical cancer.
Assuntos
Apoptose , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Inativação Gênica , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/fisiologia , RNA Interferente Pequeno/farmacologia , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia , Animais , Células COS , Chlorocebus aethiops , Feminino , Regulação Viral da Expressão Gênica , Células HeLa , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Activin A, one member of the transforming growth factor (TGF)-beta superfamily, is known to be a commitment factor for cell death and differentiation. In the present study, we demonstrate that human chronic myeloid leukemia (CML) cell lines, KU812 and K562 cells, either induced apoptosis or differentiation, respectively, by treatment with activin A. During these cell fate decisive events caused by activin A, rapid and transient up-regulation of Mcl-1 was observed in both cell lines. In activin A-induced apoptosis of KU812 cells, continuous up-regulation of Bax was observed. After the decrease in Mcl-1 expression had occurred, activation of caspase-9 and caspase-3 and cleavage of DFF45 were shown to take place in KU812 cells, resulting in the fragmentation of the genomic DNA of the cells. In contrast, the down-regulation of Mcl-1 without up-regulation of Bax caused accumulation of hemoglobin (Hb) contents in activin A-treated K562 cells. Interestingly, erythropoietin (EPO) prevented activin A-induced apoptosis with continuous expression of Mcl-1 and caused KU812 cells to undergo erythroid differentiation. To address the role of Mcl-1 in activin A-treated CML cells, KU812 and K562 cells were stably transfected with cDNA encoding Mcl-1 (designated as KU812/mcl and K562/mcl cells). As in combined effect of activin A and EPO on the parental KU812 cells, activin A induced differentiation, but not apoptosis, of KU812/mcl cells without modulating Bax levels. Activin A-treated K562/mcl cells, as well as parental cells, were only differentiated to erythroid cells. These results suggest that Mcl-1 is an early inducible gene activated by the activin A signaling pathway for both cellular differentiation and apoptosis, and continuous expression of Mcl-1 may be contributed to differentiation signals to the erythroid lineage in CML cells.
Assuntos
Apoptose , Inibinas/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Ativinas , Inibidores de Caspase , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Interações Medicamentosas , Eritropoetina/farmacologia , Proteína de Sequência 1 de Leucemia de Células MieloidesRESUMO
BMPs exert a negative growth effect on various types of cells. We have previously reported that BMP-2 inhibited the growth of HS-72 mouse hybridoma cells by inducing p21(CIP1/WAF1) expression. In the present study, we demonstrated that BMP-2 activated the mouse p21(CIP1/WAF1) promoter in HS-72 cells, and that a 29-base pair (b) region of the promoter (-1928/-1900 relative to the TATA box), conserved between mice and humans, was responsive to BMP-2 as well as expression of Smad1, Smad4, and constitutively active mutants of BMP type I receptors. Furthermore, an oligonucleotide containing the 29-b region was found to be associated with Smad4 and phosphorylated Smad1 in the nuclear extract of BMP-2-stimulated HS-72 cells. These results suggested that BMP-2 might activate p21(CIP1/WAF1) transcription by inducing a binding of Smad4 and Smad1 to the 29-b region in HS-72 cells.
Assuntos
Linfócitos B/metabolismo , Proteínas Morfogenéticas Ósseas/farmacologia , Ciclinas/genética , Regiões Promotoras Genéticas , Receptores de Fatores de Crescimento , Proteínas Repressoras , Fator de Crescimento Transformador beta , Animais , Proteína Morfogenética Óssea 2 , Receptores de Proteínas Morfogenéticas Ósseas , Células COS , Linhagem da Célula , Inibidor de Quinase Dependente de Ciclina p21 , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Hibridomas , Camundongos , Proteínas Oncogênicas Virais/farmacologia , Receptores de Superfície Celular/metabolismo , Elementos de Resposta , Proteínas Smad , Proteína Smad1 , Proteína Smad4 , Células-Tronco/metabolismo , Transativadores/genética , Transativadores/metabolismo , TransfecçãoRESUMO
We have investigated the effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and several potent vitamin D3 analogs [1,25(OH)2-16-ene-23-yne-D3; 1,25(OH)2-16-ene-23-yne-26,27-F6-D3] on productive infection by human immunodeficiency virus (HIV) in human macrophages. Macrophages derived from the peripheral blood were either pretreated with the vitamin D3 analogs, washed, and exposed to HIV (pre-infection treatment) or were infected with HIV, washed, and cultured with the vitamin D3 compounds (post-infection treatment). After three days of HIV-infection, levels of p24 antigen were measured. Pretreatment of macrophages with either 1,25(OH)2D3 or 1,25(OH)2-16-ene-23-yne-26,27-F6-D3 (pre-infection treatment) increased productive HIV infection about 3.5-fold; 1,25(OH)2-16-ene-23-yne-D3 increased levels about 4.7-fold. In contrast, exposure of HIV infected macrophages to the vitamin D3 compounds (post-infection treatment) did not affect levels of HIV production compared to untreated controls. Soluble CD4 completely inhibited productive HIV infection of macrophages pretreated with vitamin D3 analogs. Also, the vitamin D3 compounds slightly decreased CD4 expression on macrophages. The mechanism of enhanced productive HIV infection by the vitamin D3 compounds is unclear, but can not be explained by either alteration of CD4 expression or entry into cells by a CD4-independent route. These studies may have implications for both the basic biology of HIV infectious production and possibly clinical treatment of AIDS patients.
Assuntos
Calcitriol/farmacologia , Infecções por HIV/tratamento farmacológico , Macrófagos/microbiologia , Monócitos/microbiologia , Antígenos CD/análise , Antígenos de Diferenciação Mielomonocítica/análise , Antígenos CD4/análise , Antígenos CD4/farmacologia , Calcitriol/análogos & derivados , Citometria de Fluxo , HIV/fisiologia , Humanos , Receptores de Lipopolissacarídeos , Macrófagos/imunologia , Monócitos/imunologia , Solubilidade , Replicação Viral/efeitos dos fármacosRESUMO
Geranylgeraniol, a polyprenylalcohol composing the side chain of vitamin K2 (VK2), was previously reported to be a potent inducer of apoptosis in tumor cell lines (Ohzumi H et al, J Biochem 1995; 117: 11-13). We examined the apoptosis-inducing ability of VK2 (menaquinone 3 (MK3), MK4 and MK5) and its derivatives such as phytonadione (VK1), as well as polyprenylalcohols with side chains of various lengths including farnesol (C15-OH; FO), geranylgeraniol (C20-OH; GGO), and geranylfarnesol (C25-OH; GFO) toward leukemia cells in vitro. MK3, MK4, MK5 and GFO (at 10 microM) showed a potent apoptosis-inducing activity for all freshly isolated leukemia cells tested and for leukemia cell lines such as NB4, an acute promyelocytic leukemia (APL)-derived cell line and MDS92, a cell line derived from a patient with myelodysplastic syndrome, although there were some differences depending on the cells tested. In contrast, VK1 showed no effect on any of the leukemia cells. The combination of MK5 plus all-trans retinoic acid (ATRA) resulted in enhanced induction of apoptosis in both freshly isolated APL cells and NB4 cells as compared to each reagent alone. These data suggest the possibility of using VK2 and its derivatives for the treatment of myelogenous leukemias, including APL.
Assuntos
Apoptose/efeitos dos fármacos , Leucemia/patologia , Tretinoína/farmacologia , Vitamina K/análogos & derivados , Vitamina K/farmacologia , Medula Óssea/patologia , Diterpenos/farmacologia , Sinergismo Farmacológico , Farneseno Álcool/farmacologia , Citometria de Fluxo , Gefarnato/análogos & derivados , Gefarnato/farmacologia , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva , Leucemia Mieloide , Leucemia Promielocítica Aguda , Estrutura Molecular , Síndromes Mielodisplásicas , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Vitamina K 1/farmacologia , Vitamina K 2/análogos & derivadosRESUMO
Arsenic trioxide (As2O3) effectively induces clinical remission via apoptosis in relapsed acute promyelocytic leukemia (APL). However, because this new anti-leukemic drug is also considered to be a poison, its possible adverse effects are a highly important issue related to its clinical use. We here investigated, both in vitro and in vivo, the effects of a combination of As2O3 and GM-CSF as a novel therapeutic approach for the treatment of APL. Treatment of both retinoic acid (RA)-sensitive and -resistant APL cell lines (NB4 and UF-1 cells, respectively), as well as primary APL cells with a combination of As2O3 and GM-CSF for 4 days resulted in inducing differentiation, but not apoptosis, to mature granulocytes. In addition, a combination of both agents induced degradation of the PML/RARalpha protein. GM-CSF was found to be associated with increased tyrosine phosphorylation of Jak2 kinase in both NB4 and UF-1 cells, and a specific inhibitor of Jak2, AG490, completely blocked the ability of GM-CSF to prevent apoptosis and induce differentiation of As2O3-treated UF-1 cells. In in vivo analysis, As2O3 induced differentiation of APL cells in a RA-resistant APL model of human GM-CSF-producing transgenic SCID mice that had a high level of human GM-CSF in their sera. In contrast, As2O3 alone diminished tumors in UF-1 cells transplanted into NOD/SCID mice via induction of apoptosis. In conclusion, a combination of As2O3 and GM-CSF appears to be a novel differentiation-inducing therapy in patients with APL, including relapsed or RA-resistant cases.
Assuntos
Antineoplásicos/farmacologia , Arsenicais/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Leucemia Promielocítica Aguda/tratamento farmacológico , Óxidos/farmacologia , Animais , Antineoplásicos/uso terapêutico , Trióxido de Arsênio , Arsenicais/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Quimioterapia Combinada , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Humanos , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patologia , Camundongos , Camundongos SCID , Proteínas de Neoplasias/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Óxidos/uso terapêutico , Células Tumorais CultivadasRESUMO
Recent clinical studies in China and USA showed that arsenic trioxide (As2O3) is an effective treatment of acute promyelocytic leukemia (APL) patients refractory to all-trans retinoic acid (RA). We here investigate the effects of As2O3 on RA-resistant APL in vivo and in vitro using our RA-resistant APL model system. As2O3 can induce inhibition of cellular growth of both RA-sensitive NB4 and RA-resistant UF-1 APL cells via induction of apoptosis in vitro. The expression of BCL-2 protein decreased in a dose- and time-dependent manner in NB4 cells. Interestingly, the levels of BCL-2 protein were not modulated by As2O3, but it did upregulate BAX protein in UF-1 cells. UF-1 cells (1x10(7)) were transplanted into hGM-CSF-producing transgenic SCID mice and successfully formed subcutaneous tumors. After 40 days of implantation, mice were treated with As2O3, all-trans RA and PBS for 21 days. In all-trans RA- and PBS-treated mice, tumors grew rapidly, with a 4.5-fold increase in volume at day 21 compared to the initial size. In marked contrast, tumor size was decreased to half of the initial size by the treatment of As2O3, which resulted in cells with the typical appearance of apoptosis. Interestingly, one of the As2O3-treated mice showed mature granulocytes in the diminished tumor, suggesting that As2O3 had dual effects on RA-resistant APL cells in vivo: both inducing apoptosis and differentiation of the leukemic cells. We conclude that our RA-resistant APL model will be useful for evaluating novel therapeutic approaches to patients with RA-resistant APL, and for further investigation of the metabolism of As2O3 in vivo.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Arsenicais/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Leucemia Promielocítica Aguda/patologia , Óxidos/farmacologia , Tretinoína/farmacologia , Animais , Antineoplásicos/uso terapêutico , Trióxido de Arsênio , Arsenicais/uso terapêutico , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Humanos , Camundongos , Camundongos SCID , Camundongos Transgênicos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Óxidos/uso terapêutico , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/fisiologia , Transplante Heterólogo , Tretinoína/uso terapêutico , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/patologia , Proteína X Associada a bcl-2 , Proteína de Morte Celular Associada a bcl , Proteína bcl-XRESUMO
Activins, members of the transforming growth factor-beta family, have been implicated in the regulation of growth and differentiation of various types of cells. We have recently found that activin A induces apoptotic cell death of plasmacytic cells including B cell hybridoma cells and myeloma cells. In the present study, we demonstrated that activin A caused cell-cycle arrest in the G1 phase before appearance of apoptotic cells in mouse B cell hybridoma cells. Phosphorylation of retinoblastoma protein (Rb) and in vitro Rb kinase activity of cyclin-dependent kinase (CDK)4 was inhibited in activin A-treated cells. Analysis of expression of genes regulating Rb phosphorylation revealed that activin A suppressed cyclin D2, the sole D-type cyclin gene expressed in the hybridoma cells, and activated p21CIP1/WAF1 but had no effect on expression of cyclin-dependent kinases (CDK2, CDK4, CDK6) and other CDK inhibitors (p27KIP1, p16INK4a, p15INK4b). Modulation of cyclin D2 and p21CIP1/WAF1 expression resulted in a decrease in level of cyclin D2-CDK4 complex and an increase in level of CDK4 complexed with p21CIP1/WAF1. Moreover, overexpression of cyclin D2 partially abrogated inhibition of Rb phosphorylation and G1 arrest in the hybridoma cells.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ciclo Celular , Ciclo Celular/efeitos dos fármacos , Ciclinas/efeitos dos fármacos , Inibinas/farmacologia , Plasmócitos/efeitos dos fármacos , Proteínas Proto-Oncogênicas , Proteínas Supressoras de Tumor , Ativinas , Animais , Apoptose/efeitos dos fármacos , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Divisão Celular/efeitos dos fármacos , Ciclina D2 , Quinase 2 Dependente de Ciclina , Quinase 4 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p15 , Inibidor p16 de Quinase Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Quinases Ciclina-Dependentes/efeitos dos fármacos , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Fase G1 , Hibridomas/efeitos dos fármacos , Camundongos , Fosforilação , Plasmócitos/patologia , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Proteína do Retinoblastoma/efeitos dos fármacos , Proteína do Retinoblastoma/metabolismoRESUMO
OBJECTIVE: All-trans retinoic acid (RA) resistance in acute promyelocytic leukemia (APL) has been a serious clinical problem in differentiation-inducing therapy. However, the mechanisms underlying acquired RA resistance in APL patients are not well understood. MATERIALS AND METHODS: We recently established a spontaneous RA-resistant APL cell line (UF-1) from a patient and used this cell line as an excellent in vitro model for RA-resistant clinical situations. We investigated the structural and functional abnormalities of chimeric PML/RARalpha gene in UF-1 cells and preserved materials from the original patient. RESULTS: A novel point mutation was detected in the ligand-binding (E) domain of the RARalpha portion of the PML/RARalpha gene in UF-1 cells. This mutation resulted in amino acid substitution of Arg611 (CGG) for Trp611 (TGG) in the short-form PML/RARalpha protein, which corresponded to Arg276 in wild-type RARalpha. Importantly, the same mutation was also detected in the preserved materials from the original patient. COS-1 cells were transiently transfected with cDNA encoding wild-type and mutant PML/RARalpha constructed by site-directed mutagenesis and performed RA-binding assay. Interestingly, RA-binding activity was dramatically decreased in the mutant PML/RARalpha compared with that of the wild-type chimeric protein, suggesting that this single amino acid substitution is critical for RA binding. CONCLUSIONS: These results strongly suggest that a novel point mutation in the ligand-binding domain of the RARalpha portion (Arg611) of the chimeric PML/RARalpha gene decreased sensitivity to all-trans RA. We conclude that acquisition of the PML/RARalpha mutation is one possible mechanism for development of RA resistance in patients with APL in vivo.
Assuntos
Leucemia Promielocítica Aguda/genética , Mutação , Proteínas de Neoplasias/genética , Proteínas de Fusão Oncogênica/genética , Sequência de Aminoácidos , Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Leucemia Promielocítica Aguda/tratamento farmacológico , Ligantes , Dados de Sequência Molecular , Proteínas de Neoplasias/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Ligação Proteica , Tretinoína/metabolismo , Tretinoína/uso terapêutico , Células Tumorais CultivadasRESUMO
Activin A, a member of the transforming growth factor-beta (TGF-beta) family, is produced by a variety of cells and implicated in the regulation of the reproductive endocrine system, mesoderm induction, and erythropoiesis. In the present study, we showed that activin A inhibited the production of interleukin-1beta (IL-1beta), a potent proinflammatory cytokine, and enhanced the production of IL-1 receptor antagonist (IL-1ra), in activated THP-1 and U-937 human monocytic cells, resulting in the reduction of IL-1 biologic activity. Northern blot analysis revealed that activin A had no effect on mRNA accumulation of IL-1beta and IL-1ra, indicating that activin A regulates IL-1beta and IL-1ra production at a posttranscriptional level. As it is well known that an inactive precursor form of IL-1beta (pro-IL-1beta) is converted to an active mature form (mature IL-1beta), we examined the expression levels of pro-IL-1beta and mature IL-1beta by immunoblot analysis. Although activin A inhibited the production of mature IL-1beta in activated U-937 cells, the relative protein expression of pro-IL-1beta was unaltered by activin A, suggesting that activin A inhibits IL-1beta production by blocking proteolytic conversion of pro-IL-1beta into mature IL-1beta. Taken together, these findings suggest that activin A may function as an anti-inflammatory cytokine by modulating mature IL-1beta and IL-1ra production in inflammatory sites.
Assuntos
Substâncias de Crescimento/farmacologia , Inibinas/farmacologia , Interleucina-1/biossíntese , Monócitos/efeitos dos fármacos , Receptores de Interleucina-1/antagonistas & inibidores , Sialoglicoproteínas/biossíntese , Ativinas , Linhagem Celular , Humanos , Proteína Antagonista do Receptor de Interleucina 1 , Monócitos/metabolismo , RNA Mensageiro/biossíntese , Sialoglicoproteínas/metabolismo , Células U937RESUMO
Hemostatic molecular markers were serially monitored in a prospective fashion during remission induction therapy with all-trans retinoic acid (ATRA) in sixteen patients with acute promyelocytic leukemia (APL). One patient with leukocytosis before treatment and three patients who later developed hyperleukocytosis also received chemotherapy with behenoyl Ara-C and daunorubicin. Plasma levels of E-fragment of fibrin and fibrinogen degradation product (FDP-E), FDP-D dimer (D-D), thrombin-antithrombin complex (TAT), and plasmin-alpha 2 plasmin inhibitor complex (PIC) were markedly elevated in all but one patient before treatment, and these parameters decreased to normal or near normal ranges in most patients within the first 7 days of treatment. Interestingly, we have found that these parameters were again elevated during the later course of ATRA therapy (after day +7) in eleven patients for various reasons including cytotoxic chemotherapy (3 cases), fever (5 cases; 2 cases with apparent infection, 3 cases without known etiology), Caesarean section (1 case), and no apparent etiology (2 cases). Three patients showed bleeding complications during re-elevation of molecular markers, but none developed thrombosis. Plasma elastase-alpha 1 proteinase inhibitor complex (E-alpha 1 PI) was markedly elevated in all patients at diagnosis and did not decrease significantly during ATRA therapy. Plasma tissue factor antigen was mildly elevated in one out of four patients studied, and thrombomodulin was elevated in two out of ten patients tested. These results confirmed the rapid normalization of coagulopathy during the early phase of remission induction therapy with ATRA but suggest that re-elevation of molecular markers occurs frequently during the later course of ATRA therapy.
Assuntos
Antineoplásicos/uso terapêutico , Coagulação Sanguínea/efeitos dos fármacos , Fibrinólise/efeitos dos fármacos , Hemostasia/efeitos dos fármacos , Leucemia Promielocítica Aguda/tratamento farmacológico , Tretinoína/uso terapêutico , Adolescente , Adulto , Idoso , Biomarcadores/sangue , Criança , Feminino , Seguimentos , Humanos , Leucemia Promielocítica Aguda/sangue , Leucócitos/enzimologia , Masculino , Pessoa de Meia-Idade , Elastase Pancreática/sangue , Estudos Prospectivos , Indução de Remissão/métodos , Trombomodulina/metabolismo , Tromboplastina/metabolismoRESUMO
Retinoic acid (RA) has profound effects upon the proliferation and differentiation of many hematopoietic cells. The mechanism by which RA acts is unclear. Recently, several retinoic acid receptors (RAR) have been cloned. We studied expression of RAR-alpha mRNA by RNA blots in hematopoietic cells blocked at different stages of differentiation. All hematopoietic cells expressed RAR-alpha mRNA (3.4, 4.5 kb) including KG-1 (myeloblasts); HL-60 (promyelocytes); ML3, THP-1, U937 (myelomonoblasts and monoblasts); K562 (erythroblasts); and S-LB1 (T-lymphocytes). In addition, transformed cells from four non-hematopoietic tissues also expressed RAR-alpha mRNA. Steady-state levels of RAR-alpha mRNA were not affected by induction of terminal differentiation of HL-60 cells to either granulocytes or macrophages. Furthermore, both actively proliferating and resting lymphocytes from the same individuals expressed equal concentrations of RAR-alpha mRNA. Taken together, data suggest that level of expression of RAR-alpha mRNA is not related to cellular proliferation. We also showed that exposure to ligand (all-trans retinoic acid) did not change levels of RAR-alpha mRNA in three different cell types. Half-life of RAR-alpha mRNA was short (0.7 h) as determined by measuring decay of message after addition of actinomycin D. Consistent with this finding, accumulation of RAR-alpha mRNA increased in cells of three lines as their protein synthesis was inhibited. In summary, hematopoietic cells of different lineages and stages of differentiation constitutively express RAR-alpha mRNA. This expression is unaffected either by terminal differentiation or cell cycle. The RAR-alpha mRNA is short-lived and super-inducible by a protein synthesis inhibitor.
Assuntos
Proteínas de Transporte/genética , Células-Tronco Hematopoéticas/análise , RNA Mensageiro/análise , Diferenciação Celular , Linhagem Celular , Cicloeximida/farmacologia , Meia-Vida , Humanos , Leucemia/metabolismo , Linfócitos/análise , Receptores do Ácido RetinoicoRESUMO
Retinoic acid (RA) and 1,25-dihydroxyvitamin D3 (D3) are well known for inducing differentiation in many leukemic cell lines. The nuclear signalling pathways of RA and D3 are mediated through their cognate receptors, the retinoic acid receptor (RAR) and vitamin D3 receptor (VDR), respectively. Retinoid X receptor (RXR) is an auxiliary factor that forms a heterodimer with RAR and VDR, enabling their efficient transcriptional activation. 9-cis RA, a high-affinity ligand for RXR, greatly enhanced D3-induced CD14 expression in U937 cells, while RA alone did not induce CD14 expression. 9-cis RA also resulted in morphological changes of U937 cells to macrophage-like cells when combined with D3, while RA alone resulted in granulocyte-like cells. RA and D3 together enhanced c-fms expression, phagocytic activity, and acted synergistically to promote nitroblue tetrazolium reduction activity and inhibit proliferation. Northern analysis showed that U937 cells constitutively expressed RAR-alpha, VDR and RXR-alpha mRNAs. RA or D3 alone or in combination did not affect RAR-alpha and VDR expression, while 9-cis RA and 9-cis RA plus all-trans RA significantly reduced RXR-alpha expression. Interestingly, D3 could restore the down-regulation of RXR-alpha mRNA by 9-cis RA. These findings suggest that there is crossover of the nuclear signalling pathways of RA and D3. This may have clinical implications in that RA and D3 may be used in combination for differentiation-inducing therapy in acute myelogenous leukemia and myelodysplastic syndrome.
Assuntos
Antineoplásicos/farmacologia , Calcitriol/farmacologia , Diferenciação Celular/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Tretinoína/farmacologia , Linhagem Celular , Granulócitos/citologia , Humanos , Receptores de Lipopolissacarídeos/efeitos dos fármacos , Receptores de Lipopolissacarídeos/metabolismo , Monócitos/citologia , RNA Mensageiro/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/efeitos dos fármacos , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Estereoisomerismo , Tretinoína/análogos & derivadosRESUMO
Retinoic acid (RA) induces HL-60 cells to differentiate terminally into mature granulocytes, which subsequently die by apoptosis. The biological effects of RA are mediated by two distinct families of transcription factors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs). RARs and RXRs form heterodimers and regulate retinoid-mediated gene expression. We have recently developed a novel RAR-selective antagonist (ER27191) which prevents RAR activation by retinoids. Using this RAR-selective antagonist, and RXR and RAR agonist, we demonstrate the RAR-mediated signaling pathway is important for differentiation and apoptosis of myeloid leukemic cells. Simple activation of RXRs is not sufficient to induce apoptosis of the cells. Interestingly, the combination of the RAR-selective antagonist and 9-cis RA resulted in partial differentiation and apoptosis of HL-60 and NB4 cells, whereas the RAR antagonist completely blocked all-trans RA-induced differentiation and apoptosis of the cells. Additional experiments showed that levels of BCL-2 protein decreased during differentiation of myeloid leukemic cells. Furthermore, HL-60 cells transduced with a bcl-2 expression vector showed the same differentiation response to retinoids as did parental HL-60 cells even though apoptosis was inhibited in these bcl-2-transduced cells, suggesting that differentiation and apoptosis are regulated independently in myeloid leukemic cells.
Assuntos
Antracenos/farmacologia , Apoptose/efeitos dos fármacos , Células HL-60/citologia , Células HL-60/efeitos dos fármacos , Células HL-60/fisiologia , Pirróis/farmacologia , Receptores do Ácido Retinoico/antagonistas & inibidores , Alitretinoína , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Resistência a Medicamentos , Humanos , Leucemia Mieloide/patologia , Leucócitos/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/farmacologia , Receptores do Ácido Retinoico/metabolismo , Receptores do Ácido Retinoico/fisiologia , Receptor alfa de Ácido Retinoico , Transdução de Sinais , Tretinoína/metabolismo , Tretinoína/farmacologiaRESUMO
A 45-year-old woman with acute lymphoblastic leukemia (ALL) who failed to achieve complete remission (CR) after one course of induction chemotherapy with vincristine, daunorubicin, prednisolone and l-asparaginase was successfully treated with a high dose of cytosine arabinoside (Ara-C) and mitoxantrone. The leukemic blasts were CD7, 19, 33, and 38 antigens positive, and had a rearrangement in the T-cell receptor delta chain gene. The karyotype was normal. Primary induction failure and positivity for myeloid antigens are both reported to be poor prognostic factors for ALL. Nevertheless, this patient was successfully treated with the high dose Ara-C and mitoxantrone, and she remains in CR for over 20 months. Combination chemotherapy with high dose Ara-C and mitoxantrone may be of benefit for refractory ALL with both CD7 and myeloid antigens.
Assuntos
Antígenos CD7 , Antígenos de Neoplasias , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Citarabina/administração & dosagem , Feminino , Humanos , Pessoa de Meia-Idade , Mitoxantrona/administração & dosagem , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Indução de RemissãoRESUMO
A case of acute promyelocytic leukemia (APL) developed in a patient with congenital antithrombin III (AT-III) deficiency is reported. Despite the presence of disseminated intravascular coagulation (DIC), plasma AT-III activity was not decreased at the diagnosis of APL compared to the patient's baseline level (approximately 50% of normal). He was successfully treated with all-trans retinoic acid (ATRA) to achieve complete remission without the use of heparin. Although he developed phlebitis at the site of insertion of the intravenous catheter during remission-induction, no major thrombotic episode was noted. Coagulation parameters including fibrin and fibrinogen degradation products (FDP-E), thrombin-antithrombin complex (TAT), FDP-D dimer (D-D dimer), and plasmin-alpha 2 plasmin inhibitor complex (PIC) improved rapidly after initiation of ATRA. This case is a clear demonstration of the characteristics of DIC developing in APL, i.e. no or minimal decrease in the level of AT-III activity and a predominant increase in the fibrinolytic system, rather than hypercoagulability.
Assuntos
Deficiência de Antitrombina III , Leucemia Promielocítica Aguda/fisiopatologia , Doenças Hematológicas/congênito , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
To elucidate the physiologic role of thrombopoietin (TPO) for hematologic reconstitution following allogeneic bone marrow transplantation (BMT), serum TPO levels as well as interleukin-3 (IL-3), IL-6 and IL-11 were serially measured in 55 samples from 3 patients who underwent allogeneic BMT using an enzyme-linked immunosorbent assay (ELISA). The TPO level was higher in the serum taken during marrow aplasia than in the pretransplant serum. The serum TPO levels and platelet counts showed a strong inverse relationship in all patients examined. We also sequentially measured endogenous serum TPO levels before and within 36 h after platelet transfusions. Endogenous serum TPO levels were inversely correlated with platelet mass following platelet transfusions. Serum levels of IL-3 had no apparent correlation with platelet counts and serum levels of IL-11 remained below the detection levels (31.3 pg/ml) in all samples. Serum levels of IL-6 were high during myeloaplasia and more upregulated in the febrile period. These findings support the view that TPO is the central regulator for megakaryopoiesis in vivo and the rationale for its clinical use after allogeneic BMT.
Assuntos
Transplante de Medula Óssea/fisiologia , Interleucinas/sangue , Trombopoetina/sangue , Adulto , Feminino , Humanos , Interleucina-11/sangue , Interleucina-3/sangue , Interleucina-6/sangue , Masculino , Pessoa de Meia-Idade , Transplante HomólogoRESUMO
Recent studies have shown that a high proportion of patients with acute promyelocytic leukemia (APL) achieve complete remission after treatment with all-trans retinoic acid (RA). Nevertheless, despite an initial good response, most patients who received continuous treatment with all-trans RA relapsed and develop RA-resistant disease. The detailed mechanisms for this development of RA resistance by APL cells are still unclear. Several possible mechanisms have been considered to explain in vitro resistance to RA. One obvious explanation is the generation of new mutations in the retinoid receptors. However, UF-1 cells (the first permanent APL cell line with RA-resistant features) had no point mutations in the ligand-binding domain of the RAR-alpha gene. Another potential mechanism for clinical RA resistance is the pharmacologic alteration in the metabolism of all-trans RA. Continuous treatment with all-trans RA in APL is associated with a progressive reduction of the plasma concentrations of RA. Induction of cytochrome P-450, cellular RA-binding protein (CRABP) and P-glycoprotein resulted in lower plasma and cellular levels of active retinoids. Thus, acquired resistance to RA may be explained at least in part by drug metabolism in leukemic cells.
Assuntos
Antineoplásicos/farmacologia , Leucemia Promielocítica Aguda/tratamento farmacológico , Tretinoína/farmacologia , Resistencia a Medicamentos Antineoplásicos , HumanosRESUMO
Despite its recent cloning and characterization, the physiological role of FemA remains unclear. To easily and reliably identify femA in staphylococci, we analysed 45 blood isolates of staphylococci using enzymatic detection of polymerase chain reaction (ED-PCR). ED-PCR is a new method that detects amplified PCR products using biotin-streptavidin affinity and an enzyme-linked antibody. Of 45 samples, 34 strains contained the femA gene as detected by ED-PCR. Phenotyping analysis showed that these 34 strains (femA-positive) were Staphylococcus aureus and that the other 11 (femA-negative) were S. epidermidis. These results were completely consistent with the results of femA detection using standard PCR and subsequent Southern blot hybridization. The ED-PCR procedure was complete within 4 h and could be done in one tube. We conclude that ED-PCR is a rapid, simple and reliable method for detecting the femA gene and that it provides an important means of classifying staphylococcal strains in the clinical field.