RESUMO
We used nuclear transfer (NT) to develop transgenic female pigs harboring goat beta-casein promoter/human granulocyte-macrophage colony stimulating factor (hGM-CSF). The expression of hGM-CSF was specific to the mammary gland, and the glycosylation-derived size heterogeneity corresponded to that of the native human protein. Although various cell types have been used to generate cloned animals, little is currently known about the potential use of fibroblasts derived from a cloned fetus as donor cells for nuclear transfer. The developmental potential of porcine cloned fetal fibroblasts transfected with hGM-CSF was evaluated in the present study. Cloned fetal fibroblasts were isolated from a recipient following the transplantation of NT embryos. The cells were transfected with both hGM-CSF and the neomycin resistance gene in order to be used as donor cells for NT. Reconstructed embryos were implanted into six sows during estrus; two of the recipient sows delivered seven healthy female piglets with the hGM-CSF gene (confirmed with PCR and fluorescent in situ hybridization) and microsatellite analysis confirmed that the clones were genetically identical to the donor cells. The expression of hGM-CSF was strong in the mammary glands of a transgenic pig that died a few days prior to parturition (110 d after AI). These results demonstrated that somatic cells derived from a cloned fetus can be used to produce recloned and transgenic pigs.
Assuntos
Animais Geneticamente Modificados , Fibroblastos/citologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Técnicas de Transferência Nuclear/veterinária , Suínos/genética , Animais , Clonagem de Organismos , Feminino , Humanos , GravidezRESUMO
We investigated the in vitro effect of As2O3 on proliferation, cell cycle regulation, and apoptosis in human myeloma cell lines. As2O3 significantly inhibited the proliferation of all of eight myeloma cell lines examined in a dose-dependent manner with IC50 of approximately 1-2 microM. DNA flow cytometric analysis indicated that As2O3 (2 microM) induced a G1 and/or a G2-M phase arrest in these cell lines. To address the mechanism of the antiproliferative effect of As2O3, we examined the effect of As2O3 on cell cycle-related proteins in MC/CAR cells in which both G1 and G2-M phases were arrested. Western blot analysis demonstrated that treatment with As2O3 (2 microM) for 72 h did not change the steady-state levels of CDK2, CDK4, cyclin D1, cyclin E, and cyclin B1 but decreased the levels of CDK6, cdc2, and cyclin A. The mRNA and protein levels of CDKI, p21 were increased by treatment with As2O3, but those of p27 were not. In addition, As2O3 markedly enhanced the binding of p21 with CDK6, cdc2, cyclin E, and cyclin A compared with untreated control cells. Furthermore, the activity of CDK6-associated kinase was reduced in association with hypophosphorylation of Rb protein. The activity of cdc2-associated kinase was decreased, which was accompanied by the up-regulation of cdc2 phosphorylation (cdc2-Tyr15 phosphorylation) resulting from reduction of cdc25B and cdc25C phosphatases. As2O3 also induced apoptosis in MC/CAR cells as evidenced by flow cytometric detection of sub-G1 DNA content and annexin V binding assay. This apoptotic process was associated with down-regulation of Bcl-2, loss of mitochondrial transmembrane potential (delta psi(m)), and an increase of caspase-3 activity. These results suggest that As2O3 inhibits the proliferation of myeloma cells, especially MC/CAR cells, via cell cycle arrest in association with induction of p21 and apoptosis.
Assuntos
Arsenicais/farmacologia , Ciclo Celular/efeitos dos fármacos , Ciclinas/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Óxidos/farmacologia , Apoptose/efeitos dos fármacos , Trióxido de Arsênio , Northern Blotting , Western Blotting , Caspase 3 , Caspases/metabolismo , Divisão Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21 , Relação Dose-Resposta a Droga , Citometria de Fluxo , Humanos , Concentração Inibidora 50 , Testes de Precipitina , Fatores de Tempo , Células Tumorais CultivadasRESUMO
The seco-steroid 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) induces differentiation and inhibits clonal proliferation of HL-60 cells. We analyzed the effect of a novel vitamin D3 analog, EB1089, on normal myeloid and leukemic cells as well as CD34+ cells. EB1089 showed an extraordinary inhibition of clonal growth of HL-60 cells (ED50 = 5 x 10(-11) M) and AML blast cells (ED50 = 9 x 10(-10) M) compared to 1,25(OH)2D3 without suppression of growth of normal human bone marrow CFU-GM. The CD34+ cells from acute myeloid leukemia (AML) blasts were inhibited in a dose-dependent fashion by 1,25(OH)2D3 with an ED50 of 1.2 x 10(-9) M; and even more strikingly, 10(-10) M of EB1089 inhibited all clonal growth of human CD34+ leukemic colony-forming cells. In contrast, both EB1089 and 1,25(OH)2D3 (10(-8) M) showed little or only mild inhibition of CD34+ clongenic hematopoietic cells from normal human peripheral blood (PB); and in liquid culture, EB1089 stimulated the proliferation of normal human CD34+ cells about 2.5 times as compared to control cultures. In order to evaluate the potential use of EB1089 for purging leukemic cells from normal CD34+ progenitor cells for PB stem cell transplantation (PBSCT), normal human PB mononuclear cells (PBMNC) were contaminated with HL-60 cells, and then CD34+ cells purified and treated with EB1089. We found that CD34+ purification and EB1089 purging was able to eliminate approximately 100% of HL-60 leukemic cells with no toxicity to normal CD34+ hematopoietic progenitor cells. These data suggested that purification of CD34+ cells and ex vivo treatment with EB1089 might provide an effective therapeutic approach for PBSCT.
Assuntos
Antineoplásicos/farmacologia , Calcitriol/análogos & derivados , Células-Tronco Hematopoéticas/efeitos dos fármacos , Leucemia Mieloide/patologia , Leucócitos Mononucleares/efeitos dos fármacos , Antígenos CD34 , Calcitriol/farmacologia , Divisão Celular/efeitos dos fármacos , Separação Celular , Células HL-60 , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/patologia , Humanos , Leucemia Mieloide/terapia , Leucócitos Mononucleares/citologiaRESUMO
The purpose of this study was to assess the therapeutic potential of injecting the gene for HLA-B7/beta2-microglobulin into the subcutaneous metastatic nodules of patients who are refractory to conventional treatments. The nine patients evaluated were divided into three groups and given escalating doses of DNA (20, 40, and 100 microg of the HLA-B7 plasmid DNA/lipid complex for each group) every 2 weeks. Biopsy specimens from the treated tumor nodules of all nine patients were positive for the presence of DNA and for HLA-B7 mRNA expression. Moreover, in six of the nine patients, immunohistology of tumor biopsy samples revealed the expression of recombinant HLA-B7 protein. Also, all nine patients showed an increase in NK activity in their circulating peripheral blood lymphocytes. In two lung cancer patients, one partial and one mixed response was observed after gene transfer. These responses were confined to the treated nodules and the untreated locoregional lymph nodes; the lung masses showed no regression. Remission durations were 14 and 6 weeks, respectively, and in a total of 35 cycles no significant toxicities were observed. Immunohistologic analysis revealed an increased infiltration of CD4+ T cells, macrophages, and NK cells after therapy. In two responding cases, direct intratumoral injection of an allogeneic class I gene could elicit an antitumor response in locoregional areas, possibly through the activation of NK cells.
Assuntos
Terapia Genética/métodos , Antígenos HLA-B/genética , Células Matadoras Naturais/imunologia , Neoplasias/genética , Microglobulina beta-2/genética , Adulto , Idoso , Antígenos CD/imunologia , Feminino , Expressão Gênica/genética , Técnicas de Transferência de Genes , Genes MHC Classe I/genética , Humanos , Imuno-Histoquímica , Injeções , Lipossomos/metabolismo , Masculino , Pessoa de Meia-Idade , Neoplasias/terapia , RNA Mensageiro/genéticaRESUMO
EB1089 is a novel 1,25(OH)2D3 analog that has more potent antitumor properties with reduced hypercalcemic effects than 1,25(OH)2D3. We investigated the role of transforming growth factor-beta1 (TGF-beta1) in the growth inhibition of human acute myeloid leukemia cell line, HL-60, by EB1089. Clonal growth of HL-60 cells was inhibited in a dose-dependent manner by EB1089. Although TGF-beta1 alone slightly inhibited proliferation of HL-60 cells, the addition of TGF-beta1 into culture treated with 10(-8) M of EB1089 showed a significant synergistic antiproliferative effect in a dose-dependent manner. EB1089 up-regulated the expression of TGF-beta receptor type I (TGF-beta RI), type II (TGF-beta RII) and TGF-beta1. Antiproliferative effect of EB1089 was partially reversed by TGF-beta1 neutralizing antibody (anti-TGF-beta1). Vitamin D receptor (VDR) expression was increased by TGF-beta1, suggesting synergistic action of TGF-beta1 and EB1089. Combined treatment of EB1089 and TGF-beta1 resulted in an increased expression of cyclin-dependent kinase inhibitor (CDKI), p27 protein, compared to either ligand alone. Up-regulation of p27 protein expression by either TGF-beta1 or EB1089 was reduced by anti-TGF-beta1. These findings suggest that TGF-beta1 is involved in the antiproliferative effect of EB1089 on HL-60 cells.
Assuntos
Receptores de Ativinas Tipo I , Calcitriol/análogos & derivados , Proteínas de Ciclo Celular , Inibidores do Crescimento/farmacologia , Células HL-60/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/biossíntese , Receptores de Fatores de Crescimento Transformadores beta/biossíntese , Fator de Crescimento Transformador beta/biossíntese , Proteínas Supressoras de Tumor , Calcitriol/farmacologia , Inibidor de Quinase Dependente de Ciclina p27 , Humanos , Proteínas Serina-Treonina Quinases/genética , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Calcitriol/biossíntese , Receptores de Calcitriol/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/farmacologia , Ensaio Tumoral de Célula-TroncoRESUMO
Arsenic trioxide (As2O3) has been shown to inhibit the proliferation of hematologic malignant cells. Previously, we reported that As2O3 had an antitumoral effect in head and neck cancer. Here, we investigated the induction of apoptosis and its mechanism in PCI-1 head and neck squamous carcinoma cells, after treatment with As2O3. Treatment with 2 microM of As2O3 caused apoptosis in PCI-1 cells following 3 days of exposure, which was detected by the annexin V-PI and DAPI staining methods. The cell death population was markedly increased, being 88% larger than the As2O3-untreated control cells. To address the mechanism of apoptosis, a Western blot assay was performed, showing that Bax was up-regulated without a change in Bcl-2. Activation of caspase-9 during As2O3-induced apoptosis was substantiated by monitoring the proteolysis of the caspase-9, which was associated with an increase of Apaf-1 and cytochrome c protein. PCI-1 cells rapidly changed the mitochondria membrane potential (DeltaPsim) after addition of As2O3. Furthermore, activation of caspase-3 was demonstrated by monitoring the proteolysis of the caspase-3 and by measuring caspase-3 activity with a fluorogenic substrate, which was associated with the cleavage of poly(ADP-ribose) polymerase. To examine the in vivo effect of As2O3, C3H mouse inoculated with syngenic SCC7 cells was treated by intratumoral injection of As2O3 (300 microg) every day, demonstrating that tumor mass was dramatically reduced on day 4, and revealed induction of apoptosis by TUNEL assay. These results suggest that apoptosis of PCI-1 cells by As2O3 is induced by activation of caspase-3 via cytochrome c, caspase-9 and Apaf-1 complex.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Arsenicais/farmacologia , Carcinoma de Células Escamosas/metabolismo , Caspases/efeitos dos fármacos , Neoplasias de Cabeça e Pescoço/metabolismo , Óxidos/farmacologia , Animais , Antineoplásicos/uso terapêutico , Apoptose/fisiologia , Fator Apoptótico 1 Ativador de Proteases , Trióxido de Arsênio , Arsenicais/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Caspase 3 , Caspase 9 , Caspases/metabolismo , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Humanos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Óxidos/uso terapêutico , Proteínas/efeitos dos fármacos , Proteínas/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismoRESUMO
Progression of cell cycle in eukaryotes is regulated by a series of the cyclin-dependent kinases (CDKs) and cyclin-dependent kinase inhibitors (CDKIs). It has been shown that 1,25(OH)2D3 is able to arrest cell cycle at G1 phase in malignant cells including HL-60 cells. EB1089 is a novel 1,25(OH)2D3 analog that has more potent antileukemic properties with reduced hypercalcemic effect in vitro and in vivo than 1,25(OH)2D3. In the present study, we examined the effect of EB1089 on HL-60 cells at the protein levels of several G1 regulatory proteins. Exposure of HL-60 cells to EB1089 (1x10-8 M) for 3 days showed the G1 block by FACS analysis. The level of p21 was markedly induced in HL-60 cells treated with EB1089 at 24 h, and p27 were progressively increased in a time-dependent manner. The expressions of CDK2 and CDK6 were down-regulated during G1 block of HL-60 cells, and CDK4 is progressively elevated. In addition, level of cyclin D1 was increased in a time-dependent manner, however, no change of cyclin E was noted through the G1 to S traverse. Immunoprecipitation study demonstrated that p27 did not bind to CDK2, CDK4 and CDK6 in EB1089-treated HL-60 cell extracts. In contrast, complexes immunoprecipitated from EB1089-treated HL-60 cells with antibodies CDK2 and CDK6 contained higher amounts of immunodetectable p21 protein compared to untreated HL-60 cells, whereas no detectable change was noted with anti-CDK4 antibody. Furthermore, the kinase activities of CDK2 and CDK6 were decreased while little change was observed in CDK4 activity. These data indicated that p21 protein is a strong candidate for the control of G1 progression in EB1089-treated HL-60 cells, and its major target molecules are CDK2 and CDK6.
Assuntos
Antineoplásicos/farmacologia , Calcitriol/análogos & derivados , Proteínas de Ciclo Celular/efeitos dos fármacos , Quinases Ciclina-Dependentes/efeitos dos fármacos , Fase G1/efeitos dos fármacos , Células HL-60/efeitos dos fármacos , Calcitriol/farmacologia , Proteínas de Ciclo Celular/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Fase G1/fisiologia , Células HL-60/metabolismo , HumanosRESUMO
An inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, lovastatin, induces growth arrest and cell death in a wide variety of malignant cells in vitro. We analyzed the effect of lovastatin on myeloid leukemic cell lines. Lovastatin significantly inhibited the proliferation of 7 cell lines among 11 myeloid leukemic cell lines in a dose-dependent manner. In order to address the mechanism of antileukemic effect of lovastatin, cell cycle analysis was attempted in HL-60 cells, showing that lovastatin induced G1 arrest in HL-60 cells following 72 h of drug exposure (1.5 microM, 5 microM and 10 microM) in a dose-dependent manner. Analysis of G1 regulatory proteins demonstrated that the protein levels of cyclin-dependent kinase (CDK) 2, CDK4, CDK6 and cyclin E were decreased after treatment with lovastatin (10 microM) in a time-dependent manner, but not cyclin D1. In addition, lovastatin increased the protein level of the cyclin-dependent kinase inhibitor (CDKI), p27, and markedly enhanced the binding of p27 with CDK2 and CDK4 more than CDK6 after 24 h exposure. At higher doses of lovastatin (50 mM, 100 mM, 200 mM), a significant apoptosis was observed as evidenced by FACS analysis with annexin V staining, which was associated with downregulation of Bcl-2 protein. These results suggest that lovastatin inhibits the proliferation of myeloid leukemic cells via G1 arrest in association with p27 induction and is an effective inducer of apoptosis in HL-60 cells.
Assuntos
Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Fase G1/efeitos dos fármacos , Lovastatina/farmacologia , Proteínas de Ciclo Celular/metabolismo , Células HL-60 , Humanos , Células Tumorais CultivadasRESUMO
Since hepatocellular carcinoma (HCC) is frequently presented at an advanced stage, only a small portion of patients with HCC can be treated with local modalities. Gene therapy is, therefore, one of the more promising approaches for patients with advanced HCC. To develop a new strategy for targeting gene delivery to the hepatic tumor, the efficiency of the transarterial delivery of liposome-DNA complex was evaluated in VX2 carcinoma implanted into the liver of rabbits. A mixture of pSV-beta galactosidase plasmid (40 micrograms), lipofectin (80 microliters), and transferrin (852 micrograms), the optimal proportion of which determined in vitro, was infused via the hepatic artery of a rabbit with VX2 hepatic tumors. The efficiency of trans-arterial gene delivery was compared to that of intra-tumoral injection. Rabbits (5 in each group) were sacrificed 48 hours after gene delivery and hepatic tissues were examined using X-gal staining. beta-galactosidase staining was observed exclusively within the tumor following the trans-arterial gene transfer. In contrast, adjacent peritumoral cells in addition to hepatic tumor cells were transfected by the intra-tumoral injection of transgene. These data indicate that enhanced gene expression in hepatic tumors is possible using trans-arterial delivery of the liposome-DNA complex.
Assuntos
DNA/genética , Neoplasias Hepáticas Experimentais/patologia , Fígado/citologia , Transfecção/métodos , Transferrina/genética , Animais , Linhagem Celular Transformada , Transplante de Células/métodos , Portadores de Fármacos , Terapia Genética/métodos , Humanos , Lipossomos , Fígado/ultraestrutura , Neoplasias Hepáticas Experimentais/terapia , Coelhos , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Células Tumorais Cultivadas , beta-Galactosidase/análise , beta-Galactosidase/genéticaRESUMO
Xenotransplantation using porcine organs may resolve the chronic shortage of donor organs for clinical transplantation if significant immunologic barriers can be overcome. A xenograft can be rejected by T cells, especially CD8(+) cytotoxic T lymphocytes (CTL)-mediated responses, as these cells show cytotoxicity against xenografts by recognition of swine leukocyte antigen (SLA)-I. Peptide translocation is inhibited by the endoplasmic reticulum-resident human cytomegalovirus (HCMV) glycoprotein unique short (US) 6, due to alterations of the transporter associated with antigen processing loading onto MHC class I for antigen presentation to CD8(+) CTL. In this study we transfected the US6 gene into minipig fetal fibroblasts establishing three US6 clonal cell lines. Flow cytometry analysis of US6 clonal cell lines demonstrated a substantial reduction in SLA-I expression. The level of SLA-I expression in US6 clones was decreased to 56.3% compared with the control 42.7%. In CTL assays, the rate of CD8(+) CTL-mediated cytotoxicity was significantly reduced to 35.2% ± 11.7% compared with the control, 79.9% ± 6.5%, (P < .01). These results suggested that HCMV viral protein US6 suppresses the presentation of SLA-I on pig fetal fibroblast cells. This strategy might be used in transgenic pig production to protect porcine organs from CTL-mediated immune rejection.
Assuntos
Antígenos de Histocompatibilidade Classe II/imunologia , Proteínas de Ligação a RNA/genética , Proteínas Virais/genética , Animais , Animais Geneticamente Modificados , Linhagem Celular , Antígenos de Histocompatibilidade Classe I , Humanos , Suínos , Porco Miniatura , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologiaRESUMO
REAH is a rare benign lesion of the sinonasal tract. The nasal cavity, particularly the posterior nasal septum, is the most common site of involvement. It usually occurs unilaterally and can be cured with conservative surgical resection. We present an unusual case of adenomatoid hamartoma involving bilateral olfactory recesses and discuss the importance of distinguishing this from other neoplastic processes that may lead to overly aggressive treatment.
Assuntos
Adenocarcinoma/patologia , Hamartoma/patologia , Cavidade Nasal/patologia , Neoplasias Nasais/patologia , Doenças dos Seios Paranasais/patologia , Biópsia , Diagnóstico Diferencial , Feminino , Humanos , Imageamento por Ressonância Magnética , Pessoa de Meia-Idade , Mucosa Respiratória/patologiaRESUMO
Inflammatory pseudotumor is a rare non-neoplastic mass that may clinically and radiologically mimic a spectrum of benign and malignant neoplasms. It is uncommon in the head and neck and particularly rare at the skull base. We present a case of pseudotumor originating from the trigeminal nerve in a patient who presented with headache and facial numbness. A high index of suspicion is necessary to diagnose this benign but locally aggressive entity.
Assuntos
Neoplasias dos Nervos Cranianos/diagnóstico por imagem , Granuloma de Células Plasmáticas/diagnóstico por imagem , Tomografia Computadorizada por Raios X , Nervo Trigêmeo/diagnóstico por imagem , Neoplasias dos Nervos Cranianos/patologia , Diagnóstico Diferencial , Feminino , Granuloma de Células Plasmáticas/patologia , Humanos , Imageamento por Ressonância Magnética , Nervo Trigêmeo/patologia , Adulto JovemRESUMO
We examined telomerase activity in myeloid leukaemic cell lines, normal haemopoietic cells, and leukaemic blasts from acute myelogenous leukaemia (AML) patients. Normal bone marrow mononuclear (BMNC) cells expressed low telomerase activity. Higher telomerase activity was detected in 10 myeloid leukaemic cell lines compared to normal BMNC cells. Treatment with 1,25(OH)2D3, and vitamin D3 analogues, EB1089 and KH1060, reduced telomerase activity in vitamin D3-sensitive HL-60 cells, whereas vitamin D3 insensitive K562 cells did not change its activity. This down-regulation of telomerase activity by EB1089 was associated with induction of p21 protein. The rank order of telomerase activity was leukaemic CD34- cells > leukaemic CD34+ cells > normal CD34- cells > normal CD34+ cells. Telomerase activity was positive in all of the AML patients tested; however, heterogeneity of telomerase activity was found amongst this group. Therefore we compared telomerase activity with clinical response. Unexpectedly, we found that a higher rate of complete remission was noted in AML patients with higher telomerase activity. No association between telomerase activity and biological parameters including percentage of S-phase, cytotoxicity to cytosine arabinoside and percentage of CD34+ cells in AML blasts was found. These results suggest that telomerase activity in AML patients is detected with high frequency, but is heterogenous. Expression level of telomerase activity may have a clinical implication in AML patients regarding clinical response.
Assuntos
Leucemia Mieloide Aguda/enzimologia , Telomerase/metabolismo , Adulto , Idoso , Antígenos CD34/metabolismo , Colecalciferol/análogos & derivados , Feminino , Células HL-60 , Humanos , Masculino , Pessoa de Meia-Idade , Células Tumorais CultivadasRESUMO
Arsenic trioxide (As(2)O(3)) has been shown to inhibit the proliferation of hematologic malignant cells. However, little is known about the effect of As(2)O(3) on solid tumor. In this study, we investigated the antitumoral effect of As(2)O(3) on head and neck cancer cell lines in vitro. Treatment of As(2)O(3) inhibited the proliferation of all of 4 cell lines examined in a dose-dependent manner. To address the mechanism of antitumoral effect of As(2)O(3), cell cycle analysis was attempted in As(2)O(3)-most sensitive PCI-1 cells. Treatment of As(2)O(3) (2 microM) induced efficiently G2/M arrest in PCI-1 cells following 3 days of exposure. During the G2/M arrest, cyclin-dependent kinase inhibitor, p21, was increased in a time-dependent manner. Analysis of cell cycle regulatory proteins demonstrated that As(2)O(3) (2 microM) did not change the steady-state levels of CDK2, CDK4, CDK6, cyclin D1, cyclin E and cyclin A, but decreased the protein levels of cdc2 and cyclin B1. Furthermore, treatment of As(2)O(3) markedly enhanced the binding of p21 with cdc2, and the activity of cdc2 kinase was decreased in a time-dependent manner. These results suggest that As(2)O(3) inhibits the proliferation of head and neck cancer cells via G2/M arrest in association with the induction of p21 and the reduction of cdc2 kinase activity.
Assuntos
Antineoplásicos/farmacologia , Arsenicais/farmacologia , Ciclo Celular/efeitos dos fármacos , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Óxidos/farmacologia , Trióxido de Arsênio , Proteína Quinase CDC2/metabolismo , Divisão Celular/efeitos dos fármacos , Ciclina B/metabolismo , Ciclina B1 , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Fase G2/efeitos dos fármacos , Inibidores do Crescimento/farmacologia , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Mitose/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Chronic myelogenous leukaemia (CML) is a haematological malignant disorder characterized by the Philadelphia chromosome (Ph) and BCR-ABL gene rearrangement. This abnormal fusion gene can be considered to serve as a marker for the transformed cell clone in CML and is found in all cells arising from the same malignant precursor cell. It has been detected in CML cells of the myeloid, monocytic, erythroid and B-lymphocytic lineages. However, it is still arguable as to whether T lymphocytes or natural killer (NK) cells carry this marker. Answering this question would clarify the ontogenic relationship between NK cells and T cells. We examined 12 CML patients and studied the expression of BCR-ABL rearrangement by fluorescence in situ hybridization (FISH) in both NK cells and T cells sorted by flow cytometry. The purity of T cells was 95.6-99.8% and that of NK cells was 95.3-99.3% after sorting. Neither NK cells nor T cells showed any positive BCR-ABL signal with the exception of one patient who recovered from a lymphoid blastic crisis. We speculate that T cells and NK cells originate from BCR-ABL-negative stem cells.
Assuntos
Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Linfócitos T/metabolismo , Adulto , Idoso , Feminino , Citometria de Fluxo , Expressão Gênica , Rearranjo Gênico , Marcadores Genéticos , Humanos , Hibridização in Situ Fluorescente , Células Matadoras Naturais/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/imunologia , Masculino , Pessoa de Meia-IdadeRESUMO
EB1089, a novel 1,25-dihydroxyvitamin D3 analogue, has been known to have potent antiproliferative properties in a variety of malignant cells both in vitro and in vivo. In the present study, we analysed the effect of EB1089 on NCI-H929 human myeloma cells. EB1089 inhibited cell growth of NCI-H929 and efficiently induced the G1 phase arrest of the cell cycle in a dose-dependent manner. We could also detect apoptosis in NCI-H929 cells exposed to EB1089 (1 x 10-7 M for 72 h) using the sub-G1 group of the cell cycle by FACS and annexin V binding assays. Induction of apoptosis by EB1089 was associated with down-regulation of the Bcl-2 protein without change of the Bax protein. Regarding caspase activity, which plays a crucial role in apoptosis, EB1089-treated NCI-H929 cells revealed an increased activity of caspase 3 protease accompanied by degradation of the PARP protein in a dose- and time-dependent manner. In addition, EB1089 caused the down-regulation of p44 extracellular signal-related kinase (ERK) activity and up-regulation of the p38 kinase activity during apoptosis of NCI-H929 cells. These results suggest that EB1089 inhibits growth of NCI-H929 cells via G1 cell cycle arrest as well as apoptosis by activating p38 kinase and suppressing ERK activity.
Assuntos
Antineoplásicos/uso terapêutico , Calcitriol/análogos & derivados , Caspases/metabolismo , Colecalciferol/análogos & derivados , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Apoptose/efeitos dos fármacos , Western Blotting , Calcitriol/uso terapêutico , Caspase 3 , Ativação Enzimática/efeitos dos fármacos , Humanos , Mieloma Múltiplo/enzimologia , Células Tumorais Cultivadas , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
EB1089, a 1,25-dihydroxyvitamin D(3) analog, has been known to have potent antiproliferative properties in a variety of malignant cells in vitro and in vivo. In the present study, we analyzed the effect of EB1089 on human myeloma cell lines. EB1089 inhibited the proliferation of NCI-H929 cells and RPMI8226 cells in a dose-dependent manner among three myeloma cell lines tested. The antiproliferative effect of EB1089 on myeloma cells was related to the expression level of vitamin D receptor. To investigate the mechanism of the antiproliferative effect of EB1089, cell cycle analysis was attempted in EB1089-sensitive NCI-H929 cells. EB1089 (1 x 10(-8) M) efficiently induced G(1) arrest of the cell cycle. Analysis of G(1) regulatory proteins demonstrated that protein levels of CDK2, CDK4, cyclin D1, and cyclin A were decreased in a time-dependent manner, but not those of CDK6 and cyclin E, by EB1089. In addition, EB1089 (1 x 10(-8) M, 72 h) increased the protein level of the CDKI p27 and markedly enhanced the binding of p27 with CDK2 compared to EB1089-untreated cells. Furthermore, the activity of CDK2-associated cyclin kinase was decreased, which was accompanied by the reduction of cyclin-D1-, cyclin-E-, and cyclin-A-associated kinase activities, resulting in the hypophosphorylation of Rb protein. These results suggest that EB1089 can inhibit the proliferation of human myeloma cells, especially NCI-H929 cells, via a G(1) block in association with the induction of p27 and the reduction of CDK2 activity.