RESUMEN
Many proteins require the binding of trace metals such as Ca, Fe, Cu, or Zn, which may modulate their structure, function, or activity. To determine if there were any overall changes in metalloprotein distribution or metal concentration during the process of macrophage differentiation we induced human myeloid HL-60 leukemia cells with phorbol 12-myristate 13-acetate (PMA) and quantitatively mapped their metal content using hard X-ray fluorescence micro-analysis. We found a transient increase in the zinc content of HL-60 cell nuclei during the early stages of differentiation induction. This finding was confirmed by spectrofluorometry in HL-60 cells and extended to U-937 leukemia cells. A role for protein kinase C-beta (PKC-beta) in this process was established by examining zinc content in an HL-60 variant, HL-525, which is PKC-beta deficient, and in HL-525 cells in which PKC-beta was restored by stable overexpression. Chemical chelation of both Cu and Zn served to inhibit macrophage differentiation in HL-60 cells, indicating a requirement for these metals during this process. Finally, we demonstrate that growth of HL-60 cells in a low-zinc environment removes their susceptibility to PMA-induced differentiation, and that this capacity can be partially restored by the addition of exogenous zinc.
Asunto(s)
Diferenciación Celular , Leucemia/metabolismo , Macrófagos/fisiología , Zinc/fisiología , Transporte Activo de Núcleo Celular , Microanálisis por Sonda Electrónica , Perfilación de la Expresión Génica , Variación Genética , Células HL-60 , Humanos , Macrófagos/química , Microscopía Fluorescente , Células Mieloides/fisiología , Proteína Quinasa C/fisiología , Proteína Quinasa C beta , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Espectrometría de Fluorescencia , Acetato de Tetradecanoilforbol/farmacología , Distribución Tisular , Oligoelementos/metabolismo , Zinc/metabolismoRESUMEN
Inosine 5'-monophosphate dehydrogenase inhibitors including mycophenolic acid (MPA) are effective inducers of terminal differentiation in a variety of distinct human tumor cell types. Here, we report that MPA also induces such a differentiation in the androgen-independent prostate cancer derived cell line DU145. MPA evoked replication arrest and accumulation of the DU145 cells in the S-phase of the cell cycle. The inhibitor also induced the expression of CD55, clusterin, granulophysin, glucose-regulated protein 78, vasoactive intestinal polypeptide and prostate-specific transglutaminase, which are differentiation markers associated with the phenotype of normal prostate cells. We suggest that inosine 5'-monophosphate dehydrogenase inhibitors, which are already used for the treatment of other diseases, may be used as potential differentiation therapy drugs to control prostate cancer.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Diferenciación Celular/efectos de los fármacos , Transformación Celular Neoplásica/efectos de los fármacos , Ácido Micofenólico/farmacología , Neoplasias de la Próstata/patología , Antagonistas de Andrógenos/farmacología , Biomarcadores/análisis , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Humanos , Masculino , Fenotipo , Células Tumorales CultivadasRESUMEN
Recently, we have reported that inosine 5'-monophosphate dehydrogenase inhibitors, such as mycophenolic acid (MPA), induce the differentiation of PC-3 cells, which are derived from a human androgen-independent prostate cancer, into cells with a phenotype resembling maturing prostate secretory cells. Here, we describe such differentiation induced by the histone deacetylase inhibitor tributyrin. The maturation was defined by cytoplasmic vacuole production and induction of CD10, CD46, CD55, GRP78, keratin 17, and zinc-alpha-2-glycoprotein. To identify additional genes associated with tributyrin-induced PC-3 cell differentiation and to gain some insight into the mechanism that underlies this differentiation, we have, by means of microarray analyses, compared tributyrin-induced gene expression patterns with those of MPA, which initiates PC-3 cell differentiation by a dissimilar mode of action. We suggested that genes induced by both tributyrin and MPA would be most likely associated with differentiation rather than with the unique action of each particular inducer. Our results indicated that tributyrin or MPA induced the expression of a large number of common genes, including genes known or assumed to be NF-kappaB dependent. The NF-kappaB dependency of a group of these genes, which included the PC-3 cell differentiation marker keratin 17, was confirmed by using two common NF-kappaB activation inhibitors, Bay11-082 and TMB-8, and p65 subunit of NF-kappaB complex specific small interfering RNA. Taken together, our results implicate both NF-kappaB-dependent and NF-kappaB-independent genes in the processes leading to PC-3 cell differentiation induced by tributyrin and MPA.
Asunto(s)
Andrógenos/fisiología , Apigenina/farmacología , Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , FN-kappa B/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Antibióticos Antineoplásicos/farmacología , Transporte Biológico/efectos de los fármacos , Bloqueadores de los Canales de Calcio/farmacología , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Quimioterapia Combinada , Chaperón BiP del Retículo Endoplásmico , Ácido Gálico/análogos & derivados , Ácido Gálico/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ácido Micofenólico/farmacología , FN-kappa B/genética , Neoplasias Hormono-Dependientes/tratamiento farmacológico , Neoplasias Hormono-Dependientes/genética , Neoplasias Hormono-Dependientes/metabolismo , Neoplasias de la Próstata/genética , Transfección , Vacuolas/metabolismoRESUMEN
The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent stimulator of differentiation and apoptosis in myeloid leukemia cells. In the present study, we investigated the role of the transcription factor NF-kappaB in TPA-induced growth inhibition and apoptosis in the myeloid leukemia HL-60 cell line and its TPA-resistant cell variant HL-525. Unlike the parental cell line, HL-525 cells are protein kinase C (PKC)-beta deficient and resistant to TPA-induced differentiation and apoptosis. We found that treatment of HL-60 cells with TPA resulted in a concentration-dependent growth inhibition and an increase in apoptotic cells. TPA only had a small effect on growth and apoptosis in HL-525 cells. Treatment of HL-60 cells with TPA (0.64-3.2 nM) caused a rapid activation of NF-kappaB as determined by electrophoresis mobility shift assay (EMSA) and immunocytochemistry. Although the basal level of NF-kappaB activity was low in HL-60 cells, TPA-resistant HL-525 cells had a high basal level of NF-kappaB activity. Treatment of HL-525 cells with higher concentrations of TPA (16-80 nM) resulted in a further increase in NF-kappaB activity. (E)3-[(4-methylphenyl)-sulfonyl]-2-propenenitrile (BAY 11-7082; BAY), which inhibits IkappaB alpha phosphorylation and thus decreases NF-kappaB activation, was found to decrease TPA-induced nuclear translocation of NF-kappaB. Furthermore, BAY enhanced TPA-induced growth inhibition and apoptosis in both HL-60 and HL-525 cells. Results from the present study indicate that inhibition of NF-kappaB by BAY was associated with enhanced TPA-induced growth inhibition and apoptosis in human myeloid leukemia cells. TPA in combination with pharmacological inhibitors of NF-kappaB may improve the therapeutic efficacy of TPA and overcome the resistance to TPA in some myeloid leukemia patients.
Asunto(s)
Apoptosis , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Leucemia Mieloide/tratamiento farmacológico , FN-kappa B/antagonistas & inhibidores , Nitrilos/farmacología , Sulfonas/farmacología , Acetato de Tetradecanoilforbol/farmacología , Transporte Activo de Núcleo Celular , Adhesión Celular , Diferenciación Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Supervivencia Celular , ADN/química , Daño del ADN , Relación Dosis-Respuesta a Droga , Células HL-60 , Humanos , Proteínas I-kappa B/metabolismo , Inmunofenotipificación , Inhibidor NF-kappaB alfa , FN-kappa B/metabolismo , Fosforilación , Propidio/farmacología , Proteína Quinasa C/metabolismo , Proteína Quinasa C beta , Factores de TiempoRESUMEN
The JAK-STAT signal transduction cascade participates in various cellular processes, including immune response, cell replication, differentiation and oncogenesis. Here, we report that this cascade is induced in two human myeloid HL-60 leukemia cell variants by the granulocyte differentiation inducer dimethyl sulfoxide (DMSO) and macrophage differentiation inducer phorbol 12-myristate 13-acetate (PMA). DMSO and PMA also induced the expression and catalytic activity of 2'-5' oligoadenylate synthetase (2-5A synthetase), a known interferon (IFN) inducible enzyme. The HL-60 cell variants included HL-205, which is susceptible to DMSO- and PMA-induced differentiation, and HL-525, which is susceptible to DMSO- but not to PMA-induced differentiation. Treatment of HL-205 and HL-525 cells with DMSO and HL-205 cells with PMA-induced JAK1 phosphorylation, JAK1/STAT1 association, formation of STAT1-STAT2 heterodimers, and the binding of the active IFN stimulating growth factor 3 (ISGF3) to the IFN-stimulated response element (ISRE) fragment isolated from the 2-5A synthetase promoter. These events were either reduced or absent in the resistant HL-525 cells treated with PMA. Taken together, our data implicate the above signaling cascade in DMSO- and PMA-induced 2-5A synthetase expression and catalytic activity in the HL-60 cell system.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/genética , Proteínas de Unión al ADN/metabolismo , Dimetilsulfóxido/farmacología , Leucemia Mieloide/enzimología , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal/fisiología , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacología , Transactivadores/metabolismo , 2',5'-Oligoadenilato Sintetasa/efectos de los fármacos , 2',5'-Oligoadenilato Sintetasa/metabolismo , Catálisis , Proteínas de Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/genética , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Regulación Enzimológica de la Expresión Génica , Genes bcl-1/efectos de los fármacos , Genes bcl-1/genética , Células HL-60 , Humanos , Janus Quinasa 1 , Leucemia Mieloide/metabolismo , Factor de Transcripción STAT1 , Transducción de Señal/efectos de los fármacosRESUMEN
Our previous studies demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) had pharmacological activity for the treatment of myeloid leukemia patients. In the present study, we investigated the effects of TPA alone or in combination with capsaicin (8-methyl-N-vanillyl-6-nonenamide) on growth and differentiation in myeloid leukemia HL-60 cells and in a TPA-resistant HL-60 variant cell line termed HL-525. Treatment of HL-60 cells with TPA (0.16-1.6 nM) for 48 h resulted in concentration-dependent growth inhibition and cell differentiation (via the macrophage pathway). Capsaicin (5-50 microM) inhibited the growth of HL-60 cells in a concentration-dependent manner. Treatment of HL-60 cells with capsaicin alone only resulted in a small increase in the number of differentiated cells but treatment of the cells with TPA in combination with capsaicin synergistically increased differentiation. Moreover, inhibitors of protein kinase C (PKC), 7-hydroxystaurosporin (UCN-01; 100 nM) and chelerythrine (0.5 microM), significantly decreased HL-60 cell differentiation induced by the combination of TPA and capsaicin. These results suggest that PKC may be involved in HL-60 cell differentiation induced by TPA in combination with capsaicin. Capsaicin alone caused a very small increase in differentiation in the TPA-resistant HL-525 cells. However, treatment of HL-525 cells with combinations of TPA (0.16 nM) and capsaicin (10-50 microM) caused a strong synergistic increase in differentiation. Results from the present study suggest that a combination of TPA and capsaicin may improve the therapeutic efficacy of TPA and overcome resistance to TPA in some myeloid leukemia patients.
Asunto(s)
Capsaicina/farmacología , Estaurosporina/análogos & derivados , Acetato de Tetradecanoilforbol/farmacología , Alcaloides , Antineoplásicos/farmacología , Benzofenantridinas , Carboxilesterasa/metabolismo , Carcinógenos , Adhesión Celular , Diferenciación Celular , Línea Celular Tumoral , Supervivencia Celular , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Células HL-60 , Humanos , Leucemia Mieloide/tratamiento farmacológico , Leucemia Mieloide/metabolismo , Macrófagos/metabolismo , Modelos Químicos , Fenantridinas/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Estaurosporina/farmacología , Factores de TiempoRESUMEN
To establish a system to study differentiation therapy drugs, we used the androgen-independent human prostate PC-3 tumor cell line as a target and mycophenolic acid (MPA), tiazofurin, or ribavirin, which are inhibitors of IMP dehydrogenase, as inducers. These inhibitors evoked replication arrest, caused an increase in cell size, and triggered vacuolization of the cytoplasm. By Northern and Western blotting and immunostaining, we demonstrated MPA-induced expression of 12 proteins reported to reside in prostasomes, organelles released by secretory luminal prostate cells. Additional MPA-induced proteins were identified by two-dimensional gel electrophoresis. Among these was keratin 17, a prostate cell differentiation marker. By Northern blotting, we also demonstrated the constitutive expression of keratins 8 and 18 and induced expression of keratin 19, three other prostate cell differentiation markers. In addition, we established that cells were committed to differentiate after the 2nd day of MPA treatment using guanosine, which can abrogate the effects of MPA. Based on the expression patterns of prostasomal proteins and keratins and the presence of tentative secretory vacuoles, we hypothesize that IMP dehydrogenase inhibitors induce androgen-independent PC-3 cells to mature into cells with a phenotype that resembles normal prostate luminal cells, but at their intermediate state of differentiation.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , IMP Deshidrogenasa/antagonistas & inhibidores , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Ribavirina/análogos & derivados , Secuencia de Aminoácidos , Antibióticos Antineoplásicos/farmacología , División Celular/efectos de los fármacos , Línea Celular Tumoral , Electroforesis en Gel Bidimensional , Humanos , Masculino , Datos de Secuencia Molecular , Ácido Micofenólico/farmacología , Neoplasias de la Próstata/enzimología , Ribavirina/farmacología , Vacuolas/efectos de los fármacosRESUMEN
The role of putrescine, spermidine and spermine in phorbol 12-myristate-13-acetate (PMA)-induced macrophage differentiation was examined in human HL-60 and U-937 myeloid leukemia cells. Unlike other polyamines, spermine affected this differentiation by acting as a negative regulator. This negative regulation was established by showing that the PMA-induced macrophage phenotype, but not PMA-associated replication arrest, was abrogated (a) by replenishing the PMA-evoked decrease in cellular spermine levels with this polyamine from an exogenous source and (b) by blocking PMA-induced expression of the polyamine catabolic enzyme N(1)-spermidine/spermine acetyltransferase (SSAT) with antisense oligonucleotides in the presence of low substrate level. The PMA-evoked reduction in cellular spermine appears to result from an increase in the activity of SSAT and a decrease in the activity of ornithine decarboxylase, the polyamine biosynthetic enzyme. To a degree, these changes are due to corresponding changes in the expression of the genes that code for these enzymes. When cell differentiation is initiated, SSAT expression is increased after PMA-evoked activation of protein kinase C-beta. The present studies raise the possibility that agents able to reduce spermine levels in patients' myeloid leukemia cells may enhance the activity of differentiation therapy drugs for this type of leukemia.
Asunto(s)
Macrófagos/patología , Espermina/fisiología , Acetiltransferasas/biosíntesis , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Poliaminas Biogénicas/biosíntesis , Diferenciación Celular/fisiología , Proteínas de Unión al ADN/fisiología , Activación Enzimática , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Guanidinas/farmacología , Células HL-60 , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Factor 2 Relacionado con NF-E2 , Proteína Quinasa C/metabolismo , Proteína Quinasa C beta , Espermina/antagonistas & inhibidores , Espermina/metabolismo , Espermina/farmacología , Acetato de Tetradecanoilforbol/antagonistas & inhibidores , Acetato de Tetradecanoilforbol/farmacología , Transactivadores/fisiología , Células U937RESUMEN
Stable cell transfection is used for the expression of exogenous genes or cDNAs in eukaryotic cells. Selection of these transfectants requires a dominant selectable marker. A variety of such markers has been identified and is currently in use. However, many of these are not suitable for all cell types or require unique conditions. Here we describe a simple and versatile dominant selectable marker that involves bacterial IMP dehydrogenase (IMPDH), an enzyme essential for the replication of mammalian and bacterial cells. Although IMPDH is evolutionarily conserved, the bacterial enzyme is orders of magnitude more resistant to the toxic effect of the drug mycophenolic acid, which is an IMPDH inhibitor. We have demonstrated that transfection of human, monkey or Chinese hamster cell lines with an expression vector containing bacterial IMPDH and mycophenolic acid treatment resulted in the selection of colonies with a strikingly increased resistance to mycophenolic acid toxicity. Analysis of cells derived from these colonies indicated that the acquisition of this resistance was associated with bacterial IMPDH protein expression. As a proof of principle, we showed that mammalian cell transfection with a bicistronic IMPDH/GFP expression vector and mycophenolic acid treatment can be used to successfully select transfectants that express the fluorescent protein. These results indicate that bacterial IMPDH is a practical dominant selectable marker that can be used for the selection of transfectants that express exogenous genes or cDNAs in mammalian cells.
Asunto(s)
Genes Bacterianos , IMP Deshidrogenasa/genética , Transfección , Animales , Secuencia de Bases , Biotecnología , Células CHO , Células COS , Línea Celular , Chlorocebus aethiops , Cricetinae , ADN Bacteriano/genética , Resistencia a Medicamentos/genética , Inhibidores Enzimáticos/farmacología , Expresión Génica , Vectores Genéticos , Proteínas Fluorescentes Verdes , Humanos , IMP Deshidrogenasa/antagonistas & inhibidores , Proteínas Luminiscentes/genética , Ácido Micofenólico/farmacologíaRESUMEN
We have identified, cultured, characterized, and propagated adult pluripotent stem cells (PSC) from a subset of human peripheral blood monocytes. These cells, which in appearance resemble fibroblasts, expand in the presence of macrophage colony-stimulating factor and display monocytic and hematopoietic stem cell markers including CD14, CD34, and CD45. We have induced these cells to differentiate into mature macrophages by lipopolysaccharide, T lymphocytes by IL-2, epithelial cells by epidermal growth factor, endothelial cells by vascular endothelial cell growth factor, neuronal cells by nerve growth factor, and liver cells by hepatocyte growth factor. The pluripotent nature of individual PSC was further confirmed by a clonal analysis. The ability to store, expand, and differentiate these PSC from autologous peripheral blood should make them valuable candidates for transplantation therapy.
Asunto(s)
Monocitos/citología , Trasplante de Células Madre/métodos , Células Madre/citología , Antígenos CD34/biosíntesis , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Células Epiteliales/citología , Hepatocitos/citología , Humanos , Interleucina-2/metabolismo , Antígenos Comunes de Leucocito/biosíntesis , Metabolismo de los Lípidos , Receptores de Lipopolisacáridos/biosíntesis , Lipopolisacáridos/metabolismo , Linfocitos/metabolismo , Neuronas/citología , Fagocitosis , Linfocitos T/citología , Factores de TiempoRESUMEN
The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent inducer of differentiation in human promyelocytic leukemia cells. Recently, TPA has been successfully administered to patients with myelocytic leukemia and has produced therapeutic effects that led to temporary remission. These studies demonstrated the potential efficacy of TPA in cancer chemotherapy. We now seek to understand the biological effects and molecular mechanisms of differentiation in response to TPA treatment in leukemia cells by expression profiling using DNA microarray. Our results show distinct temporal and coordinated gene changes that are consistent with differentiation and activation of multiple biochemical pathways in HL-60 cells exposed to TPA. Alterations of gene expression in HL-60 cells include various transcription factors, cytokines and protein markers that are consistent with the induction of differentiation elicited by TPA. These temporal patterns of gene expression were abolished or greatly diminished in an HL-60 derived TPA- resistant variant cell line (HL-525), thus revealing transcriptional and consequential biochemical changes that may be required for TPA-induced differentiation. In addition, certain genes were upregulated by TPA in TPA-resistant HL-525 cells but not in TPA-sensitive HL-60 cells suggesting that these genes may play a role in the resistant phenotype. These patterns of gene expression may be important for predicting response to TPA.