RESUMEN
Increased numbers of mast cells is a typical feature of a variety of human cancers. The major mediators in the secretory granules of the MC(TC) type of mast cells, serine proteinases tryptase and chymase, may be involved in squamous cell carcinoma (SCC) lesions by inducing matrix remodeling and epithelial cell detachment. The objective of this study was to analyze immunohistochemically whether MC(TC) mast cells as well as protease inhibitors, squamous cell carcinoma antigens (SCCAs), are present in the uterine cervical SCC. In addition, the effect of tryptase and chymase on uterine cervical SCC cell lines was studied in vitro. Here we report that tryptase- and chymase-positive mast cells are present in significant numbers in the peritumoral stroma of SCC lesions. Also, weak SCCA-2 immunoreactivity is observed in the SCC lesions, but only SCCA-1 in uterine cervical specimens with nonspecific inflammation. In cell cultures, especially chymase, but not tryptase, was shown to induce effective detachment of viable, growing and non-apoptotic SiHa SCC cells from substratum. Chymase also detached viable ME-180 SCC cells from substratum as well as degraded fibronectin. In contrast, normal keratinocytes underwent apoptotic cell death after similar prolonged chymase treatment. No inhibition of chymase was detected by SiHa cell sonicates nor did these cells express marked SCCA immunopositivity. MC(TC) mast cells containing tryptase and chymase are present in the peritumoral stroma of uterine cervical SCC and the malignant cells are only weakly immunoreactive for the chymase inhibitor SCCA-2. It is chymase that appears to be capable of inducing effective detachment of viable and growing SCC cells and therefore, it may release SCC cells from a tumor leading to spreading of malignant cells.
Asunto(s)
Carcinoma/metabolismo , Quimasas/metabolismo , Células Epiteliales/metabolismo , Mastocitos/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Apoptosis/efectos de los fármacos , Carcinoma/genética , Carcinoma/patología , Adhesión Celular/efectos de los fármacos , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Quimasas/genética , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Queratinocitos/patología , Mastocitos/efectos de los fármacos , Mastocitos/patología , Metástasis de la Neoplasia , Serpinas/genética , Serpinas/metabolismo , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patologíaRESUMEN
Despite the wide use of anti-CD20 antibody rituximab in the cancer treatment of B cell malignancies, the signalling pathways of CD20-induced apoptosis are still not understood. By using dominant negative (DN)-caspase-9 overexpressing follicular lymphoma cells we demonstrated that the activation of caspase-9 was essential for rituximab-mediated apoptosis. The death receptor pathway mediated by caspase-8 activation was not involved in rituximab-mediated apoptosis since overexpression of FLIP(short) or FLIP(long) proteins, inhibitors of caspase-8 activation, could not inhibit rituximab-induced apoptosis. However, the death receptor pathway activation by anti-Fas antibodies showed an additive effect on rituximab-induced apoptosis. The stabilisation of the mitochondrial outer membrane by Bcl-x(L) overexpression inhibited cell death, showing the important role of mitochondria in rituximab-induced apoptosis. Interestingly, the rituximab-induced release of cytochrome c and collapse of mitochondrial membrane potential were regulated by caspase-9. We suggest that caspase-9 and downstream caspases may feed back to mitochondria to amplify mitochondrial disruption during intrinsic apoptosis.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Apoptosis/efectos de los fármacos , Caspasa 9/metabolismo , Linfoma Folicular/enzimología , Linfoma Folicular/patología , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Anticuerpos Monoclonales de Origen Murino , Antígenos CD40/metabolismo , Línea Celular Tumoral , Citocromos c/metabolismo , Activación Enzimática/efectos de los fármacos , Humanos , Cinética , Receptores de Muerte Celular/metabolismo , Rituximab , Proteína bcl-X/metabolismo , Receptor fas/metabolismoRESUMEN
Based on Bcl-X(L) overexpression studies we identified type I and type II follicular lymphoma cell lines in response to TRAIL. We demonstrate here that either amount of caspase-8 activation or Bid cleavage could not define the dependence on mitochondria. Furthermore, an inhibitor of NF-kappaB, PDTC, enabled TRAIL to activate type I apoptotic pathway in type II cells. However, an inhibitor of IKK did not switch apoptosis to type I pathway in type II cells, indicating that NF-kappaB might not be responsible for the switch.
Asunto(s)
Antineoplásicos/farmacología , Linfoma Folicular/metabolismo , Prolina/análogos & derivados , Transducción de Señal , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Tiocarbamatos/farmacología , Apoptosis , Secuencia de Bases , Caspasa 8/metabolismo , Línea Celular Tumoral , Cartilla de ADN , Activación Enzimática , Citometría de Flujo , Humanos , Linfoma Folicular/patología , Mutagénesis Sitio-Dirigida , FN-kappa B/antagonistas & inhibidores , Prolina/farmacología , Proteína bcl-X/metabolismoRESUMEN
BACKGROUND: Previous reports showed that mast cells can typically be found in the peritumoral stroma of cervix carcinomas as well as in many other cancers. Both histamine and TNF-alpha are potent preformed mast cell mediators and they can act simultaneously after release from mast cells. Thus, the effect of TNF-alpha and histamine on cervical carcinoma cell lines was studied. METHODS AND RESULTS: TNF-alpha alone induced slight growth inhibition and cell cycle arrest at G0/G1 phase in SiHa cells, but increased their migration. Histamine alone had no effect on cells. In addition, TNF-alpha and histamine in combination showed no additional effect over that by TNF-alpha alone, although SiHa cells were even pretreated with a protein synthesis inhibitor. Furthermore, TNF-alpha-sensitive ME-180 carcinoma cells were also resistant to the combination effect of TNF-alpha and histamine. In comparison, TNF-alpha or histamine alone induced growth inhibition in a non-cytolytic manner in normal keratinocytes, an effect that was further enhanced to cell cytolysis when both mediators acted in combination. Keratinocytes displayed strong TNF receptor (TNFR) I and II immunoreactivity, whereas SiHa and ME-180 cells did not. Furthermore, cervix carcinoma specimens revealed TNF-alpha immunoreactivity in peritumoral cells and carcinoma cells. However, the immunoreactivity of both TNFRs was less intense in carcinoma cells than that in epithelial cells in cervical specimens with non-specific inflammatory changes. CONCLUSION: SiHa and ME-180 cells are resistant to the cytolytic effect of TNF-alpha and histamine whereas normal keratinocytes undergo cytolysis, possibly due to the smaller amount of TNFRs in SiHa and ME-180 cells. In the cervix carcinoma, the malignant cells may resist this endogenous cytolytic action and TNF-alpha could even enhance carcinoma cell migration.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Células Escamosas/tratamiento farmacológico , Resistencia a Antineoplásicos , Histamina/farmacología , Queratinocitos/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Neoplasias del Cuello Uterino/tratamiento farmacológico , Anciano , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Citotoxinas/administración & dosificación , ADN de Neoplasias/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Sinergismo Farmacológico , Femenino , Histamina/administración & dosificación , Humanos , Queratinocitos/fisiología , Masculino , Persona de Mediana Edad , Factor de Necrosis Tumoral alfa/administración & dosificaciónRESUMEN
CD95/Fas and CD40 receptors are important regulators of cell survival during germinal center reaction. In this study we used a human follicular lymphoma cell line, HF1A3, to study molecular mechanisms of CD95-mediated apoptosis and CD40-induced rescue from apoptosis. CD95 stimulation induced activation of caspase-8 and -3, collapse of mitochondrial membrane potential (DeltaPsim), release of cytochrome c and fragmentation of nuclear DNA. All these apoptotic events were abrogated, when cells were pretreated with CD40 antibodies before CD95 stimulation. CD40 induced a rapid up regulation of both short and long isoforms of c-FLIP, as these proteins were detectable 4h after receptor stimulation. The induction of c-FLIP as well as the anti-apoptotic function of CD40 was completely abolished when NF-kappaB activity was inhibited by a selective inhibitor PDTC. We conclude that the anti-apoptotic signaling of CD40 involves NF-kappaB-mediated induction of c-FLIP proteins which can interfere with caspase-8 activation. However, it remains to be seen whether c-FLIP proteins are the only one ones involved in CD40-mediated protection.
Asunto(s)
Apoptosis/inmunología , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Antígenos CD40/fisiología , Regulación hacia Arriba/inmunología , Receptor fas/fisiología , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/biosíntesis , Línea Celular Tumoral , Humanos , Cinética , Transducción de Señal/inmunologíaRESUMEN
Adenovirus-mediated gene therapy for hematopoietic malignancies, especially those derived from B cells, is difficult due to systemic nature of these diseases. More importantly, most tumor cells derived from B cell lineage express a very low level of the adenovirus receptor hCAR; thus, warranting the design of adenoviral vectors with high affinity to abundant B cell surface molecules. To mimic this approach and to test the validity of adenoviral vectors in gene therapy of disseminated malignancies, we created an hCAR-expressing follicular lymphoma B cell line. The cell line was generated with the aid of a lentivirus vector carrying a novel fusion gene with EGFP replacing the cytoplasmic domain of hCAR. After verifying that this cell line was expressing the hybrid receptor in a correct manner and enrichment of the bright EGFP positive population, the cells were transduced with adenoviruses expressing the red fluorescent protein DsRed2. It was shown that regular transduction with a low viral dose (1 pfu/cell) increased the gene transfer rate by a factor of 5. Furthermore, experiments with adenovirus vector carrying the HSV-TK-GFP transgene demonstrated that the modified follicular lymphoma B cells became sensitive to ganciclovir while the parental cells remained virtually resistant to this form of gene therapy. In summary, we show here with this simple model system that adenoviral gene therapy of B cell malignancies is possible provided that correct receptors for adenovirus attachment are present on the surface of the target cells. Thus, our results warrant further modifications of adenovirus capsid to obtain vectors with specific affinity to B cell epitopes.
Asunto(s)
Resistencia a Antineoplásicos/genética , Terapia Genética , Linfoma de Células B/terapia , Linfoma Folicular/terapia , Receptores Virales/genética , Proteínas Recombinantes de Fusión/genética , Adenoviridae/genética , Antivirales/uso terapéutico , Línea Celular Tumoral , Ganciclovir/uso terapéutico , Vectores Genéticos/genética , Humanos , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Linfoma de Células B/tratamiento farmacológico , Linfoma Folicular/tratamiento farmacológico , Timidina Quinasa/genética , Transducción GenéticaRESUMEN
The BCL-2 overexpression is a hallmark of follicular lymphoma (FL). Since patients with FL often suffer from resistant to chemotherapy refractory disease, the development of new regimens is required. Herein, we analyze for the first time the effects of a B-cell lymphoma 2 (Bcl-2) antagonist, HA14-1, alone and in combination with antineoplastic agents commonly used against follicular lymphoma, in human FL cell lines with t(14;18). All cell lines tested were sensitive to HA14-1-induced cytotoxicity and apoptosis, as depicted by morphological changes, SYTO16/PI staining, oligonucleosomal DNA fragmentation and loss of Deltapsi(m). Moreover, HA14-1 significantly enhanced dexamethasone- and doxorubicin-mediated (in schedule independent and dependent manner, respectively), but not vincristine-mediated cytotoxicity and apoptosis.
Asunto(s)
Antineoplásicos/farmacología , Benzopiranos/farmacología , Fragmentación del ADN/efectos de los fármacos , Dexametasona/farmacología , Doxorrubicina/farmacología , Linfoma de Células B/metabolismo , Linfoma Folicular/metabolismo , Nitrilos/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Benzopiranos/uso terapéutico , Línea Celular Tumoral , Dexametasona/uso terapéutico , Relación Dosis-Respuesta a Droga , Doxorrubicina/uso terapéutico , Evaluación Preclínica de Medicamentos , Sinergismo Farmacológico , Humanos , Linfoma de Células B/tratamiento farmacológico , Linfoma Folicular/dietoterapia , Potenciales de la Membrana/efectos de los fármacos , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/metabolismo , Membranas Mitocondriales/patología , Nitrilos/uso terapéutico , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Vincristina/farmacología , Vincristina/uso terapéuticoRESUMEN
Glucocorticoids are commonly used in the treatment of various lymphoid malignancies. In the present study, we show that dexamethasone (Dex) induced depolarization of mitochondrial membrane, release of cytochrome c and DNA fragmentation in a human follicular lymphoma cell line, HF28RA. New protein synthesis was required before Dex-induced mitochondrial changes, and the kinetics of the apoptotic events correlated with the upregulation of the Bim protein. Furthermore, we studied whether specific inhibitors of known survival pathways would potentiate Dex-induced apoptosis. Our results show that inhibition of PKC and ERK pathways had no effect on apoptosis. In contrast, inhibition of PI3-kinase or Akt markedly enhanced Dex-induced apoptosis. The enhancement was seen at the mitochondrial level, and the kinetics of apoptosis was notably accelerated. In addition, inhibition of PI3-kinase did not alter levels of Bax, Bcl-2, Bcl-X(L) or Bim proteins in mitochondria but caused translocation of the pro-apoptotic protein Bad to mitochondria. However, inhibition of PI3-kinase-Akt pathway and subsequent translocation of Bad to mitochondria did not induce apoptosis itself. Based on these results and our current understanding of Bim and Bad action, it seems that both proteins play a synergistic role in this process. Thus, these results indicate that inhibitors of PI3-kinase-Akt pathway might be combined in future with glucocorticoids to improve the treatment of lymphoid malignancies.
Asunto(s)
Antiinflamatorios/farmacología , Apoptosis/efectos de los fármacos , Dexametasona/farmacología , Linfoma Folicular/patología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Transducción de Señal , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Caspasas/metabolismo , Citocromos c/metabolismo , Activación Enzimática , Humanos , Linfoma Folicular/tratamiento farmacológico , Linfoma Folicular/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Células Tumorales Cultivadas , Proteína X Asociada a bcl-2/metabolismo , Proteína Letal Asociada a bcl/metabolismo , Proteína bcl-X/metabolismoRESUMEN
BACKGROUND: Retinal pigment epithelium (RPE) maintains the function of photoreceptors and eyesight and is an important target for gene delivery. Since in some diseases RPE cells proliferate uncontrollably, we investigated the role of cell cycle in non-viral gene delivery into a RPE cell line (D407). METHODS: D407 (human) cells were transfected with cationic DNA complexes. Cells were synchronized into different phases of the cell cycle and transfected using poly-L-lysine (PLL) or polyethyleneimine (PEI) carriers for 3 h. The effects of different reporters (beta-galactosidase, luciferase) or promoters (CMV, SV40, tk, PDE-beta) on gene expression were evaluated 43 h later. Cellular uptake of ethidium monoazide/DNA complexes with PLL or PEI was determined by flow cytometry. Fluorescent DNA and the complexes were localized with a confocal microscope. The role of cell cycle in transcription was evaluated by stable luciferase-expressing cells. RESULTS: PLL showed lower transfection levels than PEI in synchronized cells and only slight dependence on cell cycle. PEI showed minimal efficiency at G1 phase and maximum level at S phase. All promoters and reporter genes showed dependence on cell cycle. Cellular uptake of polyplexes was highest at S phase (80-90%) and lowest at G1 phase (5-30%). Confocal microscopy showed minor differences of free DNA between groups in the nucleus, where it was largely carrier-bound. Cell cycle effects on luciferase expression were clear in stable cell line CONCLUSIONS: Transfection by polyplexes in the RPE cell line is influenced by cellular uptake and transcription, and both processes are cell-cycle-dependent. The results have implications in retinal gene therapy.
Asunto(s)
Ciclo Celular , Técnicas de Transferencia de Gen , Epitelio Pigmentado Ocular/metabolismo , Línea Celular , Humanos , Luciferasas/genética , Epitelio Pigmentado Ocular/citología , Plásmidos , TransgenesRESUMEN
Previous works suggest the involvement of mast cells in the epithelialization of chronic wounds. Since heparin is a major mediator stored in the secretory granules of mast cells, the purpose of this work was to elucidate the function of heparin in epithelialization using in vitro culture models. For this, low- and high-calcium media in monolayer and epithelium cultures of keratinocytes were used. Also, an assay based on keratinocyte adherence onto plastic surface was used as well. Heparin (0.02-200 microg/ml) inhibited keratinocyte growth in a non-cytotoxic and dose-dependent manner in low- and high-calcium media, Keratinocyte-SFM and DMEM, in the absence of growth factors and serum. Also, heparin inhibited the growth of keratinocyte epithelium in the presence of 10% fetal calf serum and DMEM. Instead, in the presence of Keratinocyte-SFM and growth factors, heparin at 2 microg/ml inhibited the growth by 18% but at higher heparin concentrations the inhibition was reversed to baseline. TNF-alpha is another preformed mediator in mast cell granules and it inhibited keratinocyte growth in monolayer and epithelium cultures. Interestingly, heparin at 2-20 microg/ml augmented or even potentiated this growth-inhibitory effect of TNF-alpha. The association of TNF-alpha with heparin was shown by demonstrating that TNF-alpha bound tightly to heparin-Sepharose chromatographic material. However, heparin could not augment TNF-alpha-induced cell cycle arrest at G0/G1 phase or intercellular adhesion molecule-1 expression in keratinocytes. In the cell adherence assay, heparin at 2 microg/ml inhibited significantly by 12-13% or 33% the adherence of keratinocytes onto the plastic surface coated with fibronectin or collagen, respectively, but this inhibition was reversed back to baseline at 20 or 200 microg/ml heparin. Also, heparin affected the cell membrane rather than the protein coat on the plastic surface. In conclusion, heparin not only inhibits or modulates keratinocyte growth and adherence but it also binds and potentiates the growth-inhibitory function of TNF-alpha.
Asunto(s)
Heparina/farmacología , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Adhesión Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Cromatografía de Afinidad , Epitelio/efectos de los fármacos , Epitelio/crecimiento & desarrollo , Fase G1/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Heparina/metabolismo , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Queratinocitos/metabolismo , Precursores de Proteínas/metabolismo , Fase de Descanso del Ciclo Celular/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
In the present study we used a human follicular lymphoma cell line, HF1A3, as an in vitro model for the antigen-driven selection process in germinal centers. Apoptosis can be induced in HF1A3 cells by B cell receptor (BCR) stimulation, but the molecular mechanisms and kinetics of this process are largely unknown. We demonstrate here that there is over 12 h delay between receptor activation and the execution phase of apoptosis, i.e. disruption of mitochondrial membrane potential, release of cytochrome c from mitochondria, caspase-3 activation and DNA fragmentation. New protein synthesis is required for mitochondrial alterations and subsequent apoptosis to occur, as these processes are completely blocked by the protein synthesis inhibitor cycloheximide. All the apoptotic events induced by BCR triggering are completely reversed by CD40 ligation with anti-CD40 antibody. CD40 ligation can reverse the apoptotic process in HF1A3 cells almost until the first mitochondrial events take place demonstrating that CD40-mediated protection operates very fast and at or before mitochondrial phase of apoptosis. Using specific inhibitors of cell signaling we could demonstrate that Raf-extracellular signal-regulated kinase, phosphatidylinositol 3-kinase, p38 or protein kinase C activation pathways are not involved in CD40-mediated protection from BCR-induced apoptosis in HF1A3 cells.