RESUMO
Objective: To observe the effect of uniform and shift rotation culture on the formation and activity of the alginate-chitosan (AC) microencapsulated HepLL immortalized human hepatocytes and HepG2 cells aggregates. Methods: AC microcapsulated HepG2 and HepLL cells were randomly divided into two groups. Each group was divided into 3 subgroups according to uniform and shift rotation culture.The size and number of aggregates were observed and measured under laser confocal microscopy and inverted microscope dynamically. The amount of albumin synthesis was detected by ELISA, the clearance of ammonia was detected by colorimetry, and diazepam conversion function was detected by high performance liquid chromatography (HPLC). Results: On day 6, 8, 10, 12, 14 and 16, the number and size of the aggregates, albumin synthesis, diazepam clearance and ammonium clearance increased significantly in shift rotation culture group than in uniform group (all P<0.01). The albumin synthesis, diazepam clearance, and ammonium clearance in the microencapsulated HepLL groups were significantly higher than those of HepG2 cells at any time (all P<0.01). Conclusion: Shift rotation culture can significantly promote the formation and increase the activity of AC microencapsulated HepLL and HepG2 aggregates, and HepLL cells may be more suitable for bioartificial liver than HepG2.
Assuntos
Agregação Celular/fisiologia , Técnicas de Cultura de Células/métodos , Células Hep G2/fisiologia , Hepatócitos/fisiologia , Albuminas/biossíntese , Albuminas/metabolismo , Alginatos , Amônia/metabolismo , Animais , Linhagem Celular Transformada/fisiologia , Quitosana , Diazepam/metabolismo , Ácido Glucurônico , Células Hep G2/citologia , Hepatócitos/citologia , Ácidos Hexurônicos , Humanos , Fígado Artificial , RotaçãoRESUMO
We established spontaneously immortalized Schwann cell lines from long-term cultures of adult Fischer 344 rat dorsal root ganglia (DRG) and peripheral nerves. One of these cell lines, designated immortalized Fischer rat Schwann cells 1 (IFRS1), showed spindle-shaped morphology; immunoreactivity for S100, p75 neurotrophin receptor (p75(NTR) ), glial fibrillary acidic protein (GFAP), laminin, and vimentin; and mRNA expression of neurotrophic factors (NGF, GDNF, and CNTF), neurotrophin receptors (p75(NTR) , truncated TrkB, and TrkC), cell adhesion molecules (L1, NCAM, and N-cadherin), myelin proteins [P0, PMP22, and myelin-associated glycoprotein (MAG)], transcription factors (Krox20, Sox10, and Oct6), neuregulin-1 receptors (ErbB2 and ErbB3), and an orphan G protein-coupled receptor (Gpr126). Conditioned medium (CM) obtained from IFRS1 cells exhibited potent biological activity for the promotion of neuronal survival and neurite outgrowth of cultured adult rat DRG neurons. Furthermore, light and electron microscopic analyses revealed that IFRS1 cells were capable of myelinating neurites while in coculture with adult rat DRG neurons. These findings indicate that IFRS1 cells possess some biological properties of mature Schwann cells and that the coculture system with adult DRG neurons and IFRS1 cells can be a useful tool for the study of peripheral nerve degeneration and regeneration.
Assuntos
Comunicação Celular/fisiologia , Neurônios/fisiologia , Células de Schwann/fisiologia , Animais , Compostos Azo , Linhagem Celular Transformada/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura , Meios de Cultivo Condicionados/farmacologia , Relação Dose-Resposta a Droga , Gânglios Espinais/citologia , Regulação da Expressão Gênica/fisiologia , Óperon Lac/genética , Microscopia Eletrônica de Transmissão/métodos , Proteína Básica da Mielina/metabolismo , Proteína P0 da Mielina/metabolismo , Naftalenos , Fator de Crescimento Neural/genética , Fator de Crescimento Neural/metabolismo , Fator de Crescimento Neural/farmacologia , Neuritos/efeitos dos fármacos , Neuritos/fisiologia , Neurônios/efeitos dos fármacos , Nervos Periféricos/citologia , Ratos , Ratos Endogâmicos F344 , Ratos Wistar , Células de Schwann/química , Células de Schwann/ultraestrutura , Transfecção/métodos , Tubulina (Proteína)/metabolismoRESUMO
Fibrin is essential for hemostasis; however, abnormal fibrin formation is hypothesized to increase thrombotic risk. We previously showed that in situ thrombin generation on a cell's surface modulates the 3-dimensional structure and stability of the fibrin network. Currently, we compared the abilities of extravascular and intravascular cells to support fibrin formation, structure, and stability. Extravascular cells (fibroblasts, smooth muscle) supported formation of dense fibrin networks that resisted fibrinolysis, whereas unstimulated intravascular (endothelial) cells produced coarse networks that were susceptible to fibrinolysis. All 3 cell types produced a fibrin structural gradient, with a denser network near, versus distal to, the cell surface. Although fibrin structure depended on cellular procoagulant activity, it did not reflect interactions between integrins and fibrin. These findings contrasted with those on platelets, which influenced fibrin structure via interactions between beta3 integrins and fibrin. Inflammatory cytokines that induced prothrombotic activity on endothelial cells caused the production of abnormally dense fibrin networks that resisted fibrinolysis. Blocking tissue factor activity significantly reduced the density and stability of fibrin networks produced by cytokine-stimulated endothelial cells. Together, these findings indicate fibrin structure and stability reflect the procoagulant phenotype of the endogenous cells, and suggest abnormal fibrin structure is a novel link between inflammation and thrombosis.
Assuntos
Coagulação Sanguínea/fisiologia , Células Endoteliais/fisiologia , Fibrina/ultraestrutura , Fibroblastos/fisiologia , Células Musculares/fisiologia , Adulto , Linhagem Celular Transformada/fisiologia , Células Cultivadas/fisiologia , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/citologia , Fator V/fisiologia , Fator Xa/fisiologia , Feminino , Fibrina/química , Fibrinólise , Humanos , Masculino , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Complexos Multiproteicos , Músculo Liso Vascular/citologia , Proteína C/fisiologia , Trombina/biossíntese , Tromboplastina/fisiologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The molecular basis of mechanosensation in sensory neurons has yet to be defined. We found that ND-C cells, a hybrid cell line derived from neonatal rat DRG neurons, express mechanosensitive ion channels, and provide a useful expression system for testing candidate mechanosensitive ion channels. ND-C cells retain some important features of DRG neurons such as the expression of TTX-sensitive Na(+) and acid-activated currents as well as the ability to respond to mechanical stimulation with cationic currents sensitive to the analgesic peptide NMB1. ND-C cells do not respond to agonists of the 'thermoTRP' channels, suggesting that these channels are not responsible for MA currents in these cells and DRG neurons. Furthermore, transfecting ND-C cells with the candidate mechanotransducer channel TRPA1 does not increase MA current amplitudes, despite TRPA1 being functionally expressed at the plasma membrane. This correlates well with the fact that all types of MA currents can be recorded from TRPA1-negative DRG neurons.
Assuntos
Linhagem Celular Transformada/efeitos dos fármacos , Gânglios Espinais/citologia , Mecanotransdução Celular/fisiologia , Canais de Potencial de Receptor Transitório/fisiologia , Potenciais de Ação , Amilorida/farmacologia , Animais , Animais Recém-Nascidos , Anquirinas , Canais de Cálcio/genética , Canais de Cálcio/fisiologia , Linhagem Celular Transformada/fisiologia , Células Híbridas/efeitos dos fármacos , Células Híbridas/fisiologia , Camundongos , Neuroblastoma/patologia , Técnicas de Patch-Clamp , Estimulação Física , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/fisiologia , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/fisiologia , Bloqueadores dos Canais de Sódio/farmacologia , Canal de Cátion TRPA1 , Canais de Cátion TRPC , TransfecçãoRESUMO
Analysis of cells in culture has made substantial contributions to biological research. The versatility and scale of in vitro manipulation and new applications such as high-throughput gene silencing screens ensure the continued importance of cell-culture studies. In comparison to mammalian systems, Drosophila cell culture is underdeveloped, primarily because there is no general genetic method for deriving new cell lines. Here we found expression of the conserved oncogene Ras(V12) (a constitutively activated form of Ras) profoundly influences the development of primary cultures derived from embryos. The cultures become confluent in about three weeks and can be passaged with great success. The lines have undergone more than 90 population doublings and therefore constitute continuous cell lines. Most lines are composed of spindle-shaped cells of mesodermal type. We tested the use of the method for deriving Drosophila cell lines of a specific genotype by establishing cultures from embryos in which the warts (wts) tumor suppressor gene was targeted. We successfully created several cell lines and found that these differ from controls because they are primarily polyploid. This phenotype likely reflects the known role for the mammalian wts counterparts in the tetraploidy checkpoint. We conclude that expression of Ras(V12) is a powerful genetic mechanism to promote proliferation in Drosophila primary culture cells and serves as an efficient means to generate continuous cell lines of a given genotype.
Assuntos
Técnicas de Cultura de Células/métodos , Linhagem Celular Transformada/fisiologia , Drosophila/citologia , Drosophila/genética , Animais , Linhagem Celular/classificação , Proliferação de Células , Drosophila/embriologia , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Feminino , Expressão Gênica , Genótipo , Cariotipagem , Masculino , Proteína Oncogênica p21(ras)/genética , Proteína Oncogênica p21(ras)/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
Using cationic liposomes to deliver cytotoxic molecules to the tumor microvasculature is currently being developed for the treatment of cancer and other angiogenesis-related diseases. To improve on their beneficial properties, the authors have examined whether the particular cationic lipid type and lipid content employed are important factors influencing cellular interactions and formulation effects. The authors prepared different PEG (polyethylene glycol)-modified cationic liposomes (PCLs) with varying percent cationic lipid content and lipid type, and evaluated liposome size, surface charge (zeta) potential, and cellular properties in vitro. The cell lines used were human umbilical vein (HUVEC), lung microvascular (HMVEC-L and HPVE-26), coronary microvascular (HMVEC-C), dermal microvascular (HMVEC-D), and immortalized dermal microvascular (HMEC-1) endothelial cells. In vitro experiments consisted of cellular uptake and cytotoxicity studies, fluorescence-activated cell sorting (FACS) analysis, fluorescence, and transmission electron microscopic analysis. Liposome size and zeta potential analysis of five different PCLs revealed significant differences in their physicochemical properties. Some cationic lipids formed relatively toxic liposomes compared to others. The efficiency of loading chemotherapeutic drugs (doxorubicin hydrochloride, etoposide), affinity of PCLs for endothelial cells, and formulation effects varied according to cationic lipid content and the lipid type. Cellular uptake was observed in lung, dermal, and coronary endothelial cells. Heparan sulfate proteoglycans were found present on HMEC-1 cells, which may have enabled PCL uptake. In conclusion, physicochemical properties of cationic liposomes and their ability to interact with endothelial cells are important factors to consider during the early stages of formulation development for the treatment of cancer and other angiogenesis-dependent diseases.
Assuntos
Endotélio Vascular/fisiologia , Lipídeos/fisiologia , Lipossomos/metabolismo , Linhagem Celular , Linhagem Celular Transformada/citologia , Linhagem Celular Transformada/efeitos dos fármacos , Linhagem Celular Transformada/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Doxorrubicina/toxicidade , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Etoposídeo/farmacologia , Etoposídeo/toxicidade , Humanos , Fosfatidilcolinas/metabolismo , Circulação PulmonarRESUMO
The cerebellum is involved in complex physiological functions including motor control, sensory perception, cognition, language, and emotion. Humans and animals with prion diseases are characterized clinically by ataxia, postural abnormalities and cognitive decline. Pathology in the cerebellum affected by prions includes spongiform degeneration, neuronal loss, and gliosis. To develop an in vitro model system for studying prion biology in cerebellar cells, we established and characterized an immortal cell line (CRBL) isolated from the cerebellum of mice lacking expression of a protein involved in cell cycle arrest. The characteristics of the cells include morphological heterogeneity, rapid proliferation, serum responsiveness during growth, and a change in the number of chromosomes. CRBL cells expressed both neuronal and glial cell markers as well as a considerable level of cellular prion protein, PrP(C). Upon in vitro infection, CRBL cells exhibited selective susceptibility to prions isolated from different sources. These cells chronically propagated prions from SMB cells. Strain-specific prion infection in CRBL cells was not due to instability of the cell line, allelic variance, or mutations in the PrP gene. Molecular properties of prions derived from SMB cells were maintained in the infected CRBL cells. Our results suggest that the specific interaction between a prion strain and hosts determined the selective susceptibility of CRBL cells, which reflects the conditions in vivo. In addition to the future studies revealing cellular and molecular mechanism involved in prion pathogenesis, CRBL cells will contribute to the studies dealing with prion strain properties and host susceptibilities.
Assuntos
Linhagem Celular Transformada/fisiologia , Suscetibilidade a Doenças , Neurônios/fisiologia , Príons/metabolismo , Animais , Contagem de Células , Células Cultivadas , Cerebelo/citologia , Citogenética/métodos , Citometria de Fluxo , Proteína Glial Fibrilar Ácida/metabolismo , Glicosilação , Camundongos , Camundongos Knockout , Transfecção/métodos , Tubulina (Proteína)/metabolismo , Proteína Supressora de Tumor p53/deficiênciaRESUMO
Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disease of transformed hematopoietic progenitor cells. It is now clear that the chimeric bcr/abl P210(bcr/abl) fusion protein, which is generated by the reciprocal translocation t (9; 22), inhibits apoptosis and increase proliferation. P210(bcr/abl) plays a central role in the pathophysiology of CML. The purpose of this study was to construct a cell line model that bcr/abl expression can be regulated by Tet-off inducing-expression-system. The full-length b3a2 bcr/abl cDNA was subcloned into the pTRE2hyg expression vector to construct the pT2-P210 plasmid. 293 cells were firstly transfected with Tet-off plasmid and the clone that the Tet-off system can work effectively after transfected with pTRE2hyg-LUC was selected by luciferase activity assay. The pT2-P210 plasmid was then transfected into the selected clone and cells were then selected for hygromycin B and G418 resistance. The results showed that individual subclones expressing bcr/abl after withdrawing doxycycline were 293pT2-P210 cell line. In conclusion, selected 293pT2-P210 cells are cells that bcr/abl expression can be regulated by Tet-off inducing-expression-system. They are suitable thoroughly to study the function of bcr/abl fusion gene and its signal regulation mechanism.
Assuntos
Proteínas de Fusão bcr-abl/biossíntese , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Sequência de Bases , Linhagem Celular Transformada/citologia , Linhagem Celular Transformada/fisiologia , Cromossomos Humanos Par 22/genética , Cromossomos Humanos Par 9/genética , Proteínas de Fusão bcr-abl/genética , Regulação Neoplásica da Expressão Gênica , Genes abl/genética , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-bcr/genética , Transfecção , Translocação Genética , Células Tumorais CultivadasRESUMO
SWAP-70 is a phosphatidylinositol trisphosphate (PtdIns(3,4,5)P(3)) binding protein, which acts in F-actin rearrangement. The role of SWAP-70 in oncogenic transformation of mouse embryo fibroblasts (MEFs) by v-Src was examined by use of MEFs defective in SWAP-70. v-Src morphologically transformed MEFs lacking SWAP-70, but growth of the transformed cells in culture was slower than that of cells supplemented with exogenous SWAP-70. The v-Src-transformed MEFs deficient in SWAP-70 were unable to grow in soft agar while those expressing SWAP70 readily formed colonies, suggesting that SWAP-70 is required for anchorage independent growth of v-Src transformed MEFs. When transplanted in nude mice, tumors formed by the v-Src transformed SWAP-70(-/-) MEFs were smaller than those formed by cells expressing exogenous SWAP-70. These results suggest that SWAP-70 may be required for oncogenic transformation and contributes to cell growth in MEFs transformed by v-Src.
Assuntos
Linhagem Celular Transformada/fisiologia , Proteínas de Ligação a DNA/fisiologia , Embrião de Mamíferos/citologia , Fibroblastos/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Proteínas Nucleares/fisiologia , Proteína Oncogênica pp60(v-src)/fisiologia , Animais , Transformação Celular Neoplásica/patologia , Camundongos , Antígenos de Histocompatibilidade Menor , Transdução de Sinais/fisiologiaRESUMO
We demonstrate that the Src kinase can augment gap junctional communication between cells derived from homozygous null Cx43 knockout mice. The total conductance between Src transformed cells was nearly twice that of nontransformed cells. In addition, the unitary conductance of the majority of single channel events between transformed cells was about 35% greater than that of nontransformed cells. Analysis showed that both nontransformed and transformed cells expressed at least two populations of channels, suggesting that Src increased junctional conductance by up-regulating one population and/or by increasing the unitary conductance of another population of channels. Interestingly, the conductance displayed by heterologous pairs of transformed and nontransformed cells resembled that of nontransformed cells. The majority of single channel events between heterologous pairs shifted back to lower conductances that were exhibited by nontransformed cells. Thus, nontransformed cells can effectively "normalize" the conductance of gap junction channels expressed by adjacent tumor cells.
Assuntos
Comunicação Celular/fisiologia , Linhagem Celular Transformada/fisiologia , Conexina 43/deficiência , Junções Comunicantes/fisiologia , Quinases da Família src/metabolismo , Animais , Células Cultivadas , Técnicas de Cocultura , Condutividade Elétrica , Camundongos , Camundongos KnockoutRESUMO
To clarify the effects of silica and silicates on cellular features of lymphocytes, a human T-lymphotropic virus type-1-immortalized polyclonal T-cell line, MT-2, was exposed to various concentrations of chrysotile-A, an asbestos classified as silicate. MT-2 cells underwent apoptosis in a dose- and time-dependent manner. The mitochondrial apoptotic pathway was activated during chrysotile-A-induced apoptosis of MT-2 cells, because of the phosphorylation of JNK and p38, increase of BAX and release of cytochrome-c from mitochondria to cytoplasma. In addition, anti-oxidants such as hydroxyl-radical excluders and capturers of superoxide and inhibitors of superoxide production effectively reduced the size of the apoptotic fraction in MT-2 cells cultured with chrysotile-A. These results indicate that the activation of reactive oxygen species may play a central role in asbestos-induced T-cell apoptosis, and anti-oxidants may help to prevent complications of pneumoconiosis.
Assuntos
Apoptose/efeitos dos fármacos , Asbestos Serpentinas/efeitos adversos , Dimetil Sulfóxido/farmacologia , Sequestradores de Radicais Livres/farmacologia , Superóxidos/antagonistas & inibidores , Apoptose/fisiologia , Linhagem Celular Transformada/efeitos dos fármacos , Linhagem Celular Transformada/fisiologia , Relação Dose-Resposta a Droga , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Superóxidos/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/fisiologia , Fatores de TempoRESUMO
Certification of continuous cell 293 culture used for cultivation of antineoplastic preparation Cancerolysin was carried out. The seeding and working banks of cells 293 were established and deposited for storage at the Vector Centre. The cells were certified in accordance with the WHO requirements. The cell 293 culture was shown to have high proliferative activity; morphology typical of the line; its karyotype and enzymogram are typical of human cells; the culture is not contaminated with bacteria, fungi, mycoplasms and viruses including oncogenic ones; it has high virus-producing activity; it preserves stability of all the biological properties in long-term cultivation. The seeding and working cell banks were recommended for the use in production of drugs for the treatment of oncologic patients.
Assuntos
Linhagem Celular Transformada/citologia , Linhagem Celular Transformada/fisiologia , Antineoplásicos , Humanos , Controle de Qualidade , Padrões de ReferênciaRESUMO
In this paper we have explored the role of different kinase pathways of signal transduction in proliferation control of E1A + Ras transformants, using specific inhibitors of MAP-kinases ERK, JNK, p38 and PI3-kinase. According to our data, suppression of signalling cascades driven by RI3K only arrested proliferation of E1A + Ras cells, while suppression of either MAP-kinase did not lead to noticeable antiproliferative effect. We have shown that suppression of RI3K with LY294002 gave rise to accumulation of cyclin-dependent kinase inhibitor p27(KiP1) but not p21(Waf1). Accumulation of p27(KiP1) in LY294002-treated E1A + Ras cells was accompanied by a decrease in Cyclin E-Cdk2 and Cyclin A-Cdk2 activity, which caused diminution of Rb phosphorylation and strengthening of E2F-Rb binding. Binding of E2F with hypophosphorylated Rb resulted in inhibition of E2F activity and reduction of E2F-regulated gene transcription, these genes being necessary for S-phase entry and DNA synthesis. Thus, RI3K--Akt cascade plays the key role in maintenance of autonomous proliferation of cells transformed with E1A and cHa-ras oncogenes. Inhibition of PI3K leads to p27(Kip1) accumulation and cell cycle arrest, consequently.
Assuntos
Proliferação de Células , Fosfatidilinositol 3-Quinases/fisiologia , Fosfotransferases/fisiologia , Transdução de Sinais , Proteínas E1A de Adenovirus/genética , Animais , Linhagem Celular Transformada/metabolismo , Linhagem Celular Transformada/fisiologia , Transformação Celular Neoplásica , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Ciclinas/metabolismo , Embrião de Mamíferos , Inibidores Enzimáticos , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Genes ras , MAP Quinase Quinase 4/antagonistas & inibidores , MAP Quinase Quinase 4/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosfotransferases/antagonistas & inibidores , Fosfotransferases/metabolismo , Ratos , Transformação Genética , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The Epstein-Barr-transformed B lymphoblastoid cell lines, LCL, which express antigens, are potential antigen-presenting cells (APCs) for the induction of cytotoxic T lymphocytes in vitro. However, transfecting LCL with subsequent selection by antibiotics is notoriously difficult because the plating efficiencies of LCL are reported to be 1% or less. Therefore, this study investigated the optimal conditions for increasing the transduction efficiency of a recombinant adenovirus to LCL for use as a source of APCs. The transduction efficiencies were < 13% (SD +/- 2.13) at a multiplicity of infection (MOI) of 100, while it was increased to 28% (SD +/- 9.43) at an MOI of 1000. Moreover, its efficiencies to LCL that expressed the coxsackie adenovirus receptor were increased to 60% (SD +/- 6.35) at an MOI of 1000, and were further increased to 70% (SD +/- 4.56) when combined with the centrifugal method. The cationic liposome or anionic polymer had no effect on the transduction efficiency when compared to that of the centrifugal method. These results may be used as a convenient source of target cells for a CTL assay and/or autologous APCs for the induction of the in vitro CTL responses that are specific to viral and tumor antigens.
Assuntos
Adenoviridae/genética , Linfócitos B/fisiologia , Linhagem Celular Transformada/fisiologia , Herpesvirus Humano 4/genética , Transdução Genética/métodos , Adenoviridae/metabolismo , Linfócitos B/citologia , Transformação Celular Viral , Herpesvirus Humano 4/metabolismo , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T CitotóxicosRESUMO
BACKGROUND: Two features of the progression from organ-confined to metastatic prostate cancer are dysregulation of the androgen receptor (AR) and a decrease in insulin-like growth factor-type-I receptor (IGF-IR) expression. The purpose of this study was to determine the effect of changes in IGF-IR expression on AR activity. METHODS: M12 human prostate cells were stably transfected with an AR expression construct to produce the M12-AR parental (PAR) cell line. PAR cells were implanted orthotopically into nude mice and M12-AR primary (PRI) cell lines were derived from intraprostatic tumors and metastatic cell lines (MET) were derived from PRI tumors that had metastasized to diaphragm or lung. RESULTS: Tumor formation in the prostate by PAR cells was decreased significantly compared to M12 controls. PAR, PRI, and MET cells expressed equivalent amounts of AR protein; however, IGF-IR expression was increased significantly in PAR and PRI cells. IGF-IR expression decreased in MET lines to the levels seen in M12 control cells. IGF-I significantly enhanced dihydrotestosterone (DHT)-stimulated, but not basal, AR transcriptional activity in PRI cells. In MET cells, IGF-I significantly suppressed DHT-stimulated transcriptional activity. In MET cells in which the IGF-IR was re-expressed from a retroviral vector, the effects of DHT and IGF-I on AR activity were similar to those seen in PRI cells. CONCLUSIONS: This study demonstrates that the changes in IGF-IR expression exhibited by this model of metastatic progression cause significant alterations in AR signaling and suggest that this interaction may be an important aspect of the changes seen in AR function in disease progression in vivo.
Assuntos
Androgênios/farmacologia , Di-Hidrotestosterona/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Neoplasias da Próstata/secundário , Receptores Androgênicos/genética , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada/citologia , Linhagem Celular Transformada/efeitos dos fármacos , Linhagem Celular Transformada/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Masculino , Camundongos , Camundongos Nus , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Neoplasias da Próstata/fisiopatologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , RNA Mensageiro/análise , Receptor IGF Tipo 1/genética , Receptores Androgênicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/fisiologiaRESUMO
BACKGROUND AND AIMS: Liver endothelial cells (LECs) perform an essential role in important pathophysiologic functions in the liver. Establishment of a human LEC line facilitates advances in LEC research. Here, we present immortalization of human LECs using retroviral gene transfer of simian virus 40 large T antigen (SV40T) and human telomerase reverse transcriptase (hTERT). We also demonstrate excision of SV40T and hTERT with TAT-mediated Cre/loxP recombination and subsequent cell sorting. METHODS: First, human LECs were transduced with a retroviral vector somatostatin receptor (SSR)#69 expressing SV40T and hygromycin-resistance genes flanked by a pair of loxA recombination targets. Then, cells were retrovirally superinfected with SSR#197 encoding hTERT and green fluorescent protein (GFP) cDNAs that were intervened by two loxBs. One SV40T-and hTERT-immortalized LEC clone, TMNK-1, was established and analyzed for its biologic characteristics. RESULTS: The cells were hygromycin-resistant and uniformly positive for GFP expression. TMNK-1 expressed EC markers, including factor VIII, vascular endothelial growth factor receptors (flt-1, KDR/Flk-1), and CD34, showed uptake of Di-I-acetylated-low-density lipoprotein and angiogenic potential in Matrigel assays. After lipopolysaccharide treatment, TMNK-1 produced tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 and exhibited increased expression of intra-cellular adhesive molecule-1, vascular cellular adhesive molecule-1, and VE-cadherin. After treatment with TAT-Cre recombinase fusion protein, approximately 60% of TMNK-1 was negative for GFP expression, and subsequent cell sorting of this population for GFP allowed for collection of the reverted form of TMNK-1. CONCLUSIONS: This study demonstrates the utility and efficiency of the reversible immortalization procedure to expand primary human LECs for basic studies.
Assuntos
Antígenos Transformantes de Poliomavirus/genética , Linhagem Celular Transformada/citologia , Células Endoteliais/citologia , Fígado/citologia , Telomerase/genética , Animais , Antígenos CD , Biomarcadores , Caderinas/genética , Linhagem Celular Transformada/fisiologia , Separação Celular , Proteínas de Ligação a DNA , Selectina E/genética , Produtos do Gene tat/genética , Humanos , Integrases/genética , Molécula 1 de Adesão Intercelular/genética , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Glicoproteínas de Membrana/genética , Camundongos , Camundongos SCID , Transplante de Neoplasias , Neovascularização Fisiológica , Receptores de Superfície Celular/genética , Retroviridae/genética , Receptores Toll-Like , Transdução Genética , Fator de Necrose Tumoral alfa/metabolismo , Molécula 1 de Adesão de Célula Vascular/genética , Proteínas Virais/genéticaRESUMO
Activating mutations in the juxtamembrane domain (FLT3-length mutations, FLT3-LM) and in the protein tyrosine kinase domain (TKD) of FLT3 (FLT3-TKD) represent the most frequent genetic alterations in acute myeloid leukemia (AML) and define a molecular target for therapeutic interventions by protein tyrosine kinase (PTK) inhibitors. We could show that distinct activating FLT3-TKD mutations at position D835 mediate primary resistance to FLT3 PTK inhibitors in FLT3-transformed cell lines. In the presence of increasing concentrations of the FLT3 PTK inhibitor SU5614, we generated inhibitor resistant Ba/F3 FLT3-internal tandem duplication (ITD) cell lines (Ba/F3 FLT3-ITD-R1-R4) that were characterized by a 7- to 26-fold higher IC50 (concentration that inhibits 50%) to SU5614 compared with the parental ITD cells. The molecular characterization of ITD-R1-4 cells demonstrated that specific TKD mutations (D835N and Y842H) on the ITD background were acquired during selection with SU5614. Introduction of these dual ITD-TKD, but not single D835N or Y842H FLT3 mutants, in Ba/F3 cells restored the FLT3 inhibitor resistant phenotype. Our data show that preexisting or acquired mutations in the PTK domain of FLT3 can induce drug resistance to FLT3 PTK inhibitors in vitro. These findings provide a molecular basis for the evaluation of clinical resistance to FLT3 PTK inhibitors in patients with AML.
Assuntos
Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Leucemia Mieloide , Proteínas do Leite , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Estaurosporina/análogos & derivados , Doença Aguda , Animais , Antimetabólitos Antineoplásicos/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Linhagem Celular Transformada/efeitos dos fármacos , Linhagem Celular Transformada/fisiologia , Citarabina/farmacologia , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos , Genisteína/farmacologia , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Mutagênese , Fenótipo , Fosforilação/efeitos dos fármacos , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Receptores Proteína Tirosina Quinases/química , Fator de Transcrição STAT5 , Estaurosporina/farmacologia , Transativadores/metabolismo , Tirfostinas/farmacologia , Tirosina Quinase 3 Semelhante a fmsRESUMO
We developed clonal cell lines of human bronchial smooth muscle origin by retroviral transduction of temperature-sensitive simian virus 40 large tumor (T) antigen. These cells show increased growth potential at 33 degrees C, but on shift to the nonpermissive temperature (39 degrees C), they show diminished or arrested growth. In addition to the expected reduction in the level of large T antigen, cells shifted to 39 degrees C show increased expression of the cyclin-dependent kinase inhibitor p21(Waf1/Cip1), characteristic of cells arrested in G1 of the cell cycle. Shifted cells undergo a process of cell hypertrophy, as demonstrated by increased time of flight and forward scatter, as well as increased expression of the contractile proteins alpha-smooth muscle actin, myosin light chain kinase, and SM22. Changes in contractile protein expression were regulated primarily in a posttranscriptional manner. Phosphatidylinositol 3-kinase activity was increased in shifted cells, and chemical inhibition of phosphatidylinositol 3-kinase attenuated alpha-actin and myosin light-chain kinase expression. We have developed clonal cell lines of human bronchial smooth muscle origin that may be useful for the study of airway smooth muscle biology. Furthermore, we demonstrate that arrest of airway smooth muscle cell cycle traversal can induce cellular hypertrophy, which parallels changes observed in the airways of patients with severe asthma.
Assuntos
Brônquios/citologia , Brônquios/fisiologia , Linhagem Celular Transformada/fisiologia , Células Clonais/fisiologia , Miócitos de Músculo Liso/fisiologia , Antígenos Virais de Tumores , Asma/genética , Técnicas de Cultura de Células , Divisão Celular/fisiologia , Transformação Celular Viral , Humanos , Hipertrofia/genética , Oncogenes , Fenótipo , Vírus 40 dos Símios/imunologia , TemperaturaRESUMO
AIM: To develop conditionally immortalized gastric mucosal cell lines that show distinct types of cell differentiation from transgenic mice harboring temperature-sensitive simian virus 40 (tsSV40) large T antigen. METHODS: Gastric mucosal cells from the transgenic mice were cultured at a permissive temperature (33 degrees C), and proliferative cells were then cloned by colony formation. RESULTS: Eight gastric cell lines showed epithelial-like morphology and grew at 33 degrees C. Three different types of the cell lines have been established: (1) MGE12-1, MGE3-2, and MGE509 cells expressing mRNAs for pit cell markers (gastric mucin and cathepsin E); (2) MGE02, MGE503, and MGE511 cells expressing mRNAs for pit and zymogenic (pepsinogen F) cell markers, and (3) MGE507 and MGE727 cells expressing mRNAs for pit, zymogenic, and parietal (H,K-ATPase alpha-subunit) cell markers. Moreover, the TaqMan assay showed that mRNA levels of mucin, H,K-ATPase alpha-subunit, and pepsinogen F were influenced by nonpermissive temperature (39 degrees C) in MGE503 and MGE727 cells. CONCLUSION: These gastric epithelial cell lines seem to reflect different stages of development of gastric mucosal cells.
Assuntos
Antígenos Transformantes de Poliomavirus/metabolismo , Diferenciação Celular/fisiologia , Células Epiteliais/citologia , Mucosa Gástrica/citologia , Animais , Biomarcadores , Western Blotting , Catepsina E/genética , Divisão Celular , Linhagem Celular Transformada/fisiologia , Células Epiteliais/metabolismo , Feminino , Mucinas Gástricas/genética , Mucosa Gástrica/metabolismo , ATPase Trocadora de Hidrogênio-Potássio/genética , Camundongos , Camundongos Nus , Camundongos Transgênicos , Pepsinogênios/genética , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TemperaturaRESUMO
BACKGROUND: The mesenchymal-epithelial conversion of metanephric mesenchyme (MM) in the formation of nephronic tubules has long served as a paradigm for inductive signaling in morphogenesis. However, the mechanisms underlying this differentiation have remained an enigma due to insufficient numbers of primary mesenchymal cells that must be isolated manually from animal embryos. To overcome this problem, we have established a conditionally immortalized cell line, the rat-inducible metanephric mesenchyme (RIMM-18) by transfection of primary mesenchymal cells with a vector, encoding an estradiol-dependent E1A-ER fusion protein. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR), luciferase reporter assay, electrophoretic mobility shift assay, immunocytochemical, and immunohistochemical stainings were used to characterize the established cell line. RESULTS: We demonstrate that in the presence of estradiol, the RIMM-18 cell line proliferates continuously, maintaining mesenchymal characteristics for over 40 passages. These cells are vimentin-positive and cytokeratin-negative. Under inductive conditions in the absence of estradiol, they are responsive to a number of cytokines, which are established inducers of mesenchymal cells in vivo and in vitro [i.e., fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and transforming growth factor-beta 2 (TGF-beta 2)]. We show the presence in RIMM-18 cells of specific protein markers and functionally active signaling pathways required for induction of tubule formation in MM. Furthermore, induced RIMM-18 cells change morphology, acquiring epithelial-like features, and begin to express epithelial markers (e.g., E-cadherin, cytokeratin, gamma-glutamyl-transpeptidase, and secreted frizzled-related protein 2 (sFRP2). CONCLUSION: This preliminary characterization of the RIMM-18 cell line suggests that it will be useful in the study of biochemical and molecular mechanisms of nephronic development and, possibly, of some types of renal cancer such as Wilms' tumor, which caricatures the normal process of kidney development.