RESUMO
We have previously described the neoplastic transformation of immortalized human bronchial epithelial cells (BEAS-2B) by the combination of the c-raf-1 and c-myc protooncogenes and the concomitant induction of neuron-specific enolase mRNA expression (A. Pfeifer et al., Proc. Natl. Acad. Sci. USA, 86: 10075-10079, 1989). In this paper we describe the morphological, biochemical, and immunohistochemical characteristics of the primary c-raf-1/c-myc tumors, xenografts of these tumors, and tumors that originated from cell lines of the primary neoplasm. The tumors were morphologically characterized by the appearance of desmosomes and tonofilaments, microvilli, and dense core granules representing markers of squamous, glandular, and neuroendocrine differentiation, respectively. A total of 11 of 13 tumors were positive by immunohistochemical techniques for neuron-specific enolase, serotonin (nine of 13), and calcitonin (six of 13). Keratins were expressed in 11 of 13 tumors, and while specific keratins (K5, K7, K16/K17) decreased, there was an increase of vimentin in the tumor cells. Gastrin-releasing peptide immunoreactivity was detectable in a small number of tumors (five of 13). BEAS-2B cells transfected with the c-raf-1 and c-myc protooncogenes and cell lines established from the primary tumors expressed major histocompatibility Class II antigen which has been found on small cell lung carcinoma cells. The tumors induced by the c-raf-1 and c-myc protooncogenes resemble the multidifferentiated phenotype of small cell lung cancer frequently detected in vivo and present a defined model to study the relation between molecular markers, phenotypical appearance, and response to chemotherapeutic agents and radiation.
Assuntos
Adenocarcinoma/patologia , Brônquios/patologia , Carcinoma de Células Escamosas/patologia , Transformação Celular Neoplásica , Genes myc , Neoplasias Pulmonares/patologia , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Adenocarcinoma/genética , Adenocarcinoma/ultraestrutura , Animais , Antígenos de Neoplasias/análise , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/ultraestrutura , Linhagem Celular , Epitélio/patologia , Antígenos de Histocompatibilidade Classe II/análise , Humanos , Imuno-Histoquímica , Queratinas/análise , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/ultraestrutura , Camundongos , Transplante de Neoplasias , Fosfopiruvato Hidratase/análise , Proteínas Proto-Oncogênicas c-raf , Transplante HeterólogoRESUMO
Overexpression of SV40 T-antigen (SV40 T-Ag) has been widely used to overcome replicative senescence of human primary cells and to promote cell immortalization. However, in the case of certain cell types, such as preadipocytes, the differentiation process of immortalized cells is blocked by SV40 T-Ag expression. In this study, human telomerase reverse transcriptase (hTERT) and papillomavirus E7 oncoprotein (HPV-E7) genes were coexpressed in human preadipocytes to test whether this combination could maintain cell differentiation capacity after immortalization. We demonstrated that the HPV-E7/hTERT expressing preadipocytes displayed an indefinite life span. Interestingly, immortalized cells were diploid and presented no chromosomic alterations. These immortalized cells were able to accumulate and hydrolyze intracellular triglycerides and to express adipocyte markers. These data demonstrate, for the first time, that coexpression of hTERT and HPV-E7 in human preadipocytes allows cells not only to display an indefinite life span but also to retain their capacity to differentiate.
Assuntos
Adipócitos/citologia , Transformação Celular Viral , Proteínas Oncogênicas Virais/metabolismo , Células-Tronco/citologia , Telomerase/metabolismo , Adipócitos/metabolismo , Adulto , Diferenciação Celular , Linhagem Celular Transformada , Células Cultivadas , Proteínas de Ligação a DNA , Feminino , Expressão Gênica , Humanos , Cariotipagem , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus , Células-Tronco/enzimologia , Células-Tronco/virologiaRESUMO
The human respiratory epithelium is in direct contact with chemical carcinogens and toxins in inhaled air. Therefore, the activities of xenobiotic-metabolising enzymes in this epithelium could modulate respiratory toxicity and carcinogenesis. We determined the expression of several xenobiotic-metabolising enzymes, including phase I and phase II enzymes, in human bronchial mucosa and peripheral lung tissues. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of phase I enzymes showed CYP1A1 and CYP2C (CYP2C8 and CYP2C18) mRNA expression in all of the 14 bronchial mucosa specimens. CYP2A6 and CYP2B6 mRNAs were found in 85% of the samples, whereas 50 and 90% of the tissues displayed CYP2E1 and CYP3A5 expression, respectively. However, CYP1A2, CYP2D6 and CYP3A4 mRNAs were not detected in all samples analysed. Normal human bronchial epithelial cells (NHBE cells) cultured in serum-free conditions showed reduced P450 expression in comparison with the bronchial mucosal samples. Similar to the bronchial mucosa, the peripheral lung tissues expressed CYP1A1, CYP2A6, CYP2B6, CYP2C (CYP2C8 and CYP2C18), CYP2E1 and CYP3A5 mRNAs, but did not show detectable levels of CYP2D6. Additional P450s, such as CYP1A2 and CYP3A4, were detected. The expression of CYP1A1, CYP1A2, CYP2B6, CYP2E1 and CYP3A4/5 in peripheral lung tissues was confirmed at the protein level, whereas CYP2A6 protein was undetectable. The use of specific primers for the detection of the phase II isoenzymes belonging to the glutathione S-transferase mu (GST mu) and N-acetyl transferase (NAT) families showed that GSTM1 was expressed in 40% of the bronchial mucosa and 25% of the peripheral lung tissues, whereas GSTM3 and NAT1 mRNAs were found in all bronchial and lung samples. Finally, NAT2 expression was detected in all peripheral lung tissues, but was not detected in the bronchus. In conclusion, these results describing the diversity of the xenobiotic-metabolising enzymes expressed in the bronchus and lung tissues indicate that the human respiratory system could significantly and specifically contribute to the activation and metabolism of several environmental procarcinogens.
Assuntos
Brônquios/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , Pulmão/enzimologia , Xenobióticos/metabolismo , Adolescente , Adulto , Idoso , Linhagem Celular , Feminino , Regulação Enzimológica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Mucosa/enzimologia , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Especificidade por SubstratoRESUMO
PURPOSE: To develop a metabolically competent, human immortalized corneal epithelial cell line for use in toxicity and inflammation studies. METHODS: Primary corneal epithelial cells (P-CEPI) were immortalized by a recombinant simian virus (SV)40 T antigen retroviral vector defective for viral replication. The cells were grown in serum-free medium with the addition of bovine pituitary extract, cloned at passage 15 and one of the best-growing clones, CEPI-17-CL4, was extensively characterized for differentiation and metabolic characteristics of the human corneal epithelium. Methods used were immunostaining, reverse transcription-polymerase chain reaction (RT-PCR), northern blot analysis, and enzyme assays. RESULTS: The CEPI-17-CL4 cells showed a typical cobblestone morphology, grew to more than 200 passages and expressed the SV40 T antigen in the nucleus of every cell. Immunofluorescence staining for CEPI-17-CL4 cells was strongly positive for keratins (K)8, K18, and K19 and vimentin; weakly positive for K3, K13, and K17; and negative for K4, K7, and K14. Expression of cytokines (interleukin [IL]-1alpha, IL-1beta, IL-6, IL-8, tumor necrosis factor-alpha, and IL-ra), growth factors (transforming growth factor [TGF]-alpha, epidermal growth factors [EGF], EGF receptor [EGFR], TGF-beta1, TGF-beta2, and platelet-derived growth factor-beta) and cytochrome P450 enzymes (1A1, 2C, 2E1, and 3A5) was similar in CEPI-17-CL4 cells and human corneal epithelial samples obtained in biopsy. The CEPI-17-CL4 cells were metabolically competent for enzymes glutathione S-transferase, quinone reductase, aflatoxin aldehyde reductase, glutathione peroxidase, glutathione reductase, superoxide dismutase, and catalase. CONCLUSIONS: The CEPI-17-CL4 cells are truly immortal and express an extensive array of cytokines, growth factors, and metabolic enzymes that resemble the original tissue. These characteristics, which remain stable up to high passage, will allow reproducible, mechanistic studies on toxicity, inflammation, and wound healing.
Assuntos
Linhagem Celular Transformada , Epitélio Corneano/citologia , Epitélio Corneano/metabolismo , Citocinas/metabolismo , Epitélio Corneano/enzimologia , Epitélio Corneano/fisiologia , Olho/efeitos dos fármacos , Substâncias de Crescimento/metabolismo , Humanos , Ceratite/patologia , Ceratite/fisiopatologia , Fenótipo , Testes de ToxicidadeRESUMO
PURPOSE: To investigate the epithelial nature of primary and SV40 virus-immortalized human corneal epithelial (CEPI) cells and to study a variety of functional responses to some key inflammatory agents (bradykinin [BK], histamine, and platelet-activating factor [PAF]) and their antagonists in these cells. METHODS: Primary CEPI (P-CEPI) and clone 4 of the SV40 virus-immortalized (CEPI-17-CL4) cells were analyzed for their interaction with several monoclonal antibodies selective for various cytokeratins to define their immunocytochemical characteristics and phenotypic traits. Both cell types were tested for their ability to respond to BK, histamine, and PAF and their antagonists, using the production of [3H]inositol phosphates ([3H]IPs) as an index of receptor activation. The ability of BK, PAF, and histamine to stimulate cytokine release and the induction of mRNA for matrix metalloproteinase-1 (MMP-1) were also studied using enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction techniques, respectively. RESULTS: P-CEPI and CEPI-17-CL4 cells were both shown to possess the epithelial cell cytokeratins labeled with AE1 and AE3 antibodies. The potencies (EC50s) of BK, histamine, and PAF were similar for stimulating [3H]IPs production in P-CEPI and CEPI-17-CL4 cells: BK = 2.27 to 2.99 nM, PAF = 17.1 to 18.26 nM, and histamine = 1.65 to 5.74 microM (all n = 3 to 6). Both cell types also responded similarly to receptor-selective antagonists for BK, PAF, and histamine (Hoe-140: Ki = 10.1 to 11.9 nM; PCA-4248: Ki = 315 to 421 nM; triprolidine: Ki = 0.8 to 4.76 nM; all n = 5 to 10). Histamine (100 microM) and interleukin-1alpha (IL-1alpha, 10 ng/ml) significantly stimulated IL-6 and granulocyte macrophage colony-stimulating factor release, and histamine, BK, and PAF stimulated the mRNA for MMP-1 in these cells. CONCLUSIONS: These studies have shown that the primary and immortalized human corneal epithelial cells express functional BK (a B2 subtype), histamine (an H1 subtype), and PAF receptors and exhibit very similar immunocytochemical, signal transduction, and pharmacological properties. Therefore, the CEPI-17-CL4 cells (currently at passage 220) appear to provide a useful representative in vitro model system to study the physiological and pathologic aspects of the human corneal epithelium.
Assuntos
Bradicinina/farmacologia , Epitélio Corneano/fisiologia , Antagonistas dos Receptores Histamínicos/farmacologia , Histamina/farmacologia , Fator de Ativação de Plaquetas/farmacologia , Receptores de Superfície Celular , Receptores Acoplados a Proteínas G , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Bradicinina/antagonistas & inibidores , Linhagem Celular Transformada/citologia , Linhagem Celular Transformada/efeitos dos fármacos , Linhagem Celular Transformada/metabolismo , Células Cultivadas , Criança , Pré-Escolar , Colagenases/biossíntese , Citocinas/metabolismo , Primers do DNA/química , Ensaio de Imunoadsorção Enzimática , Epitélio Corneano/citologia , Epitélio Corneano/efeitos dos fármacos , Humanos , Fosfatos de Inositol/metabolismo , Queratinas/metabolismo , Metaloproteinase 1 da Matriz , Pessoa de Meia-Idade , Fator de Ativação de Plaquetas/antagonistas & inibidores , Glicoproteínas da Membrana de Plaquetas/metabolismo , RNA Mensageiro/metabolismo , Receptores da Bradicinina/metabolismo , Receptores Histamínicos H1/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Natural polyphenols found in rosemary have not only potent antioxidant activities but also anticarcinogenic properties. We have studied some of the molecular mechanisms involved in their chemopreventive action using in vitro human liver and bronchial cell models. Rosemary extract, or its active components, carnosol or carnosic acid are potent inhibitors of DNA adduct formation induced by benzo(a)pyrene or aflatoxin B1. At least two mechanisms are involved in the anticarcinogenic action of rosemary extract: (i) inhibition of the metabolic activation of procarcinogens catalysed by the phase I cytochrome P450 enzymes; (ii) induction of the detoxification pathway catalysed by the phase II enzymes such as glutathione S-transferase.
Assuntos
Brônquios/enzimologia , Citocromo P-450 CYP1A2/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Fígado/enzimologia , Oxigenases de Função Mista/metabolismo , Especiarias , Aflatoxina B1/farmacologia , Western Blotting , Brônquios/efeitos dos fármacos , Células Cultivadas , Citocromo P-450 CYP3A , Adutos de DNA/biossíntese , Adutos de DNA/efeitos dos fármacos , Glutationa Transferase/metabolismo , Humanos , Fígado/efeitos dos fármacos , Mutagênicos/farmacologia , Extratos Vegetais/farmacologiaRESUMO
The majority of human lung cancers arise from bronchial epithelial cells. The normal pseudostratified bronchial epithelium is composed of basal, mucous, and ciliated cells. This multi-differentiated epithelium usually responds to xenobiotics and physical injury by undergoing basal cell hyperplasia, mucous cell hyperplasia, and squamous metaplasia. One step of the multistage process of carcinogenesis is thought to involve aberrations in control of the squamous metaplastic processes. Decreased responsiveness to regulators of terminal squamous differentiation may confer a selective clonal expansion advantage to an initiated cell. We studied the effects of endogenous [e.g., transforming growth factor beta 1 (TGF-beta 1) and serum] and exogenous [e.g., 12-O-tetradecanoyl-13-phorbol-acetate (TPA), tobacco smoke condensate, and aldehydes] modifiers of normal human bronchial epithelial (NHBE) cell in a serum-free culture system. NHBE cells are growth inhibited by all of these compounds and induced to undergo squamous differentiation by TGF-beta 1 or TPA. In contrast, lung carcinoma cell lines are relatively resistant to inducers of terminal squamous differentiation which may provide them with a selective growth advantage. Chemical agents and activated protooncogenes (ras,raf,myc) altered the response to endogenous and exogenous inducers of squamous differentiation and caused extended cellular lifespan, aneuploidy, and/or tumorigenicity. The data suggest a close relationship between dysregulation of terminal differentiation pathways and neoplastic transformation of human bronchial epithelial cells.
Assuntos
Brônquios/citologia , Diferenciação Celular , Cálcio/fisiologia , Divisão Celular , Células Cultivadas , AMP Cíclico/fisiologia , Células Epiteliais , Humanos , Técnicas In Vitro , Níquel/farmacologia , Proto-Oncogenes , Fumaça , Acetato de Tetradecanoilforbol/farmacologia , Transfecção , Fatores de Crescimento Transformadores/fisiologiaRESUMO
This report characterized one of the major cytochrome P450 isozyme involved in thiabendazole metabolism. This study was undertaken by using both cultured rabbit hepatocytes treated or not with drugs known to specifically induced various cytochromes P450 isoenzymes (i.e., P450 1A1/2 by beta-naphthoflavone, P450 2B4 by phenobarbital, P450 3A6 by rifampicine and P450 4A by clofibrate) and human liver (THLE-5) and bronchial (BEAS-2B) epithelial cells expressing or not the major constitutive human cytochromes P450 (i.e., CYP1A2, 2A6, 2B6, 2C9, 2D6, 2E1 or 3A4). Only hepatocytes exposed to beta-naphthoflavone and clofibrate significantly metabolized thiabendazole to 5-hydroxythiabendazole. Extensive biotransformation of this anthelmintic only occurred in human cells expressing CYP1A2. Moreover, experiments performed on rabbit preparations showed good correlations between thiabendazole 5-hydroxylase activity and both ethoxyresorufin and methoxyresorufin O-dealkylase activities. Thus, CYP1A2 is a major isoenzyme involved in thiabendazole 5-hydroxylation.
Assuntos
Brônquios/efeitos dos fármacos , Citocromo P-450 CYP1A2/metabolismo , Inibidores Enzimáticos/farmacologia , Isoenzimas/metabolismo , Fígado/efeitos dos fármacos , Oxigenases de Função Mista/metabolismo , Tiabendazol/metabolismo , Animais , Anticolesterolemiantes/farmacologia , Brônquios/citologia , Brônquios/enzimologia , Células Cultivadas , Clofibrato/farmacologia , Citocromo P-450 CYP1A2/biossíntese , Indução Enzimática/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Humanos , Hidroxilação , Isoenzimas/biossíntese , Fígado/citologia , Fígado/enzimologia , Masculino , Oxigenases de Função Mista/química , Coelhos , Especificidade da Espécie , Tiabendazol/análogos & derivados , Tiabendazol/química , beta-Naftoflavona/farmacologiaRESUMO
The production of DNA strand breaks by N-nitrosodimethylamine (NDMA) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) has been observed in T5-2E1 (expressing human CYP2E1) and T5-1A2 (expressing human CYP1A2) human liver cells respectively, using the Comet assay. Responses were statistically significant (P<0.05) and concentration dependent (0.01-1 microg ml-1 NDMA and 0.1-10 microg ml-1 IQ) and were not observed in T5-neo cells devoid of cytochrome P450 activity. Sulforaphane (1-isothiocyanate-4-methylsulfinylbutane) (0.1-10 microM) gave a marked inhibition of DNA strand breakage by these carcinogens (P<0.05 linear regression). This was seen in the absence of cytotoxicity and in the absence of an inhibition of H2O2-induced DNA strand breakage. The ability of sulforaphane to inhibit both CYP2E1 and CYP1A2-mediated genotoxicity therefore is relevant to human isoforms of these enzymes and may contribute to a chemopreventative activity.
Assuntos
Antimutagênicos/farmacologia , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Dano ao DNA , Dimetilnitrosamina/toxicidade , Mutagênicos/toxicidade , Quinolinas/toxicidade , Tiocianatos/farmacologia , Linhagem Celular , Inibidores do Citocromo P-450 CYP1A2 , Inibidores do Citocromo P-450 CYP2E1 , Inibidores Enzimáticos/farmacologia , Humanos , Isotiocianatos , SulfóxidosRESUMO
The commonly used spice and flavouring agent, rosemary, derived from the leaves of the plant Rosmarinus officinalis L., displays antioxidant properties in foods and in biological systems. Moreover, in animal models rosemary components were found to inhibit the initiation and tumour promotion phases of carcinogenesis. In this work, we studied the mechanisms by which rosemary components block initiation of carcinogenesis by the procarcinogen benzo[a]pyrene (B[a]P) in human bronchial epithelial cells (BEAS-2B). Whole rosemary extract (6 micrograms/ml) or an equivalent concentration of its most potent antioxidant constituents, carnosol or carnosic acid, inhibited DNA adduct formation by 80% after 6 h co-incubation with 1.5 muM B[a]P. Under similar conditions, cytochrome P450 (CYP) 1A1 mRNA expression was 50% lower in the presence of rosemary components, and CYP1A1 activity was inhibited 70-90%. The observed reduction of DNA adduct formation by rosemary components may mostly result from the inhibition of the activation of benzo[a]pyrene to its ultimate metabolites. Carnosol also affected expression of the phase II enzyme glutathione-S-transferase which is known to detoxify the proximate carcinogenic metabolite of B[a]P. Treatment of BEAS-2B cells with carnosol (1 microgram/ml) for 24 h resulted in a 3- to 4-fold induction of GST pi mRNA. Moreover, expression of a second important phase II enzyme, NAD(P)H: quinone reductase, was induced by carnosol in parallel with GST pi. Therefore, rosemary components have the potential to decrease activation and increase detoxification of an important human carcinogen, identifying them as promising candidates for chemopreventive programs.
Assuntos
Anticarcinógenos/uso terapêutico , Benzo(a)pireno/antagonistas & inibidores , Benzo(a)pireno/toxicidade , Brônquios/efeitos dos fármacos , Carcinógenos/toxicidade , Adutos de DNA/biossíntese , Mutagênicos/toxicidade , Extratos Vegetais/uso terapêutico , Brônquios/enzimologia , Brônquios/metabolismo , Neoplasias Brônquicas/induzido quimicamente , Neoplasias Brônquicas/prevenção & controle , Células Cultivadas , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/genética , Adutos de DNA/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Indução Enzimática , Inibidores Enzimáticos/uso terapêutico , Glutationa Transferase/biossíntese , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Magnoliopsida/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Regenerative or hyperplastic growth promotes carcinogenesis and can be induced by many nongenotoxic carcinogens. The mitogenic potential of the rodent liver tumor promoters, cyproterone acetate and phenobarbital was investigated in primary rat hepatocyte cultures. Two premitotic markers were analyzed, the expression of two immediate-early genes (c-fos and c-myc) and the decrease in the nuclear quinacrine dihydrochloride fluorescence indicative for a G0-G1 cell cycle shift. C-fos expression and decrease in nuclear fluorescence could be induced by both chemicals, phenobarbital being the lesser potent, whereas c-myc expression was only inducible by cyproterone acetate. In situ hybridization with c-myc revealed that both chemicals enhanced c-myc mRNA levels in individual cells, however the number of responding hepatocytes was increased by cyproterone acetate only. The chemical-induced premitotic changes in hepatocytes were highly specific in terms of affected genes and ploidy levels of responding hepatocytes.
Assuntos
Carcinógenos/farmacologia , Acetato de Ciproterona/farmacologia , Expressão Gênica/efeitos dos fármacos , Genes fos , Genes myc , Fígado/metabolismo , Matriz Nuclear/metabolismo , Fenobarbital/farmacologia , Proteínas Proto-Oncogênicas c-fos/biossíntese , Proteínas Proto-Oncogênicas c-myc/biossíntese , Animais , Células Cultivadas , Hibridização In Situ , Cinética , Fígado/citologia , Fígado/efeitos dos fármacos , Masculino , Mitose , Matriz Nuclear/efeitos dos fármacos , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Many natural dietary phytochemicals found compounds found in fruits, vegetables, spices and tea have been shown in recent years to be protective against cancer in various animal models. In the light of the potential impact of these compounds on human health it is important to elucidate the mechanisms involved. We therefore developed and characterized relevant in vitro models using immortalized human epithelial cell lines derived from target tissues in carcinogenesis, such as lung, liver and colon. Assays were established, allowing the evaluation of the cytotoxic and genotoxic effects of various procarcinogens, including nitrosamines, mycotoxins and heterocyclic amines on these metabolically-competent human epithelial cell lines. These cellular models appeared to be a useful tool to study the capacity of certain food components to block the initiation stage of carcinogenesis. The ability of carnosol and carnosic acid from rosemary as well as the synthetic dithiolethione, oltipraz, to block the formation of DNA adducts, and their effects on the expression of phase I and phase II enzymes was investigated. We have observed that both rosemary extracts and oltipraz inhibited benzo(a)pyrene- or aflatoxin B1-induced DNA adduct formation by strongly inhibiting CYP450 activities and inducing the expression of glutathione S-transferase. These results in human cell models give some insight into the different mechanisms involved in the chemopreventive action of both natural and synthetic compounds in relation to phase I and phase II enzymes.
Assuntos
Brônquios/efeitos dos fármacos , Carcinógenos/toxicidade , Fígado/efeitos dos fármacos , Testes de Toxicidade/métodos , Linhagem Celular , Epitélio/efeitos dos fármacos , HumanosRESUMO
Sparse (75 to 2000 cells/cm2) density cultures of normal human bronchial epithelial cells uniformly undergo terminal squamous differentiation when incubated in medium containing serum (fetal bovine serum [FBS]) or transforming growth factor beta-1 (TGF-beta 1). It was found that the cell density of the culture affects the probability that a cell will respond to these differentiation-inducing agents. Thus whereas irreversible inhibition of DNA synthesis occurs in sparse cell-density cultures within 24 hours after exposure, only a transient (less than 36 hours) depression in DNA synthesis was seen in high (more than 10,000 cells/cm2) density cultures. In addition, although phase microscopic image analysis revealed that virtually all of the cells displayed a squamous morphology within 1 hour after exposure to FBS or TGF-beta 1, observations made 48 to 72 hours later showed the presence of clusters of small prolate spheroid-shaped cells surrounded by many involucrin-positive squamous-appearing cells. Only the small cells were capable of DNA synthesis and cell division as determined by autoradiography and time-lapse photomicrographic images. These replicating cells immediately undergo squamous differentiation if they are subcultured and reinoculated at low cell density and incubated in medium supplemented with FBS or TGF-beta 1. Therefore the probability that a human bronchial epithelial cell will be refractive to FBS- or TGF-beta 1 induced terminal squamous differentiation is solely a function of the cell density of the culture.
Assuntos
Brônquios/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Sangue Fetal , Fator de Crescimento Transformador beta/farmacologia , Autorradiografia , Brônquios/química , Brônquios/citologia , Contagem de Células , Ciclo Celular , Linhagem Celular/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , DNA/biossíntese , Células Epiteliais , Epitélio/efeitos dos fármacos , Humanos , Precursores de Proteínas/análiseRESUMO
Overexpression of c-raf-1 and the myc family of protooncogenes is primarily associated with small cell carcinoma, which accounts for approximately 25% of human lung cancer. To determine the functional significance of the c-raf-1 and/or c-myc gene expression in lung carcinogenesis and to delineate the relationship between protooncogene expression and tumor phenotype, we introduced both protooncogenes, alone or in combination, into human bronchial epithelial cells. Two retroviral recombinants, pZip-raf and pZip-myc, containing the complete coding sequences of the human c-raf-1 and murine c-myc genes, respectively, were constructed and transfected into simian virus 40 large tumor antigen-immortalized bronchial epithelial cells (BEAS-2B); this was followed by selection for G418 resistance. BEAS-2B cells expressing both the transfected c-raf-1 and c-myc sequences formed large cell carcinomas in athymic nude mice with a latency of 4-21 weeks, whereas either pZip-raf- or pZip-myc-transfected cells were nontumorigenic after 12 months. Cell lines established from tumors (designated RMT) revealed the presence of the cotransfected c-raf-1 and c-myc sequences and expressed morphological, chromosomal, and isoenzyme markers, which identified BEAS-2B cells as the progenitor line of the tumors. A significant increase in the mRNA levels of neuron-specific enolase was detected in BEAS-2B cells containing both the c-raf-1 and c-myc genes and derived tumor cell lines. The data demonstrate that the concomitant expression of the c-raf and c-myc protooncogenes causes neoplastic transformation of human bronchial epithelial cells resulting in large cell carcinomas with certain neuroendocrine markers. The presented model system should be useful in studies of molecular events involved in multistage lung carcinogenesis.
Assuntos
Antígenos Transformantes de Poliomavirus/genética , Transformação Celular Neoplásica , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Vírus 40 dos Símios/genética , Animais , Southern Blotting , Brônquios , Linhagem Celular , Quimera , Epitélio , Expressão Gênica , Humanos , Imunoensaio , Camundongos , Camundongos Nus , Peso Molecular , Transplante de Neoplasias , Proteínas Proto-Oncogênicas/isolamento & purificação , Proteínas Proto-Oncogênicas c-myc , Proteínas Proto-Oncogênicas c-raf , Vírus 40 dos Símios/imunologia , Transfecção , Transplante HeterólogoRESUMO
BACKGROUND AND AIM: Intestinal epithelial cells (IEC) are thought to participate in the mucosal defence against bacteria and in the regulation of mucosal tissue homeostasis. Reactivity of IEC to bacterial signals may depend on interactions with immunocompetent cells. To address the question of whether non-pathogenic bacteria modify the immune response of the intestinal epithelium, we co-cultivated enterocyte-like CaCO-2 cells with human blood leucocytes in separate compartments of transwell cultures. METHODS: CaCO-2/PBMC co-cultures were stimulated with non-pathogenic bacteria and enteropathogenic Escherichia coli. Expression of tumour necrosis factor alpha (TNF-alpha), interleukin (IL)-1beta, IL-8, monocyte chemoattracting protein 1 (MCP-1), and IL-10 was studied by enzyme linked immunosorbent assays (cytokine secretion) and by semiquantitative reverse transcription-polymerase chain reaction. RESULTS: Challenge of CaCO-2 cells with non-pathogenic E coli and Lactobacillus sakei induced expression of IL-8, MCP-1, IL-1beta, and TNF-alpha mRNA in the presence of underlying leucocytes. Leucocyte sensitised CaCO-2 cells produced TNF-alpha and IL-1beta whereas IL-10 was exclusively secreted by human peripheral blood mononuclear cells. CaCO-2 cells alone remained hyporesponsive to the bacterial challenge. Lactobacillus johnsonii, an intestinal isolate, showed reduced potential to induce proinflammatory cytokines but increased transforming growth factor beta mRNA in leucocyte sensitised CaCO-2 cells. TNF-alpha was identified as one of the early mediators involved in cellular cross talk. In the presence of leucocytes, discriminative activation of CaCO-2 cells was observed between enteropathogenic E coli and non-pathogenic bacteria. CONCLUSION: The differential recognition of non-pathogenic bacteria by CaCO-2 cells required the presence of underlying leucocytes. These results strengthen the hypothesis that bacterial signalling at the mucosal surface is dependent on a network of cellular interactions.
Assuntos
Bactérias/imunologia , Citocinas/biossíntese , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Leucócitos Mononucleares/imunologia , Aderência Bacteriana , Células CACO-2 , Comunicação Celular/imunologia , Citocinas/genética , Células Epiteliais/imunologia , Escherichia coli/patogenicidade , Expressão Gênica , Humanos , Interleucina-1/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Normal human bronchial epithelial (NHBE) cells are the putative progenitor cells of all types of lung cancer. NHBE cells immortalized by SV40 T-antigen retain many characteristics of the primary cells and are a useful model for investigating the role of oncogenes, tumor suppressor genes, and certain chemical carcinogens in the molecular pathogenesis of lung cancer. In this study, SV40 T-antigen-positive cells (BEAS-2B) were characterized for their metabolic functions and were shown to continue to express epoxide hydrolase, glutathione S-transferase pi, glutathione peroxidase, and catalase. To increase their metabolic activity towards human procarcinogens, human cytochrome P450 1A2 (CYP1A2) was stably expressed by introducing CYP1A2 cDNA into BEAS-2B cells either by infection with a high-titer recombinant retrovirus (pXT-1A2) or by transfection with a CYP1A2 expression vector (pCMV1A2), which produced the cell lines B-1A2 and B-CMV1A2, respectively. Cell lines established with either expression system expressed enzymatically active CYP1A2 protein and were 50- to 400-fold more sensitive to the cytotoxic effect of the carcinogen aflatoxin B1 (AFB1) than the corresponding control cell lines. The cytotoxic effects of AFB1 were paralleled by increased metabolism of AFB1 and enhanced formation of the AFB1-N7 guanine adduct in B-CMV1A2 cells. Cytotoxicity and adduct formation correlated with a significantly higher protein expression of CYP1A2 by the cytomegalovirus promoter-driven plasmid. Since this human epithelial cell line is the precursor cell type of lung cancer, has normal phase II enzymes, and exhibits highly reproducible expression of phase I enzymes, this in vitro model should aid in the evaluation of putative human carcinogens and anticarcinogens.
Assuntos
Antineoplásicos/farmacologia , Brônquios/metabolismo , Carcinógenos/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Avaliação Pré-Clínica de Medicamentos , Modelos Biológicos , Oxirredutases/metabolismo , Aflatoxina B1/metabolismo , Aflatoxina B1/farmacologia , Linhagem Celular , Transformação Celular Viral , Cloranfenicol O-Acetiltransferase/análise , Citocromo P-450 CYP1A2 , Sistema Enzimático do Citocromo P-450/genética , Adutos de DNA , Técnicas de Transferência de Genes , Humanos , Immunoblotting , Mutagênicos , Oxirredutases/genética , Plasmídeos , Retroviridae , Vírus 40 dos Símios , TransfecçãoRESUMO
Epidemiological evidence has been supporting a relationship between dietary aflatoxin B1 (AFB1) exposure, development of human primary hepatocellular carcinoma (HCC) and mutations in the p53 tumor suppressor gene. However, the correlation between the observed p53 mutations, the AFB1 DNA adducts and their activation pathways has not been elucidated. Development of relevant cellular in vitro models, taking into account species and tissue specificity, could significantly contribute to the knowledge of cytotoxicity and genotoxicity mechanisms of chemical procarcinogens, such as AFB1, in humans. For this purpose a non-tumorigenic SV40-immortalized human liver epithelial cell line (THLE cells) which retained most of the phase II enzymes, but had markedly reduced phase I activities was used for stable expression of the human CYP1A2, CYP2A6, CYP2B6 and CYP3A4 cDNA. The four genetically engineered cell lines (T5-1A2, T5-2A6, T5-2B6 and T5-3A4) produced high levels of the specific CYP450 proteins and showed comparable or higher catalytic activities related to the CYP450 expression when compared to human hepatocytes. The T5-1A2, T5-2A6, T5-2B6 and T5-3A4 cell lines exhibited a very high sensitivity to the cytotoxic effects of AFB1 and were approximately 125-, 2-, 2- and 15-fold, respectively, more sensitive than the control T5-neo cells, transfected with an expressing vector which does not contain CYP450 cDNA. In the CYP450-expressing cells, nanomolar doses of AFB1-induced DNA adduct formation including AFB1-N7-guanine, -pyrimidyl and -diol adducts. In addition, the T5-1A2 cells showed AFM1-DNA adducts. At similar levels of total DNA adducts, both the T5-1A2 and T5-3A4 cells showed, at codon 249 of the p53 gene, AGG to AGT transversions at a relative frequency of 15x10(-6). In contrast, only the T5-3A4 cells showed CCC to ACC transversion at codon 250 at a high frequency, whereas the second most frequent mutations found in the T5-1A2 cells were C to T transitions at the first and second position of the codon 250. No significant AFB1-induced p53 mutations could be detected in the T5-2A6 cells. Therefore, the differential expression of specific CYP450 genes in human hepatocytes can modulate the cytotoxicity, DNA adduct levels and frequency of p53 mutations produced by AFB1.
Assuntos
Aflatoxina B1/análise , Aflatoxina B1/toxicidade , Sistema Enzimático do Citocromo P-450/metabolismo , Adutos de DNA/análise , Genes p53/efeitos dos fármacos , Fígado/efeitos dos fármacos , Mutação , Adulto , Aflatoxina B1/metabolismo , Western Blotting , Linhagem Celular , Humanos , Fígado/metabolismoRESUMO
Normal human liver tissue and cultured human hepatocytes are valuable models to study xenobiotic metabolism and toxicity, but they only have a limited in vitro life-span and are not readily available. This report describes the establishment of replicative cultures of human adult liver epithelial cells in serum-free medium. The longevity of three of these cultures, derived from different donors, was extended by introduction of the simian virus 40 large T antigen gene. Two cell lines, THLE-2 and -3, established with a recombinant simian virus 40 large T antigen virus have undergone > 100 population doublings, are nontumorigenic when injected into athymic nude mice, have near-diploid karyotypes, and do not express alpha-fetoprotein. The cells express cytokeratin 18 and albumin in early passage, whereas higher-passage cells in logarithmic-phase growth also express cytokeratin 19. THLE-2 and -3 cells metabolize benzo[a]pyrene, N-nitrosodimethylamine, and aflatoxin B1 to their ultimate carcinogenic metabolites that adduct DNA, which indicates functional cytochrome P450 pathways. Other enzymes involved in metabolism of chemical carcinogens, such as epoxide hydrolase, NADPH cytochrome P450 reductase, superoxide dismutase, catalase, glutathione S-transferases, and glutathione peroxidase are also retained by THLE cells. Thus, these immortalized human liver cells constitute an in vitro model for pharmacotoxicological studies and for the investigation of etiology and pathogenesis of human hepatocellular carcinoma.
Assuntos
Antígenos Transformantes de Poliomavirus/genética , Carcinógenos/metabolismo , Fígado/metabolismo , Vírus 40 dos Símios/genética , Adulto , Biotransformação , Southern Blotting , Linhagem Celular Transformada , Células Cultivadas , DNA/genética , DNA/isolamento & purificação , DNA/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Células Epiteliais , Epitélio/metabolismo , Humanos , Cariotipagem , Fígado/citologia , Fenótipo , RNA/genética , RNA/isolamento & purificação , RNA Mensageiro/metabolismo , Mapeamento por Restrição , Albumina Sérica/biossíntese , Albumina Sérica/isolamento & purificação , TransfecçãoRESUMO
Loss of normal functions and gain of oncogenic functions when the p53 tumor suppressor gene is mutated are considered critical events in the development of the majority of human cancers. Human bronchial epithelial cells (BEAS-2B) provide an in vitro model system to study growth, differentiation, and neoplastic transformation of progenitor cells of lung carcinoma. When wild-type (WT) or mutant (MT; codon 143Val-Ala) human p53 cDNA was transfected into nontumorigenic BEAS-2B cells, we observed that (i) transfected WT p53 suppresses and MT p53 enhances the colony-forming efficiency of these cells, (ii) MT p53 increases resistance to transforming growth factor beta 1, and (iii) clones of MT p53 transfected BEAS-2B cells are tumorigenic when inoculated into athymic nude mice. These results are consistent with the hypothesis that certain mutations in p53 may function in multistage lung carcinogenesis by reducing the responsiveness of bronchial epithelial cells to negative growth factors.
Assuntos
Carcinoma Broncogênico/patologia , Fator de Crescimento Transformador beta/farmacologia , Proteína Supressora de Tumor p53/genética , Animais , Divisão Celular/efeitos dos fármacos , Genes p53 , Humanos , Técnicas In Vitro , Camundongos , Camundongos Nus , Transplante de Neoplasias , Transfecção , Células Tumorais CultivadasRESUMO
The ability to establish long-term cell lines of small-cell lung cancer (SCLC) has provided an in vitro model for the disease. We report on the characterization of 10 new human SCLC cell lines established from 34 cytopathologically positive specimens. Based on morphologic and biochemical characterization, growth properties, and expression of MYC and neuroendocrine properties, eight cell lines were categorized as "classic" and two cell lines as "variant". Cytogenetic examination revealed loss of all or part of 3p in all nine SCLC cell lines analyzed. The smallest deletion in common was found at 3p21-3p25. Restriction fragment length polymorphism (RFLP) analyses with probes for 3p were performed for correlation with karyotypic data and supported the cytogenetic findings. In 21 SCLC specimens (cell lines and tumor tissue) with normal DNA, used for comparison, we observed loss of heterozygosity at RAF1 (3p25) in ten of ten informative pairs by using two RFLPs from the RAF1 locus. In addition, loss of heterozygosity was noted in nine of 10 pairs examined with DNF15S2 (3p21) and four of four with D3S3 (3p14). Analysis of cell lines and tumor specimens that lacked paired normal tissue showed a homozygous pattern with the RAF1 probes in all 18 cases. Northern blots revealed significant expression of RAF1 in all cell lines tested. The transcript size was normal. The cytogenetic and RFLP data suggest that the RAF1 locus at 3p25 is involved in the chromosomal deletion of SCLC.