RESUMEN
Strategies that enable E1-defective recombinant adenoviruses to selectively undergo replication in neoplastic tissue may be useful for future investigations or therapies of malignancies. A growing body of evidence suggests that some molecular alterations commonly associated with malignancies, such as p53 mutations, can modify the specific E1 requirements for replication of human serotype adenoviruses. In the studies reported here, a panel of human non-small cell lung cancer cell lines with previously defined p53 status were characterized for basal interleukin-6 (IL-6) and bcl-2 content because previous studies have indicated both proteins can functionally substitute for the replication requirements provided by native E1 viral proteins. Cell lines were infected with E1-defective adenovirus 5 and simultaneously transfected with different combinations of E1 plasmids, or a bcl-2 expression plasmid, and adenovirus present in the cells was quantified 6 days later. These assays demonstrated that E1A with both 19- and 55-kDa E1B-encoding plasmids were required for maximal adenoviral replication, independent of the varying p53/IL-6/basal bcl-2 phenotypes of the host cell lines. E1A was required for maximal replication enablement, independent of the basal IL-6 content of these cell lines, and exogenous IL-6 also did not obviate the E1A requirement. Interestingly, the bcl-2 expression plasmid did not consistently substitute for the 19-kDa expression plasmid in the context of this replication complementation assay. These results suggest that (1) basal levels of IL-6 greater than that present in these cell lines are necessary for functional replacement of the E1A replication function and (2) bcl-2 does not predictably substitute for the 19-kDa E1B replication function in the context of trans complementation.
Asunto(s)
Adenoviridae/fisiología , Virus Defectuosos/fisiología , Replicación Viral/fisiología , Proteínas E1A de Adenovirus/fisiología , Proteínas E1B de Adenovirus/fisiología , Genes bcl-2/fisiología , Genes p53/fisiología , Prueba de Complementación Genética , Humanos , Interleucina-6/metabolismo , Células Tumorales CultivadasRESUMEN
n-Butyrate (butyrate) has been shown to amplify transgene expression in cells infected with E1-defective adenoviruses. The present studies were undertaken in order to better define the actions of butyrate in the context of adenovirus gene expression, and to attempt to elucidate the mechanism by which butyrate mediates the transgene amplification. It was found that butyrate amplified viral transgene expression over a concentration range of 0.5-5 mM, and that the amplification required an exposure of 12-24 hr for maximal effect. Western blot analysis of representative viral proteins showed that butyrate treatment amplified DNA-binding protein, but not fiber protein. A transient adenoviral replication system suggested that butyrate had a modest inhibitory effect on replication of the E1-defective adenovirus. Use of a specific inhibitor of histone deacetylase, trichostatin A (TSA), reproduced the amplification of the viral transgene product achieved with the butyrate. In contrast, adenoviral transgene expression could not be amplified by TSA treatment in a cell line known to have a TSA-resistant histone deacetylase. Butyrate amplified steady-state gene expression of the viral transgene, but had no detectable effects on either DNA-binding protein or fiber steady-state gene expression. Nuclear run-off experiments showed that both butyrate and TSA caused an increase in the viral transgene transcription. It was concluded that inhibitors of histone deacetylase amplify adenoviral transgene expression at the transcriptional level.
Asunto(s)
Adenovirus Humanos/genética , Butiratos/farmacología , Inhibidores de Histona Desacetilasas , Virus Defectuosos/genética , Inhibidores Enzimáticos/farmacología , Regulación Viral de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Ácidos Hidroxámicos/farmacología , ARN Viral/biosíntesis , Proteínas Recombinantes/genética , Transgenes , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Previous work by this group has established that E1-defective, recombinant adenoviruses can be replication-enabled by the codelivery of a plasmid encoding the deleted E1 functions, a strategy now designated conditional replication-enablement system for adenovirus (CRESA). In the studies reported here, the original replication-enabling plasmid was replaced by two separate plasmids that encoded the necessary E1A and E1B functions, respectively. An RNA transcript encoding the requisite E1A functions was shown to substitute functionally for the E1A plasmid without significant loss of new adenovirus production in in vitro experiments. No replication competent adenovirus was detectable in the cells treated with the plasmids, or the RNA and plasmid combinations. Subcutaneous human tumor nodules containing a fraction of cells cotransduced with the replication-enabling RNA + DNA and an adenovirus containing a herpes simplex virus thymidine kinase (HSVtk) expression cassette were reduced to a greater extent than control nodules containing the same fraction of cells cotransduced with the virus and an irrelevant plasmid. These experiments show that an E1-defective adenovirus can be conditionally replication-enabled by an RNA transcript encoding the required E1 functions, and that the replication-enablement is sufficient to produce an augmentation of an adenovirus-mediated therapeutic effect in vivo.
Asunto(s)
Adenoviridae/genética , Proteínas E1A de Adenovirus/genética , Terapia Genética/métodos , Vectores Genéticos , Neoplasias Experimentales/terapia , ARN Viral , Animales , Ganciclovir/uso terapéutico , Células HeLa , Humanos , Ratones , Timidina Quinasa/genética , Células Tumorales CultivadasRESUMEN
Achieving limited recombinant viral replication may provide a means of amplifying viral-mediated gene transfer in vivo. We have previously shown that cotransduction of an E1-defective adenovirus with a plasmid containing the deleted E1 functions into prostate carcinoma cells resulted in E1-defective virus production by those cells. The studies described here have extended these findings to more firmly establish the capacity of the trans complementation approach to achieve amplification of recombinant viral transgene expression. The recombinant virus used for all the studies was AdCMV-luc which contained a luciferase expression cassette; the replication-enabling plasmid, pE1, encoded the E1 functions deleted from AdCMV-luc. Quantitative in vitro studies with the HeLa cell line showed that for each plaque forming unit of AdCMV-luc originally exposed to the cells, 0.54 x 10(3) new replication-defective viruses were detected in supernatants and lysates over the following 4 days. Multiple cell lines were shown to support new virus production following cotransduction of AdCMV-luc and pE1. Small numbers of replication-competent viruses were detected in the lysates and supernatants from the cotransduced cells such that for every 10(5) replication-defective viruses approximately two replication-competent viruses were produced. Tumor nodules produced by engrafting a mixture of AdCMV-luc/pE1-cotransduced HeLa cells with uninfected HeLa cells yielded much higher levels of luciferase expression than control tumors containing mixtures of cells infected with AdCMV-luc alone. In total, these results demonstrate new virus production by cells receiving a replication-defective adenovirus and a replication-enabling plasmid are capable of amplifying recombinant viral transgene expression in vivo.
Asunto(s)
Adenoviridae/fisiología , Virus Defectuosos/genética , Plásmidos , Transducción Genética , Replicación Viral/genética , Adenoviridae/genética , Animales , Regulación Viral de la Expresión Génica/genética , Células HeLa , Humanos , Ratones , Recombinación Genética , TransgenesRESUMEN
Replication-incompetent adenovirus is conventionally produced by cells that supply replication-enabling proteins from viral sequences present in trans. As an alternative means of recombinant adenovirus production, replication-enabling E1A sequences were cotransduced into human prostate carcinoma cells infected with an E1A-deleted adenovirus containing a luciferase expression cassette. The replication-enabling plasmid was cotransduced by ionic linkage to the recombinant adenovirus exterior. Cells cotransduced with the replication-enabling plasmid made new adenovirus with titers up to 8 x 10(6) in the supernatants 72-120 hr after transduction. Like the parent virus, the virus present in the cotransduced supernatants and lysates was capable of transferring luciferase activity to new cells. The virus present in the cotransduced cell supernatants was amplified and shown to be identical to the parent virus by genomic analysis. It was concluded that simultaneous addition of a replication-defective adenovirus and a replication-enabling gene sequence in a trans configuration converts some of the cotransduced cells into recombinant adenovirus-producing cells.
Asunto(s)
Proteínas E1A de Adenovirus/genética , Adenovirus Humanos/genética , Virus Defectuosos/genética , Vectores Genéticos/genética , Plásmidos/genética , Proteínas E1A de Adenovirus/fisiología , Adenovirus Humanos/fisiología , Carcinoma/patología , Virus Defectuosos/fisiología , Genes Reporteros , Prueba de Complementación Genética , Humanos , Luciferasas/biosíntesis , Luciferasas/genética , Masculino , Neoplasias de la Próstata/patología , Proteínas Recombinantes de Fusión/biosíntesis , Transfección/métodos , Células Tumorales Cultivadas , Replicación ViralRESUMEN
Human surfactant protein A (SPA) expression is considered a marker of respiratory epithelial differentiation. Non-small cell lung cancers (NSCLC) are respiratory epithelial derivatives, and it was previously shown that a minority of these cancers expressed SPA, presumably a consequence of their respiratory epithelial origin. In the studies reported here, SPA-I gene transcriptional regulatory sequences were localized to a 2.75-kb genomic 5'-flanking region fragment obtained by screening a human genomic library. The 2.75-kb fragment was used to direct a luciferase coding sequence transcriptionally within a plasmid construct. In plasmid transduction experiments, the SPA-directed luciferase plasmid produced significant luciferase activity in the SPA-expressing NSCLC cell line, H441, but only background levels in the non-SPA-expressing A549 cells. Because Northern blot analysis of resected NSCLC showed that the majority expressed SPA, an SPA-transcriptional targeting strategy was investigated using chimeric toxin genes comprising the coding sequence for herpes simplex virus thymidine kinase (HSV-TK) under transcriptional control of SPA or SV40 regulatory sequences. As expected, transduction of the constitutive, SV40-directed plasmid followed by ganciclovir treatment reduced numbers of both the A549 and H441 cells. In contrast, the SPA-directed plasmid reduced only the SPA-expressing H441 cells and had no significant effect on the A549 cells. The results of these in vivo experiments suggest the concept of transcriptionally directing toxin genes with SPA can produce targeted toxicity in NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/terapia , Terapia Genética , Neoplasias Pulmonares/terapia , Proteolípidos/genética , Surfactantes Pulmonares/genética , Toxinas Biológicas/genética , Humanos , Neoplasias Pulmonares/cirugía , Plásmidos , Proteínas Asociadas a Surfactante Pulmonar , Secuencias Reguladoras de Ácidos Nucleicos , Simplexvirus/enzimología , Timidina Quinasa/genética , Timidina Quinasa/uso terapéutico , Toxinas Biológicas/uso terapéutico , Transcripción Genética , Células Tumorales CultivadasRESUMEN
Carcinomas are malignancies derived from epithelial cells that frequently respond poorly to conventional chemotherapy. Selective expression or transduction of toxin genes to carcinomas, i.e. molecular chemotherapy, may offer important advantages over conventional chemotherapy. As one approach to developing a means of selectively expressing toxin genes, the transcriptional regulatory sequences of a gene expressed in multiple carcinomas were used to direct expression of the herpes simplex virus thymidine kinase (HSVtk) coding sequences. The secretory leukoprotease inhibitor (SLPI) gene was found to be expressed in lung, breast, oropharyngeal, bladder, endometrial, ovarian and colorectal carcinomas. The tissue-specific transcriptional regulatory sequences were isolated and used to construct a chimeric gene in which the SLPI sequences directed HSVtk expression. SLPI-expressing carcinomas were reduced in number by transduction of the SLPI-directed toxin plasmid plus ganciclovir, but the same treatment had no effect on a cell line that did not express SLPI. These results suggest that SLPI-directed therapeutic genes could be used for directing toxicity to carcinoma tissues, especially if combined with other targeting strategies.
Asunto(s)
Carcinoma/terapia , Terapia Genética , Toxinas Biológicas/genética , Toxinas Biológicas/uso terapéutico , Adenoviridae/genética , Secuencia de Bases , Carcinoma/genética , Cartilla de ADN/genética , Femenino , Humanos , Datos de Secuencia Molecular , Plásmidos/genética , Proteínas Inhibidoras de Proteinasas Secretoras , Proteínas/genética , Proteínas Recombinantes de Fusión/genética , Inhibidor Secretorio de Peptidasas Leucocitarias , Inhibidores de Serina Proteinasa/genética , Simplexvirus/enzimología , Simplexvirus/genética , Timidina Quinasa/genética , Células Tumorales CultivadasRESUMEN
PURPOSE, PATIENTS, AND METHODS: Since transforming growth factor beta (TGF beta) has been implicated as an important mediator of pulmonary fibrosis, we measured TGF beta protein and gene expression in alveolar epithelial lining fluid (ELF) of fibrotic scleroderma lungs sampled by bronchoalveolar lavage (BAL). TGF beta protein was qualitatively examined by Western blot analysis, and quantitatively by radioreceptor assays. Gene expression was evaluated in BAL mononuclear cells by Northern blot analysis with quantification of relative gene expression by densitometric analysis of the autoradiograms. RESULTS: Normal and scleroderma subjects had a 24-kd protein that comigrated with defined human TGF beta 1 and immunoreacted with anti-TGF beta antibody. The normal population had a significantly higher average TGF beta concentration (705 pM) compared with the scleroderma subjects (177 pM). The TGF beta 1 gene was expressed in amounts that did not significantly differ between the scleroderma and normal groups. On an individual subject basis, the TGF beta concentration variability did not correlate with variations in BAL cellularity or TGF beta 1 gene expression within the recovered mononuclear cells. CONCLUSIONS: It is concluded that both normal and fibrotic lungs have TGF beta 1 present at the alveolar epithelial surface. However, in the fibrotic scleroderma lungs, TGF beta protein content and gene expression were not increased at the alveolar epithelial surface. The simultaneous analysis of TGF beta protein content, gene expression, and cellular constituents within individual ELF specimens showed that the cellular components of the ELF do not appear to be major determinants of TGF beta protein concentration at the alveolar epithelial surface.
Asunto(s)
Líquido del Lavado Bronquioalveolar/química , Fibrosis Pulmonar/diagnóstico , Esclerodermia Sistémica/diagnóstico , Factor de Crecimiento Transformador beta/química , Adulto , Anciano , Alabama/epidemiología , Autorradiografía , Northern Blotting , Western Blotting , Estudios de Evaluación como Asunto , Femenino , Expresión Génica , Hospitales Universitarios , Humanos , Masculino , Persona de Mediana Edad , Servicio Ambulatorio en Hospital , Fibrosis Pulmonar/epidemiología , Fibrosis Pulmonar/patología , Ensayo de Unión Radioligante , Esclerodermia Sistémica/epidemiología , Esclerodermia Sistémica/patología , Sensibilidad y EspecificidadRESUMEN
Gene therapy may serve as a valuable therapeutic modality for malignancies, such as lung cancer, that are poorly responsive to conventional therapies. Although many methods for transducing new genes into cells have been described, little is known about gene transduction into lung cancer, especially under conditions that might be encountered in clinical use. As a first step in addressing this important issue, the study presented here examined the ability of a recombinant retrovirus to add a selectable marker gene to the A549 non-small cell lung cancer (NSCLC) cell line under a variety of conditions. Examination of viral exposure times ranging from 30 sec to 4 hr revealed that the number of infected cells increased with every increment in time. By increasing the multiplicity of infection to 1.0 and including a polycation, Polybrene, as an infection facilitator, 0.8% of the NSCLC cells were infected with only a 30-sec viral exposure. Nebulization, a potentially attractive route of administration for pulmonary malignancies, had no significant effect on viral titer, proviral structure, or proviral transcripts. A single lyophilization did reduce viral titer by 58 +/- 6%, but did not affect the proviral structure or transcripts produced by the surviving viruses. These results suggest that recombinant retroviruses have the potential to add new genes to malignancies accessible by the airways under conditions likely required for clinical use.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/terapia , Neoplasias Pulmonares/terapia , Retroviridae/genética , Transducción Genética , Células 3T3 , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Estudios de Evaluación como Asunto , Liofilización , Marcadores Genéticos , Terapia Genética/métodos , Humanos , Neoplasias Pulmonares/genética , Ratones , Nebulizadores y Vaporizadores , Provirus/genética , Retroviridae/fisiología , Células Tumorales Cultivadas , Replicación ViralRESUMEN
Growth factors produced by alveolar macrophages are thought to promote the fibroblast proliferation within interstitial spaces of fibrotic lungs. This study investigated the possibility that the macrophage-produced growth factors might modulate the expression of basic fibroblast growth factor (bFGF) by lung fibroblasts. To evaluate this question, bFGF gene expression and protein production were evaluated in normal adult human lung fibroblast cell lines. Under normal culture conditions, the fibroblasts expressed the bFGF gene as two major transcripts (7.1, 3.7 kb). The addition of fetal calf serum (FCS) to serum-starved fibroblasts caused a 5- to 10-fold increase in bFGF expression. Steady-state bFGF expression was increased 108% by platelet-derived growth factor (PDGF) and 602% by transforming growth factor-beta (TGF-beta). Insulin-like growth factor-1 had no significant effect on bFGF expression. Nuclear runoff studies demonstrated that both PDGF and TGF-beta increased the relative rates of bFGF transcription in the fibroblasts. Western blot analysis of lysates from fibroblasts treated with either PDGF or TGF-beta had no detectable increase in bFGF protein above unstimulated controls. However, the simultaneous addition of PDGF and TGF-beta, or FCS, produced a marked increase in bFGF. These experiments show that two growth factors present in the alveolar airspace compartment of fibrotic lungs can promote the expression of bFGF within lung fibroblasts.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/metabolismo , Pulmón/metabolismo , Factor de Crecimiento Derivado de Plaquetas/fisiología , Factor de Crecimiento Transformador beta/fisiología , Adulto , Northern Blotting , Western Blotting , Línea Celular , Femenino , Factor 2 de Crecimiento de Fibroblastos/genética , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Humanos , Factor I del Crecimiento Similar a la Insulina/fisiología , Pulmón/citología , Masculino , Persona de Mediana Edad , Transcripción GenéticaRESUMEN
This study examined the morphology, in vitro growth, and two genetic responses to serum stimulation in the nonsmall cell lung cancer (NSCLC) cell lines SK-Lu-1, SK-MES-1, A427, and A549. Morphologically, all four were NSCLC: SK-Lu-1 was undifferentiated, the remainder were adenocarcinoma variants. SK-Lu-1 and SK-MES-1 were slow growing with low-anchorage independent growth capacity; the A427 and A549 lines were fast growing with high-anchorage independent growth capacity. All of the lines expressed basic fibroblast growth factor (bFGF) as a dominant 7.1 kb transcript at amounts significantly lower than in control human lung fibroblasts. bFGF expression could be upregulated by serum exposure in several nontransformed human cell lines, but only the SK-Lu-1 NSCLC cells increased bFGF after serum exposure (482%) compared with a peak increase of 1222% in the fibroblast controls. All of the NSCLC cell lines increased c-fos in response to the same serum stimulations. These results show that growth-factor gene expression can be modulated in NSCLC, and that significant differences exist among NSCLC cell lines commonly used as laboratory correlates of human disease.
Asunto(s)
Proteínas Sanguíneas/farmacología , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/patología , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Factor 1 de Crecimiento de Fibroblastos/genética , Factor 1 de Crecimiento de Fibroblastos/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteínas Proto-Oncogénicas c-fos/sangre , Proteínas Proto-Oncogénicas c-fos/genética , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/patología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
The amino acids L-leucine, L-isoleucine, and L-arginine require a subthreshold concentration of glucose to elicit insulin release and electrical activity from B-cells. There is evidence suggesting that protons couple the metabolism of glucose to the functional response of B-cells. In view of this, a permeable weak acid, sulfamerazine, was used to determine if the generation of intracellular protons could account for the permissive action of glucose. Addition of 10 mM sulfamerazine elicited constant spike activity only with 20 mM leucine. With 20 mM arginine or isoleucine, sulfamerazine induced silent depolarization no different from that caused by sulfamerazine alone. The pattern of the electrical activity of each amino acid plus 5.6 mM glucose or alpha-ketoisocaproic acid alone was qualitatively different; addition of sulfamerazine enhanced the electrical response. The permeable weak base NH4Cl at 20 mM immediately inhibited the electrical response to each amino acid plus glucose or alpha-ketoisocaproic acid alone. The effects of the permeable weak acid and base indicate that intracellular pH is important in maintaining amino acid-induced electrical activity. The permissive role of glucose may be due to provision of protons only with leucine.
Asunto(s)
Aminoácidos/fisiología , Glucosa/fisiología , Islotes Pancreáticos/fisiología , Sulfamerazina/farmacología , Potenciales de Acción/efectos de los fármacos , Cloruro de Amonio/farmacología , Animales , Permeabilidad de la Membrana Celular , Sinergismo Farmacológico , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Cetoácidos/farmacología , Masculino , Potenciales de la Membrana/efectos de los fármacos , RatonesRESUMEN
The influence of exogenous phospholipase C and melittin on electrical activity in islet B cells was determined to assess the extent to which polyphosphatidylinositol turnover serves to generate components that influence the electrical events in the plasma membrane. Phospholipase C hydrolyzes polyphosphatidylinositol to diacylglycerol and polyphosphoinositol, whereas melittin increases the susceptibility of phospholipids to phospholipases and increases the permeability of the membrane to ions. Application of both 20 mU/ml phospholipase C or 0.5 mg/ml melittin to 11.1 mM glucose elicited a time-dependent enhancement of glucose-induced electrical activity that stabilized after 10 min. Phospholipase C increased both the active phase fraction and the burst frequency, whereas melittin only increased the burst frequency. These results indicate that both compounds, which disrupt the phospholipid environment of the plasma membrane, play a role in modulating the oscillatory pattern of electrical activity in the B cell, although melittin is obviously not influencing the factors controlling the ionic events in the same manner as phospholipase C.
Asunto(s)
Venenos de Abeja/farmacología , Glucosa/farmacología , Islotes Pancreáticos/fisiología , Meliteno/farmacología , Fosfolipasas de Tipo C/farmacología , Animales , Membrana Celular/fisiología , Electrofisiología , Islotes Pancreáticos/efectos de los fármacos , Cinética , Masculino , Ratones , Ratones Endogámicos CBA , Fosfatidilinositoles/metabolismoRESUMEN
The possible role of protein kinase c in regulating the electrical events in the B-cell plasma membrane was examined by using the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), a known activator of this enzyme. TPA has been found to enhance glucose- and sulfonylurea-induced insulin secretion with little or no effect on the fluxes of 86Rb+ or 45Ca2+ across the plasma membrane. TPA, 0.2 microM, did not influence the membrane potential from 0 to 5.6 mM glucose but increased by two- to threefold the fraction of the plateau phase of the oscillatory electrical activity induced by 7.0-11.1 mM glucose. This effect of TPA was completely blocked by 0.5 mM spermidine, an inhibitor of protein kinase c. However, spermidine had no influence on the electrical activity elicited by glucose alone. Glyburide, 10 nM, initiated slow depolarization and constant spike activity after about 18 and 25 min, respectively. TPA or 2.8 mM glucose reduced the lag period for glyburide to elicit an electrical response by about 75%. The duration of the spikes was increased two- to threefold by the presence of glucose or TPA with glyburide. There were also characteristic differences in the shape of the spikes under each experimental condition. Spermidine inhibited the influence of glucose, but not TPA, on the glyburide-induced electrical response. These results indicate that TPA may influence stimulant-induced electrical events via protein kinase c or by directly altering the ionic permeability of the plasma membrane.