RESUMEN
BACKGROUND: The epithelial cell adhesion molecule (EpCAM) is overexpressed on carcinomas, and its downregulation inhibits the oncogenic potential of multiple tumour types. Here, we investigated underlying mechanisms of epcam overexpression in ovarian carcinoma. METHODS: Expression of EpCAM and DNA methylation (bisulphite sequencing) was determined for ovarian cancer cell lines. The association of histone modifications and 16 transcription factors with the epcam promoter was analysed by chromatin immunoprecipitation. Treatment with 5-Aza-2'-deoxycytidine (5-AZAC) was used to induce EpCAM expression. RESULTS: Expression of EpCAM was correlated with DNA methylation and histone modifications. Treatment with 5-AZAC induced EpCAM expression in negative cells. Ten transcription factors were associated with the epcam gene in EpCAM expressing cells, but not in EpCAM-negative cells. Methylation of an Sp1 probe inhibited the binding of nuclear extract proteins in electromobility shift assays; such DNA methylation sensitivity was not observed for an NF-κB probe. CONCLUSION: This study provides insights in transcriptional regulation of epcam in ovarian cancer. Epigenetic parameters associated with EpCAM overexpression are potentially reversible, allowing novel strategies for sustained silencing of EpCAM expression.
Asunto(s)
Antígenos de Neoplasias/genética , Carcinoma/genética , Moléculas de Adhesión Celular/genética , Epigénesis Genética/fisiología , Marcadores Genéticos/fisiología , Neoplasias Ováricas/genética , Factores de Transcripción/fisiología , Antígenos de Neoplasias/metabolismo , Secuencia de Bases , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Carcinoma/metabolismo , Moléculas de Adhesión Celular/metabolismo , Línea Celular Tumoral , Metilación de ADN/fisiología , Molécula de Adhesión Celular Epitelial , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Neoplasias Ováricas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
The human epithelial cell adhesion molecule (hEpCAM) is involved in epithelial morphogenesis and repair of epithelial tissues. We hypothesized that changes in hEpCAM expression in vivo correlate with regeneration of renal epithelia after ischaemia/reperfusion injury (IRi). Unilateral IRi was performed on kidneys of hEpCAM transgenic mice. Changes in hEpCAM expression were investigated by quantitative RT-PCR in renal cortex and medulla dissected by laser dissection microscopy and expression patterns of hEpCAM in regenerating kidneys were assessed by immunohistochemistry. The mechanism of hEpCAM promoter activation was investigated in vitro, by real-time bioluminescent imaging in HK-2 cells and in primary tubular epithelial cells (PTECs) subjected to hypoxia and reoxygenation. In vivo, the transcription of the human epcam gene significantly increased in the renal cortex during tubular re-epithelialization (p < 0.01). Moreover, the number of tubuli that expressed hEpCAM protein more than doubled in the renal cortex during regeneration. De novo expression of hEpCAM was detected in the S1 segments of proximal tubuli. Under hypoxic conditions in vitro, activity of the hEpCAM promoter was up-regulated two-fold in the HK-2 proximal epithelial cell line. Moreover, both in primary proximal epithelial cells and in HK-2 cells, hEpCAM protein expression was increased after hypoxia and reoxygenation. The significant up-regulation of hEpCAM during post-ischaemic renal regeneration in vivo and during in vitro hypoxia indicates that hEpCAM expression is associated with renal regeneration.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Moléculas de Adhesión Celular/metabolismo , Células Epiteliales/patología , Riñón/patología , Riñón/fisiología , Regeneración , Regulación hacia Arriba , Animales , Antígenos de Neoplasias/análisis , Antígenos de Neoplasias/genética , Moléculas de Adhesión Celular/análisis , Moléculas de Adhesión Celular/genética , Línea Celular , Molécula de Adhesión Celular Epitelial , Células Epiteliales/metabolismo , Humanos , Hipoxia/metabolismo , Hipoxia/patología , Inmunohistoquímica , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patologíaRESUMEN
There have been a number of theoretical treatments of excitons in DNA, most neglecting both the intrachain and interchain wavefunction overlaps of the electron and hole, treating them as Frenkel excitons. Recently, the importance of the intrachain and interchain coupling has been highlighted. Experiments have shown that in (dA)n oligomers and in duplex (dA)n.(dT)n, to be abbreviated (A/T), where A is adenine and T is thymine, the exciton wavefunction is delocalized over several bases. In duplexes it is possible to have charge-transfer (CT) excitons. Theoretical calculations have suggested that CT excitons in DNA may have lower energy than single chain excitons. In all the calculations of excitons in DNA, the polarization of the surrounding water has been neglected. Calculations have shown, however, that polarization of the water by an excess electron or a hole in DNA lowers its energy by approximately 1/2 eV, causing it to become a polaron. It is therefore to be expected that polarization charge induced in the surrounding water has a significant effect on the properties of the exciton. In what follows, we present calculations of some properties CT excitons would have in an A/T duplex taking into account the wavefunction overlaps, the effect of the surrounding water, which results in the electron and hole becoming polarons, and the ions in the water. As expected, the CT exciton has lowest energy when the electron and hole polarons are directly opposite each other. By appropriate choice of the dielectric constant, we can obtain a CT exciton delocalized over the number of sites found in photoinduced absorption experiments. The absorption threshold that we then calculate for CT exciton creation in A/T is in reasonable agreement with the lowest singlet absorption deduced from available data.
Asunto(s)
ADN/química , Electrones , Transferencia de Energía , Fenómenos Químicos , Química Física , Modelos Moleculares , Poli dA-dT/química , Teoría Cuántica , Agua/químicaRESUMEN
Pompe disease is a rare autosomal recessive lysosomal storage disease caused by deficiency of acid-alpha-glucosidase (GAA). This deficiency results in glycogen accumulation in the lysosomes, leading to lysosomal swelling, cellular damage and organ dysfunction. In early-onset patients (the classical infantile form and juvenile form) this glycogen accumulation leads to death. The only therapy clinically available is enzyme replacement therapy, which compensates for the missing enzyme by i.v. administration of recombinant produced enzyme. The development of clinically relevant animal models gained more insight in the disease and allowed evaluation of recombinant enzyme therapy. Several therapies are currently under investigation for Pompe disease, including gene therapy. This review gives an overview of the available knockout mouse models, of the in vitro and in vivo studies performed using recombinant produced enzyme. Furthermore, it describes current therapeutic approaches for Pompe disease as well as experimental therapies like gene correction therapy.
Asunto(s)
Enfermedad del Almacenamiento de Glucógeno Tipo II/tratamiento farmacológico , Enfermedad del Almacenamiento de Glucógeno Tipo II/fisiopatología , alfa-Glucosidasas/uso terapéutico , Animales , Modelos Animales de Enfermedad , Terapia Genética , Glucano 1,4-alfa-Glucosidasa/deficiencia , Glucano 1,4-alfa-Glucosidasa/uso terapéutico , Enfermedad del Almacenamiento de Glucógeno Tipo II/enzimología , Humanos , Ratones , Ratones Noqueados , Terapias en Investigación , alfa-Glucosidasas/deficienciaRESUMEN
Although some successes have been reported using adenoviral vectors for the treatment of cancer, adenoviral cancer gene therapy is still hampered by the lack of sufficient tumor cell killing. To increase the efficiency, adenoviruses have been modified to replicate specifically in tumor tissues by using tumor specific promoters controlling genes essential for adenoviral replication. However, many conditionally replicating adenoviral vectors replicate in one tumor type only, which limits their application. The epithelial glycoprotein-2 (EGP-2) promoter is active in a broad variety of carcinomas, the most common type of cancer. We utilized this promoter to restrict adenoviral replication. In this report we demonstrate that the potency of the replication-competent adenovirus AdEGP-2-E1 to specifically lyse EGP-2 positive cells is comparable to wild-type adenovirus (AdWT). In addition, we show that in vivo AdEGP-2-E1 replicates as efficient as AdWT in EGP-2 positive tumor cells. On the contrary, in EGP-2 negative cell lines as well as in primary human liver samples, the replication was attenuated up to 4-log in comparison to wild-type virus. This report clearly shows the potency of the EGP-2 promoter to mediate highly efficient and specific adenoviral replication for carcinoma gene therapy.
Asunto(s)
Adenoviridae/genética , Antígenos de Superficie/genética , Carcinoma/genética , Replicación Viral/genética , Animales , Línea Celular Tumoral , Vectores de Enfermedades , Molécula de Adhesión Celular Epitelial , Escherichia coli/genética , Femenino , Humanos , Inmunohistoquímica , Técnicas In Vitro , Hígado/efectos de los fármacos , Hígado/virología , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Adenoviral vectors are widely used in cancer gene therapy. After systemic administration however, the majority of the virus homes to the liver and the expressed transgene may cause hepatotoxicity. To restrict transgene expression to tumor cells, tumor- or tissue-specific promoters are utilized. The tumor antigen epithelial glycoprotein-2 (EGP-2), also known as Ep-CAM, is expressed in many cancers from different epithelial origins. In this study, the EGP-2 promoter was shown to restrict the expression of luciferase and thymidine kinase in an adenoviral context in different cell lines. In vivo, the EGP-2 promoter mediated efficient expression of luciferase in tumors but showed a 3-log lower activity in liver tissue when compared with the cytomegalovirus (CMV) promoter. Similarly, the EGP-2 promoter mediated specific cell killing after ganciclovir treatment in EGP-2-positive cells. Moreover, in vivo, this treatment regiment did not cause any rise in the liver enzymes aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT), demonstrating absence of liver toxicity. In contrast, CMV-mediated expression of thymidine kinase in combination with ganciclovir treatment resulted in high ASAT and ALAT values. This study demonstrates the value of the EGP-2 promoter to restrict transgene expression to a broad range of tumor types, thereby preventing liver toxicity.
Asunto(s)
Antígenos de Neoplasias/genética , Moléculas de Adhesión Celular/genética , Regulación Neoplásica de la Expresión Génica , Terapia Genética/métodos , Neoplasias/terapia , Regiones Promotoras Genéticas/genética , Adenoviridae , Alanina Transaminasa/metabolismo , Animales , Aspartato Aminotransferasas/metabolismo , Línea Celular Tumoral , Cartilla de ADN , Molécula de Adhesión Celular Epitelial , Ganciclovir/toxicidad , Vectores Genéticos/genética , Humanos , Luciferasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Timidina Quinasa/metabolismo , Timidina Quinasa/toxicidad , Pruebas de Toxicidad , Transgenes/genéticaRESUMEN
A wide range of strategies in cancer immunotherapy has been developed in the last decade, some of which are currently being used in clinical settings. The development of these immunotherapeutical strategies has been facilitated by the generation of relevant transgenic animal models. Since the different strategies in experimental immunotherapy of cancer each aim to activate different immune system components, a variety of transgenic animals have been generated either expressing tumor associated, HLA, oncogenic or immune effector cell molecule proteins. This review aims to discuss the existing transgenic mouse models generated to study and develop cancer immunotherapy strategies and the variable results obtained. The potential of the various transgenic animal models regarding the development of anti-cancer immunotherapeutical strategies is evaluated.
Asunto(s)
Inmunoterapia/métodos , Neoplasias/terapia , Animales , Antígenos de Neoplasias/inmunología , Antígenos de Histocompatibilidad/genética , Humanos , Ratones , Ratones Transgénicos , Neoplasias ExperimentalesRESUMEN
The human pancarcinoma-associated epithelial glycoprotein-2 (EGP-2), a M(r) 38,000 transmembrane antigen also known as 17-1A or Ep-CAM, is commonly used for targeted immunotherapy of carcinomas because it is strongly expressed by most carcinomas. EGP-2 is, however, also expressed in most normal epithelia. To evaluate anti-EGP-2-directed treatment-associated effects on tumors and on EGP-2-positive normal tissue, we generated EGP-2-expressing transgenic mice. A 55-kb DNA fragment consisting of the 14-kb genomic coding sequence of the human EGP-2 gene with approximately 10-kb-upstream and approximately 31-kb-downstream sequences was isolated and used to direct EGP-2 expression in an epithelium-specific manner. In the EGP-2 transgenic mice, EGP-2 appeared to be specifically expressed in all of those epithelial tissues that also express EGP-2 in humans, whereas all of the other tissues were negative. The specific in vivo localization of the i.v. administered anti-EGP-2 monoclonal antibody MOC31 was studied in EGP-2-positive and -negative tumors induced s.c. in this EGP-2 transgenic mouse model. Immunohistochemical analysis showed specific localization of MOC31 in the EGP-2-positive tumors but not in the EGP-2-negative tumors. No anti-EGP-2 monoclonal antibody localization was observed in any of the EGP-2-positive normal mouse tissues, which indicated a limited in vivo accessibility. In conclusion, an EGP-2 transgenic mouse model has been generated that expresses the EGP-2 antigen as in humans and, therefore, can serve as a model to evaluate the efficacy and safety of a variety of anti-EGP-2-based immunotherapeutic modalities in both tumors and normal tissue.
Asunto(s)
Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/inmunología , Inmunoterapia/métodos , Melanoma Experimental/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Antígenos de Neoplasias/biosíntesis , Moléculas de Adhesión Celular/biosíntesis , Modelos Animales de Enfermedad , Molécula de Adhesión Celular Epitelial , Femenino , Humanos , Masculino , Melanoma Experimental/genética , Melanoma Experimental/terapia , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Regiones Promotoras GenéticasRESUMEN
The UBE1L gene isolated from the chromosome 3p21 region has an extremely reduced level of mRNA in lung cancer. Sequence analysis showed a 45% homology to the human ubiquitin-activating enzyme E1 at the amino acid level. To further characterize the protein product, we generated UBE1L protein-specific antibodies. Immunoblot analysis revealed a full-length gene product of approximately 112 kDa. Assessment of the level and distribution pattern of the UBE1L protein in normal and tumor tissue using the generated antibodies showed that the UBE1L protein was present in normal lung cells and non-lung cancer cell lines, but was undetectable in all 14 human lung cancer cell lines analyzed. This difference in expression of the UBE1L protein between normal lung tissue and lung tumor-derived cell lines suggests a possible involvement of an E1-like protein in the origin and/or progression of lung tumors.
Asunto(s)
Ligasas/metabolismo , Neoplasias Pulmonares/enzimología , Western Blotting , Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Ligasas/genética , Ligasas/inmunología , Neoplasias Pulmonares/patología , Células Tumorales Cultivadas , Enzimas Activadoras de Ubiquitina , Ubiquitina-Proteína LigasasRESUMEN
The human pancarcinoma-associated epithelial glycoprotein-2 (EGP-2), also known as 17-1A or Ep-CAM, is a 38-kDa transmembrane antigen, commonly used for targeted immunotherapy of carcinomas. Although strongly expressed by most carcinomas, EGP-2 is also expressed in most simple epithelia. To evaluate treatment-associated effects and side-effects on tumor and normal tissue respectively, we generated an EGP-2-expressing transgenic Wistar rat. To express the cDNA of the EGP-2 in an epithelium-specific manner, the 5' and 3' distal flanking regions of the human keratin 18 (K18) gene were used. EGP-2 protein expression was observed in the liver and pancreas, whereas EGP-2 mRNA could also be detected in lung, intestine, stomach and kidney tissues. In this rat, EGP-2-positive tumors can be induced by injecting a rat-derived carcinoma cell line transfected with the GA733-2 cDNA encoding EGP-2. Transgenic rats were used to study specific in vivo localization of an i.v. anti-EGP-2 monoclonal antibody, MOC31, applied i.v. Immunohistochemical analyses showed the specific localization of MOC31 in s.c. induced EGP-2-positive tumors, as well as in the liver. In contrast, in EGP-2-transgenic rats, MOC31 did not bind to EGP-2-negative tumors, the pancreas, or other normal tissues in vivo. In conclusion, an EGP-2-transgenic rat model has been generated that serves as a model to evaluate the efficacy and safety of a variety of anti-EGP-2-based immunotherapeutic modalities.
Asunto(s)
Anticuerpos Monoclonales/farmacocinética , Antígenos de Neoplasias/genética , Carcinoma/terapia , Moléculas de Adhesión Celular/genética , Inmunización Pasiva , Animales , Animales Modificados Genéticamente , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Antígenos de Neoplasias/biosíntesis , Antígenos de Neoplasias/inmunología , Carcinoma/inmunología , Carcinoma/patología , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/inmunología , ADN Complementario/genética , Molécula de Adhesión Celular Epitelial , Células Epiteliales/metabolismo , Femenino , Humanos , Técnicas para Inmunoenzimas , Queratinas/genética , Masculino , Trasplante de Neoplasias , Especificidad de Órganos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Wistar , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Transfección , Células Tumorales Cultivadas , Vísceras/metabolismoRESUMEN
The mechanism of activation of glutamine production by the hindlimb during diabetic ketoacidosis (DKA) was investigated in rats. Muscle glutamine production was estimated to account for over 90% of the total glutamine produced by the hindlimb. DKA produced significant increases in the concentrations of NH4+ and IMP in hindlimb muscles, suggesting that AMP deaminase is activated by DKA. NH4Cl- and HCl-induced acidosis did not produce these changes, indicating either that acidosis itself is not the stimulus for increased AMP deaminase activity or that the more severe degree of acidosis accompanying DKA is necessary for activation. Muscle glutamine concentrations were depressed in DKA. Experiments with isolated epitrochlearis muscle showed that the transport and permeability properties of the muscle cells (as judged by uptake and release of alpha-aminoisobutyrate and glutamine) were not altered by DKA. However, glutamine uptake by muscle cells was significantly inhibited by L-leucine, the concentration of which, along with other branched-chain amino acids, is markedly elevated in DKA.
Asunto(s)
Cetoacidosis Diabética/metabolismo , Glutamina/metabolismo , Músculos/metabolismo , Tejido Adiposo/metabolismo , Alanina/metabolismo , Aminoácidos/sangre , Ácidos Aminoisobutíricos/metabolismo , Animales , Agua Corporal/metabolismo , Miembro Posterior , Técnicas In Vitro , Leucina/farmacología , Masculino , Músculos/citología , Ratas , Ratas Endogámicas , Piel/metabolismo , Inanición/metabolismoRESUMEN
beta-Hydroxybutyrate (but not acetoacetate) caused marked inhibition of ammonia production and glutamine extraction in isolated perfused kidneys from normal rats. Glutamine synthesis was not affected by beta-hydroxybutyrate (BHB). Measurement of metabolite levels in freeze-clamped kidneys showed that BHB increased glutamine concentration, decreased ammonia concentration, and reduced the mitochondrial NAD+/NADH ratio (calculated) in perfused kidneys. BHB inhibited flux through the glutamate dehydrogenase pathway, probably as a result of reduction in the NAD+/NADH ratio, in isolated renal mitochondria. In isolated perfused kidneys from acidotic rats, ammonia production and mitochondrial NAD+/NADH were both elevated and BHB did not inhibit renal ammoniagenesis. Although ammonia production in the acidotic kidneys was not directly related to the mitochondrial NAD+/NADH ratio, the elevation of this ratio may have permitted a normal rate of oxidation of glutamine end products--which is essential for maintaining the elevated ammoniagenesis--to take place in the presence of BHB.