RESUMEN
Immune responses against oncolytic adenovirus (Ad) vectors are thought to limit vector anti-tumor efficacy. With Syrian hamsters, which are immunocompetent and whose tumors and normal tissues are permissive for replication of Ad5-based oncolytic Ad vectors, treating with high-dose cyclophosphamide (CP) to suppress the immune system and exert chemotherapeutic effects enhances Ad vector anti-tumor efficacy. However, long-term CP treatment and immunosuppression can lead to anemia and vector spread to normal tissues. Here, we employed three cycles of transient high-dose CP administration plus intratumoral injection of the oncolytic Ad vector VRX-007 followed by withdrawal of CP. Each cycle lasted 4-6 weeks. This protocol allowed the hamsters to remain healthy so the study could be continued for ~100 days. The tumors were very well suppressed throughout the study. With immunocompetent hamsters, the vector retarded tumor growth initially, but after 3-4 weeks the tumors resumed rapid growth and further injections of vector were ineffective. Preimmunization of the hamsters with Ad5 prevented vector spillover from the tumor to the liver yet still allowed for effective long-term anti-tumor efficacy. Our results suggest that a clinical protocol might be developed with cycles of transient chemotherapy plus intratumoral vector injection to achieve significant anti-tumor efficacy while minimizing the side effects of cytostatic treatment.
Asunto(s)
Adenoviridae/fisiología , Ciclofosfamida/administración & dosificación , Vectores Genéticos/efectos de los fármacos , Neoplasias Experimentales/prevención & control , Viroterapia Oncolítica/métodos , Virus Oncolíticos/fisiología , Adenoviridae/genética , Adenoviridae/inmunología , Animales , Antineoplásicos Alquilantes/administración & dosificación , Línea Celular Tumoral , Cricetinae , Inmunosupresores/administración & dosificación , Mesocricetus , Neoplasias Experimentales/genética , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/virología , Virus Oncolíticos/genética , Virus Oncolíticos/inmunologíaRESUMEN
The adenovirus death protein (ADP) is expressed at late times during a lytic infection of species C adenoviruses. ADP promotes the release of progeny virus by accelerating the lysis and death of the host cell. Since some human lymphocytes survive while maintaining a persistent infection with species C adenovirus, we compared ADP expression in these cells with ADP expression in lymphocytes that proceed with a lytic infection. Levels of ADP were low in KE37 and BJAB cells, which support a persistent infection. In contrast, levels of ADP mRNA and protein were higher in Jurkat cells, which proceed with a lytic infection. Epithelial cells infected with an ADP-overexpressing virus died more quickly than epithelial cells infected with an ADP-deleted virus. However, KE37, and BJAB cells remained viable after infection with the ADP-overexpressing virus. Although the levels of ADP mRNA increased in KE37 and BJAB cells infected with the ADP-overexpressing virus, the fraction of cells with detectable ADP was unchanged, suggesting that the control of ADP expression differs between epithelial and lymphocytic cells. When infected with an ADP-deleted adenovirus, Jurkat cells survived and maintained viral DNA for greater than 1 month. These findings are consistent with the notion that the level of ADP expression determines whether lymphocytic cells proceed with a lytic or a persistent adenovirus infection.
Asunto(s)
Infecciones por Adenoviridae/virología , Proteínas E3 de Adenovirus/metabolismo , Adenovirus Humanos/metabolismo , Linfocitos/virología , Proteínas E3 de Adenovirus/genética , Adenovirus Humanos/genética , Línea Celular , Humanos , Liberación del Virus , Replicación ViralRESUMEN
We report that radiation enhances the antitumor efficacy of the oncolytic adenovirus vector VRX-007 in Syrian hamster tumors. We used tumor-specific irradiation of subcutaneous tumors and compared treatment options of radiation alone or combined with VRX-007 and cyclophosphamide (CP). Radiation therapy further augmented the VRX-007-mediated inhibition of tumor growth, in both CP-treated and non-CP-treated hamsters, even though radiation did not lead to increased viral replication in tumors when compared with those treated with VRX-007 alone. Moreover, tumor growth inhibition was similar in tumors irradiated either 1 week before or after injection with VRX-007, which suggests that radiation exerts its antitumor effect independently from vector therapy. Thus, our results demonstrate that these two therapies do not have to be provided simultaneously to enhance their combined effectiveness against subcutaneous hamster tumors.
Asunto(s)
Adenoviridae/fisiología , Vectores Genéticos/fisiología , Neoplasias , Virus Oncolíticos/fisiología , Radiación , Animales , Antineoplásicos Alquilantes/administración & dosificación , Antineoplásicos Alquilantes/farmacología , Línea Celular Tumoral , Cricetinae , Ciclofosfamida/administración & dosificación , Ciclofosfamida/farmacología , Femenino , Vectores Genéticos/administración & dosificación , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Neoplasias/radioterapia , Neoplasias/terapia , Viroterapia Oncolítica , Carga Tumoral/efectos de los fármacos , Carga Tumoral/efectos de la radiación , Replicación Viral/efectos de la radiaciónRESUMEN
We have previously reported that intratumoral injection of VRX-007--an Ad5 (a species C adenovirus)-based vector overexpressing adenovirus death protein--can suppress the growth of subcutaneous HaK (hamster renal cancer) tumors. VRX-007 replication and tumor growth inhibition are enhanced when the hamsters are immunosuppressed by a high dose of cyclophosphamide (CP), an immunosuppressive and chemotherapeutic agent. Here, we report that continuous immunosuppression with CP was not required for increased oncolytic activity of VRX-007 because short-term dosing or continuous dosing with the drug yielded similar antitumor results. Prolonged viral replication was found only in animals on continuous CP treatment. We used 007-Luc, a replication-competent, luciferase-expressing vector similar to VRX-007, to investigate the replication of the vector over time. Tumor growth inhibition was similar in hamsters given CP treatment either 1 week before or 1 week after 007-Luc injection, which suggests that CP exerts its antitumor efficacy independently of vector therapy. 007-Luc did not spread far from the inoculation site, even in immunosuppressed, CP-treated animals. Our results indicate that the enhanced effectiveness that is produced by the combination of VRX-007 and CP therapies is due to their two independent mechanisms and that they do not have to be given simultaneously for the improved outcome.
Asunto(s)
Adenoviridae/genética , Antineoplásicos Alquilantes/farmacología , Ciclofosfamida/farmacología , Vectores Genéticos/genética , Neoplasias/genética , Virus Oncolíticos/genética , Animales , Antineoplásicos Alquilantes/administración & dosificación , Línea Celular Tumoral , Cricetinae , Ciclofosfamida/administración & dosificación , Femenino , Expresión Génica/efectos de los fármacos , Orden Génico , Genes Reporteros , Vectores Genéticos/administración & dosificación , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/terapia , Viroterapia Oncolítica , Replicación Viral/efectos de los fármacosRESUMEN
We have studied the oncolytic efficacy of two adenovirus vectors named KD3 and INGN 007, which differ from each other only in that whereas KD3 has two small deletions in its e1a gene that restrict its replication to rapidly cycling cells, INGN 007 has wild-type e1a gene. Both vectors overexpress the adenovirus death protein (ADP). Both KD3 and INGN 007 effectively suppressed the growth of subcutaneous human A549 and Hep3B tumors in nude mice upon intratumoral injection, and contained the growth of subcutaneous LNCaP tumors after intravenous injection, making some tumors shrink or disappear. However, in a more demanding model, intravenous injections of neither KD3 nor wild-type Ad5 were effective against subcutaneous A549 tumors, whereas INGN 007 increased the mean survival time by 35%. INGN 007 was also effective in suppressing tumor growth in a challenging A549 orthotopic lung cancer model. INGN 007 was superior to dl1520 (ONYX-015) in repressing subcutaneous A549 tumors. Our results suggest that vectors such as INGN 007 might provide better antitumor efficacy in the clinic as well.
Asunto(s)
Adenoviridae/fisiología , Vectores Genéticos/metabolismo , Neoplasias Experimentales/terapia , Virus Oncolíticos/genética , Replicación Viral , Adenoviridae/genética , Animales , Línea Celular Tumoral , Cricetinae , Femenino , Terapia Genética , Humanos , Ratones , Ratones Desnudos , Viroterapia Oncolítica , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Syrian hamster is a practical animal model for studying the systemic effects of oncolytic vectors derived from adenovirus serotype 5 (Ad5). Ad5 replicates well in Syrian hamster tissues, and Syrian hamster cell lines are available that are known to support Ad5 replication. In this study, we established four new Syrian hamster cell lines from transplantable pancreatic, renal, hepatic and lung tumors. The pancreatic cell line (SHPC6) and the renal cell line were highly permissive for Ad5 replication. The SHPC6 cell line formed disseminated intraperitoneal tumors when cells were injected into the peritoneal cavity. INGN 007, an oncolytic Ad5-based vector, completely reversed the growth of disseminated intraperitoneal SHPC6 tumor nodules following intraperitoneal injection of the vector, leading to 100% survival of the treated animals. SHPC6 cells also formed subcutaneous tumors, whose growth was suppressed by INGN 007 following intratumoral injection. INGN 007 replicated in both the intraperitoneal and subcutaneous SHPC6 tumors. Following intraperitoneal injection, INGN 007 did not replicate in the livers of hamsters with intraperitoneal SHPC6 tumors, and was not hepatotoxic. These studies suggest that the SHPC6 cell line may be useful as a model for disseminated pancreatic cancer, and that INGN 007 may be a safe and effective vector to treat these tumors.
Asunto(s)
Carcinoma Ductal Pancreático/terapia , Carcinoma Ductal Pancreático/virología , Modelos Animales de Enfermedad , Viroterapia Oncolítica/métodos , Neoplasias Pancreáticas/terapia , Neoplasias Pancreáticas/virología , Adenoviridae/fisiología , Animales , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Cricetinae , Femenino , Humanos , Mesocricetus , Virus Oncolíticos/fisiología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Preclinical biodistribution studies with INGN 007, an oncolytic adenovirus (Ad) vector, supporting an early stage clinical trial were conducted in Syrian hamsters, which are permissive for Ad replication, and mice, which are a standard model for assessing toxicity and biodistribution of replication-defective (RD) Ad vectors. Vector dissemination and pharmacokinetics following intravenous administration were examined by real-time PCR in nine tissues and blood at five time points spanning 1 year. Select organs were also examined for the presence of infectious vector/virus. INGN 007 (VRX-007), wild-type Ad5 and AdCMVpA (an RD vector) were compared in the hamster model, whereas only INGN 007 was examined in mice. DNA of all vectors was widely disseminated early after injection, but decayed rapidly in most organs. In the hamster model, DNA of INGN 007 and Ad5 was more abundant than that of the RD vector AdCMVpA at early times after injection, but similar levels were seen later. An increased level of INGN 007 and Ad5 DNA but not AdCMVpA DNA in certain organs early after injection, and the presence of infectious INGN 007 and Ad5 in lung and liver samples at early times after injection, strongly suggests that replication of INGN 007 and Ad5 occurred in several Syrian hamster organs. There was no evidence of INGN 007 replication in mice. In addition to providing important information about INGN 007, the results underscore the utility of the Syrian hamster as a permissive immunocompetent model for Ad5 pathogenesis and oncolytic Ad vectors.
Asunto(s)
Adenoviridae/fisiología , Vectores Genéticos/farmacocinética , Animales , Cricetinae , ADN Viral/aislamiento & purificación , Femenino , Terapia Genética , Vectores Genéticos/administración & dosificación , Inyecciones Intravenosas , Hígado/virología , Pulmón/virología , Masculino , Mesocricetus , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Neoplasias/terapia , Virus Oncolíticos , Especificidad de la Especie , Replicación ViralRESUMEN
Oncolytic (replication-competent) adenoviruses as anticancer agents provide new, promising tools to fight cancer. In support of a Phase I clinical trial, here we report safety data with INGN 007 (VRX-007), an oncolytic adenovirus with increased anti-tumor efficacy due to overexpression of the adenovirus-encoded ADP protein. Wild-type adenovirus type 5 (Ad5) and a replication-defective version of Ad5 were also studied as controls. A parallel study investigating the biodistribution of these viruses is described elsewhere in this issue. The toxicology experiments were conducted in two species, the Syrian hamster, which is permissive for INGN 007 and Ad5 replication and the poorly permissive mouse. The studies demonstrated that the safety profile of INGN 007 is similar to Ad5. Both viruses caused transient liver damage upon intravenous injection that resolved by 28 days post-infection. The No-Observable-Adverse-Effect-Level (NOAEL) for INGN 007 in hamsters was 3 x 10(10) viral particles per kg. In hamsters, the replication-defective vector caused less toxicity, indicating that replication of Ad vectors in the host is an important factor in pathogenesis. With mice, INGN 007 and Ad5 caused toxicity comparable to the replication-defective adenovirus vector. Partially based on these results, the FDA granted permission to enter into a Phase I clinical trial with INGN 007.
Asunto(s)
Adenoviridae/fisiología , Vectores Genéticos/efectos adversos , Proteínas E3 de Adenovirus/biosíntesis , Animales , Recuento de Células Sanguíneas , Línea Celular , Cricetinae , Eritropoyesis , Vectores Genéticos/administración & dosificación , Humanos , Inyecciones Intravenosas , Hígado/patología , Hígado/virología , Mesocricetus , Ratones , Ratones Endogámicos C57BL , Virus Oncolíticos , Replicación ViralRESUMEN
We have previously described oncolytic adenovirus (Ad) vectors KD3 and KD3-interferon (IFN) that were rendered cancer-specific by mutations in the E1A region of Ad; these mutations abolish binding of E1A proteins to p300/CBP and pRB. The antitumor activity of the vectors was enhanced by overexpression of the Adenovirus Death Protein (ADP, E3-11.6K) and by replication-linked expression of IFN-alpha. We hypothesized that the anticancer efficacy of the KD3-IFN vector could be further improved by expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). E1-deleted Ad vectors were constructed carrying reporter genes for enhanced green fluorescent protein or secreted placental alkaline phosphatase (SEAP) and a therapeutic gene for TRAIL under control of the TetON system. Expression of the genes was increased in the presence of a helper virus and the inducer doxycycline such that up to 231-fold activation of expression for the TetON-SEAP vector was obtained. Coinfection with TetON-TRAIL augmented oncolytic activity of KD3 and KD3-IFN in vitro. Induction of TRAIL expression did not reduce the yield of progeny virus. Combination of TetON-TRAIL and KD3-IFN produced superior antitumor activity in vivo as compared with either vector alone demonstrating the efficacy of a four-pronged cancer gene therapy approach, which includes Ad oncolysis, ADP overexpression, IFN-alpha-mediated immunotherapy, and pharmacologically controlled TRAIL activity.
Asunto(s)
Adenoviridae/genética , Proteínas E3 de Adenovirus/genética , Terapia Genética , Vectores Genéticos , Interferón-alfa/genética , Neoplasias/terapia , Ligando Inductor de Apoptosis Relacionado con TNF/biosíntesis , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Adenoviridae/fisiología , Proteínas E3 de Adenovirus/biosíntesis , Proteínas E3 de Adenovirus/fisiología , Animales , Apoptosis/genética , Línea Celular Tumoral , Doxiciclina/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Interferón-alfa/biosíntesis , Interferón-alfa/fisiología , Ratones , Mutación , Neoplasias/genética , Neoplasias/patología , Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Tetraciclina/farmacologíaRESUMEN
We have constructed a novel oncolytic adenovirus (Ad) vector, named VRX-011, in which the replication of the vector is targeted to cancer cells by the replacement of the wild-type Ad E4 promoter with the human telomerase reverse transcriptase (hTERT) promoter. Genes in the Ad E4 transcription unit are essential for Ad replication; therefore, VRX-011 will grow efficiently only in cells in which the hTERT promoter is active, that is, in a wide range of cancer and immortalized cells but not in most somatic cells. Consistent with these expectations, VRX-011 replicated efficiently in all cancer cell lines examined, while its growth was restricted in various primary and normal cells. VRX-011 overexpresses ADP (also known as E3-11.6K), an Ad protein required for efficient cell lysis and release of virions from cells at late stages of infection. This overexpression enhances cell-to-cell spread and could significantly increase antitumor efficacy. In a xenograft model in nude mice, both intratumoral and intravenous administration of VRX-011 effectively suppressed the growth of subcutaneous Hep3B human liver tumors. Also, intravenous delivery of VRX-011 greatly reduced the number and size of A549 human lung cancer cell nodules in a disseminated lung tumor model in nude mice. Importantly, tail vein administration of different doses of VRX-011 in C57BL/6 mice showed minimal liver toxicity. Considering its broad range of lytic replication in cancer cells, its attenuated phenotype in primary cells, its efficacy in suppressing xenografts, and its low toxicity in mouse liver, VRX-011 is a promising candidate for further evaluation as an anticancer therapeutic.
Asunto(s)
Adenoviridae/genética , Proteínas E4 de Adenovirus/genética , Proteínas de Unión al ADN/genética , Terapia Genética/métodos , Vectores Genéticos/genética , Neoplasias/terapia , Telomerasa/genética , Adenosina Difosfato/metabolismo , Adenoviridae/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Regulación Viral de la Expresión Génica , Ingeniería Genética , Vectores Genéticos/administración & dosificación , Humanos , Ratones , Ratones Endogámicos , Ratones Desnudos , Neoplasias/virología , Neoplasias Experimentales , Regiones Promotoras Genéticas , Transducción Genética/métodos , Transgenes , Replicación ViralRESUMEN
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis through two receptors, TRAIL-R1 (also known as death receptor 4) and TRAIL-R2 (also known as death receptor 5), that are members of the TNF receptor superfamily of death domain-containing receptors. We show that human adenovirus type 5 encodes three proteins, named RID (previously named E3-10.4K/14.5K), E3-14.7K, and E1B-19K, that independently inhibit TRAIL-induced apoptosis of infected human cells. This conclusion was derived from studies using wild-type adenovirus, adenovirus replication-competent mutants that lack one or more of the RID, E3-14.7K, and E1B-19K genes, and adenovirus E1-minus replication-defective vectors that express all E3 genes, RID plus E3-14.7K only, RID only, or E3-14.7K only. RID inhibits TRAIL-induced apoptosis when cells are sensitized to TRAIL either by adenovirus infection or treatment with cycloheximide. RID induces the internalization of TRAIL-R1 from the cell surface, as shown by flow cytometry and indirect immunofluorescence for TRAIL-R1. TRAIL-R1 was internalized in distinct vesicles which are very likely to be endosomes and lysosomes. TRAIL-R1 is degraded, as indicated by the disappearance of the TRAIL-R1 immunofluorescence signal. Degradation was inhibited by bafilomycin A1, a drug that prevents acidification of vesicles and the sorting of receptors from late endosomes to lysosomes, implying that degradation occurs in lysosomes. RID was also shown previously to internalize and degrade another death domain receptor, Fas, and to prevent apoptosis through Fas and the TNF receptor. RID was shown previously to force the internalization and degradation of the epidermal growth factor receptor. E1B-19K was shown previously to block apoptosis through Fas, and both E1B-19K and E3-14.7K were found to prevent apoptosis through the TNF receptor. These findings suggest that the receptors for TRAIL, Fas ligand, and TNF play a role in limiting virus infections. The ability of adenovirus to inhibit killing through these receptors may prolong acute and persistent infections.
Asunto(s)
Infecciones por Adenoviridae/virología , Adenoviridae/fisiología , Proteínas E3 de Adenovirus/fisiología , Apoptosis/fisiología , Glicoproteínas de Membrana/fisiología , Receptores del Factor de Necrosis Tumoral/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Infecciones por Adenoviridae/metabolismo , Infecciones por Adenoviridae/patología , Proteínas Reguladoras de la Apoptosis , Línea Celular , Humanos , Ligandos , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF , Ligando Inductor de Apoptosis Relacionado con TNF , Replicación ViralRESUMEN
We have previously described two replication-competent adenovirus vectors, named KD1 and KD3, for potential use in cancer gene therapy. KD1 and KD3 have two small deletions in the E1A gene that restrict efficient replication of these vectors to human cancer cell lines. These vectors also have increased capacity to lyse cells and spread from cell to cell because they overexpress the adenovirus death protein, an adenovirus protein required for efficient cell lysis and release of adenovirus from the cell. We now describe a new vector, named KD1-SPB, which is the KD1 vector with the E4 promoter replaced by the promoter for surfactant protein B (SPB). SPB promoter activity is restricted in the adult to type II alveolar epithelial cells and bronchial epithelial cells. Because KD1-SPB has the E1A mutations, it should replicate within and destroy only alveolar and bronchial cancer cells. We show that KD1-SPB replicates, lyses cells, and spreads from cell to cell as well as does KD1 in H441 cells, a human cancer cell line where the SPB promoter is active. KD1-SPB replicates, lyses cells, and spreads only poorly in Hep3B liver cancer cells. Replication was determined by expression of the E4ORF3 protein, viral DNA accumulation, fiber synthesis, and virus yield. Cell lysis and vector spread were measured by lactate dehydrogenase release and a "vector spread" assay. In addition to Hep3B cells, KD1-SPB also did not express E4ORF3 in HT29.14S (colon), HeLa (cervix), KB (nasopharynx), or LNCaP (prostate) cancer cell lines, in which the SPB promoter is not expected to be active. Following injection into H441 or Hep3B tumors growing in nude mice, KD1-SPB caused a three- to fourfold suppression of growth of H441 tumors, similar to that seen with KD1. KD1-SPB had only a minimal effect on the growth of Hep3B tumors, whereas KD1 again caused a three- to fourfold suppression. These results establish that the adenovirus E4 promoter can be replaced by a tissue-specific promoter in a replication-competent vector. The vector has three engineered safety features: the tissue-specific promoter, the mutations in E1A that preclude efficient replication in nondividing cells, and a deletion of the E3 genes which shield the virus from attack by the immune system. KD1-SPB may have use in treating human lung cancers in which the SPB promoter is active.
Asunto(s)
Adenovirus Humanos/genética , Terapia Genética , Vectores Genéticos , Neoplasias/terapia , Replicación Viral , Proteínas E1A de Adenovirus/genética , Proteínas E4 de Adenovirus/biosíntesis , Adenovirus Humanos/fisiología , Animales , Replicación del ADN , Humanos , Ratones , Especificidad de Órganos , Regiones Promotoras Genéticas , Células Tumorales CultivadasRESUMEN
The ELL gene encodes an RNA polymerase II transcription factor that frequently undergoes translocation with the MLL gene in acute human myeloid leukemia. Here, we report that ELL can regulate cell proliferation and survival. In order to better understand the physiological role of the ELL protein, we have developed an ELL-inducible cell line. Cells expressing ELL were uniformly inhibited for growth by a loss of the G(1) population and an increase in the G(2)/M population. This decrease in cell growth is followed by the condensation of chromosomal DNA, activation of caspase 3, poly(ADP ribose) polymerase cleavage, and an increase in sub-G(1) population, which are all indicators of the process of programmed cell death. In support of the role of ELL in induction of cell death, expression of an ELL antisense RNA or addition of the caspase inhibitor ZVAD-fmk results in a reversal of ELL-mediated death. We have also demonstrated that the C-terminal domain of ELL, which is conserved among the ELL family of proteins that we have cloned (ELL, ELL2, and ELL3), is required for ELL's activity in the regulation of cell growth. These novel results indicate that ELL can regulate cell growth and survival and may explain how ELL translocations result in the development of human malignancies.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Leucemia Mieloide Aguda/metabolismo , Proteínas de Neoplasias , Factores de Elongación de Péptidos , Factores de Transcripción/fisiología , Clorometilcetonas de Aminoácidos/farmacología , Antígenos de Diferenciación , Apoptosis , Western Blotting , Caspasa 3 , Inhibidores de Caspasas , Caspasas/metabolismo , Proteínas de Ciclo Celular , Muerte Celular , División Celular , Línea Celular , Supervivencia Celular , Inhibidores de Cisteína Proteinasa/farmacología , ADN/metabolismo , Proteínas de Unión al ADN/genética , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Citometría de Flujo , Fase G1 , Fase G2 , Humanos , Leucemia Mieloide Aguda/genética , Microscopía Fluorescente , Microscopía de Contraste de Fase , Mitosis , Oligonucleótidos Antisentido/metabolismo , Plásmidos/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Propidio/farmacología , Proteína Fosfatasa 1 , Proteínas/metabolismo , ARN Mensajero/metabolismo , Factores de Tiempo , Factores de Transcripción/genética , Factores de Elongación Transcripcional , Transfección , Translocación Genética , Células Tumorales CultivadasRESUMEN
Human adenovirus type 4 (Ad4), the sole member of subgroup E, contains an open reading frame in the E3 region predicted to encode a unique 30-kDa protein (named Ad4E3-30K). Ad4E3-30K is predicted to be an integral membrane protein containing an N-terminal signal sequence, a lumenal domain, a transmembrane domain near the C-terminus, and a 37-amino-acid cytoplasmic tail. To determine whether this protein is expressed, rabbit polyclonal antisera were raised against 30K-containing fusion proteins expressed in bacteria. A 30K protein was detected by immunoprecipitation from cell-free translation products and from Ad4-infected A549 cells radiolabeled in the presence of tunicamycin. The protein was detected at only low levels in infected cells. It was not synthesized by a mutant with a large E3 deletion that includes the Ad4E3-30K gene. This mutant grows as well as wild-type Ad4 in culture. Features of Ad4E3-30K were characterized in different transient expression systems. The protein underwent glycosylation by addition of approximately six asparagine-linked oligosaccharides. These glycans were sensitive to endoglycosidase H, indicating that they were either high-mannose or hybrid types, but not complex types, and that the protein did not pass through the Golgi apparatus. Immunofluorescence staining of transfected cells revealed that Ad4E3-30K was localized primarily in the endoplasmic reticulum and nuclear envelope.
Asunto(s)
Proteínas E3 de Adenovirus/genética , Proteínas E3 de Adenovirus/metabolismo , Adenovirus Humanos/química , Proteínas E3 de Adenovirus/química , Proteínas E3 de Adenovirus/inmunología , Adenovirus Humanos/genética , Adenovirus Humanos/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Línea Celular , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Glicosilación , Hexosaminidasas/metabolismo , Humanos , Sueros Inmunes/inmunología , Datos de Secuencia Molecular , Peso Molecular , Pruebas de Precipitina , Biosíntesis de Proteínas , Factores de Tiempo , Transfección , Virus Vaccinia/fisiologíaRESUMEN
We have constructed two novel adenovirus (Ad) replication-competent vectors, named KD1 and KD3, that may have use in anticancer therapy. The vectors have two key features. First, they markedly overexpress the Ad death protein (ADP), an Ad nuclear membrane glycoprotein required at late stages of infection for efficient cell lysis and release of Ad from cells. Overexpression of ADP was achieved by deleting the E3 region and reinserting the adp gene. Because ADP is overexpressed, KD1 and KD3 are expected to spread more rapidly and effectively through tumors. Second, KD1 and KD3 have two E1A mutations (from the mutant dl1101/1107) that prevent efficient replication in nondividing cells but allow replication in dividing cancer cells. These E1A mutations preclude binding of E1A proteins to p300 and pRB. As a result, the virus should not be able to drive cells from G(0) to S phase and therefore should not be able to replicate in normal tissues. We show that KD1 and KD3 do not replicate well in quiescent HEL-299 cells or in primary human bronchial epithelial cells, small airway epithelial cells, or endothelial cells; however, they replicate well in proliferating HEL-299 cells and human A549 lung carcinoma cells. In cultured A549 cells, KD1 and KD3 lyse cells and spread from cell to cell more rapidly than their control virus, dl1101/1107, or wild-type Ad. They are also more efficient than dl1101/1107 or wild-type Ad in complementing the spread from cell to cell of an E1(-) E3(-) replication-defective vector expressing beta-galactosidase. A549 cells form rapidly growing solid tumors when injected into the hind flanks of immunodeficient nude mice; however, when A549 cells were infected with 10(-4) PFU of KD3/cell prior to injection into mice, tumor formation was nearly completely suppressed. When established A549 tumors in nude mice were examined, tumors injected with buffer grew 13.3-fold over 5 weeks, tumors injected with dl1101/1107 grew 8-fold, and tumors injected with KD1 or KD3 grew 2.6-fold. Hep 3B tumors injected with buffer grew 12-fold over 3.5 weeks, whereas tumors injected with KD1 or KD3 grew 4-fold. We conclude that KD1 and KD3 show promise as anticancer therapeutics.
Asunto(s)
Proteínas E3 de Adenovirus/fisiología , Adenovirus Humanos/fisiología , Carcinoma Hepatocelular/fisiopatología , Vectores Genéticos/fisiología , Neoplasias Hepáticas/fisiopatología , Replicación Viral , Proteínas E3 de Adenovirus/genética , Adenovirus Humanos/genética , Animales , División Celular , Línea Celular , Células Cultivadas , Expresión Génica , Vectores Genéticos/genética , Células HeLa , Humanos , Ratones , Ratones Desnudos , Neoplasias Experimentales/fisiopatología , Células Tumorales CultivadasRESUMEN
Adenoviruses encode proteins that block responses to interferons, intrinsic cellular apoptosis, killing by CD8(+) cytotoxic T lymphocytes and killing by the death ligands TNF, Fas ligand and TRAIL. The viral proteins are believed to prolong acute and persistent adenovirus infections. The proteins may prove useful in protecting adenovirus gene therapy vectors and transplanted cells from the immune system.
Asunto(s)
Adenoviridae/inmunología , Adenoviridae/genética , Proteínas E1A de Adenovirus/fisiología , Proteínas E1B de Adenovirus/fisiología , Animales , Apoptosis , Terapia Genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Herein, we report that the adenovirus E3-14.7K protein inhibits the inflammatory response to adenovirus in transgenic mice in which the E3-14.7K gene was selectively expressed in the respiratory epithelium, using the human surfactant protein C (SP-C) promoter. E3-14.7K mRNA and protein were detected specifically in the lungs of SPC/E3-14.7K transgenic mice. Responses of the transgenic mice to Av1Luc1, an E1-E3-deleted Ad vector encoding the luciferase reporter gene, were examined, including vector transgene expression and lung inflammation. In wild-type mice, luciferase activity declined rapidly and was lost 14 days following Av1Luc1 administration. The loss of luciferase activity was associated with pulmonary infiltration by macrophages and lymphocytes. In heterozygous SPC/E3-14.7K mice, luciferase activity was increased by 7 days compared with control littermates, and pulmonary infiltration by macrophages was decreased. In homozygous (+/+) SPC/E3-14.7K mice, luciferase activity was increased 7, 14, and 21 days following administration compared with wild-type mice, and lung inflammation was markedly reduced. After Av1Luc1 administration, PCNA staining of bronchiolar and alveolar respiratory epithelial cells was decreased in SPC/E3-14.7K transgenic mice, indicating decreased epithelial cell proliferation, a finding consistent with the observed reduction in inflammation. CD4 and CD8 lymphocyte populations were only mildly altered, while humoral responses to adenoviral vectors were unchanged in the SPC/E3-14.7K mice. The E3-14.7K protein expressed selectively in respiratory epithelial cells suppresses Ad-induced pulmonary epithelial cell cytotoxicity and lung inflammation in vivo and prolongs reporter gene expression.
Asunto(s)
Adenoviridae/genética , Proteínas E3 de Adenovirus/genética , Vectores Genéticos/genética , Pulmón/inmunología , Adenoviridae/inmunología , Proteínas E3 de Adenovirus/metabolismo , Animales , Anticuerpos Antivirales/sangre , Linfocitos T CD4-Positivos , Linfocitos T CD8-positivos , Epitelio/metabolismo , Regulación de la Expresión Génica/genética , Vectores Genéticos/inmunología , Humanos , Luciferasas/metabolismo , Pulmón/metabolismo , Pulmón/patología , Macrófagos Alveolares , Ratones , Ratones Transgénicos , Especificidad de Órganos , Regiones Promotoras Genéticas/genética , Proteolípidos/genética , Surfactantes Pulmonares/genética , ARN Mensajero/análisis , Proteínas Recombinantes de Fusión , TransgenesRESUMEN
DNA viruses use elegant mechanisms to overcome the antiviral responses mediated by tumor necrosis factor (TNF), the TNF receptor family member Fas and the interferons. TNF, which is secreted by activated monocytes and lymphocytes, induces apoptosis as well as expression of genes involved in the inflammatory and immune responses. Depending on the DNA virus and the viral proteins, the following mechanisms to prevent TNF receptor- and Fas-induced apoptosis are used: (1) absorption of extracellular TNF by secreted homologs of the TNF receptor; (2) degradation of Fas; (3) inhibition of the assembly of FADD and Caspase 8 with TNFR1 and Fas; (4) direct inhibition of proapoptotic caspase enzymatic activity; and (5) inhibition of the proapoptotic members of the Bcl-2 family. Interferons induce expression of multiple antiviral genes. DNA viruses encode proteins that function in different ways to block interferon-induced transcription as well as the activity of enzymes that block viral protein synthesis. These antiviral proteins prolong acute and persistent infections.
Asunto(s)
Apoptosis/inmunología , Virus ADN/inmunología , Interferones/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Proteína Ligando Fas , Ligandos , Glicoproteínas de Membrana/inmunología , Proteínas Virales/inmunologíaAsunto(s)
Transducción de Señal , Proteínas Virales/fisiología , Animales , Muerte Celular , División Celular , HumanosRESUMEN
DNA viruses have evolved elaborate mechanisms to overcome host antiviral defences. In adenovirus-infected cells, programmed cell death (apoptosis) induced by the cytokine tumour necrosis factor (TNF) is inhibited by several adenovirus-encoded proteins. Occupation of the cell-surface receptor Fas, a member of the TNF-receptor superfamily that is expressed on most cell types, triggers apoptosis of that cell. Here we show that the adenovirus RID (for receptor internalization and degradation) protein complex, which is an inhibitor of TNF-induced apoptosis, mediates internalization of cell-surface Fas and its destruction inside lysosomes within the cell. Fas has not previously been shown to be internalized and then degraded. RID also mediates internalization of the receptor for epidermal growth factor, but it does not affect the transferrin receptor or class I antigens of the major histocompatibility complex. Removal of Fas from the surface of adenovirus-infected cells expressing RID may allow infected cells to resist Fas-mediated cell death and thus promote their survival.