RESUMO
OBJECTIVE: To identify and characterize read-through RNAs and read-through circular RNAs (rt-circ-HS) derived from transcriptional read-through hypoxia inducible factor 1α (HIF1α) and small nuclear RNA activating complex polypeptide 1 (SNAPC1) the two adjacent genes located on chromosome 14q23, in renal carcinoma cells and renal carcinoma tissues, and to study the effects of rt-circ-HS on biological behavior of renal carcinoma cells and on regulation of HIF1α. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) and Sanger sequencing were used to examine expression of read-through RNAs HIF1α-SNAPC1 and rt-circ-HS in different tumor cells. Tissue microarrays of 437 different types of renal cell carcinoma (RCC) were constructed, and chromogenic in situ hybridization (ISH) was used to investigate expression of rt-circ-HS in different RCC types. Small interference RNA (siRNA) and artificial overexpression plasmids were designed to examine the effects of rt-circ-HS on 786-O and A498 renal carcinoma cell proliferation, migration and invasiveness by cell counting kit 8 (CCK8), EdU incorporation and Transwell cell migration and invasion assays. RT-PCR and Western blot were used to exa-mine expression of HIF1α and SNAPC1 RNA and proteins after interference of rt-circ-HS with siRNA, respectively. The binding of rt-circ-HS with microRNA 539 (miR-539), and miR-539 with HIF1α 3' untranslated region (3' UTR), and the effects of these interactions were investigated by dual luciferase reporter gene assays. RESULTS: We discovered a novel 1 144 nt rt-circ-HS, which was derived from read-through RNA HIF1α-SNAPC1 and consisted of HIF1α exon 2-6 and SNAPC1 exon 2-4. Expression of rt-circ-HS was significantly upregulated in 786-O renal carcinoma cells. ISH showed that the overall positive expression rate of rt-circ-HS in RCC tissue samples was 67.5% (295/437), and the expression was different in different types of RCCs. Mechanistically, rt-circ-HS promoted renal carcinoma cell proliferation, migration and invasiveness by functioning as a competitive endogenous inhibitor of miR-539, which we found to be a potent post-transcriptional suppressor of HIF1α, thus promoting expression of HIF1α. CONCLUSION: The novel rt-circ-HS is highly expressed in different types of RCCs and acts as a competitive endogenous inhibitor of miR-539 to promote expression of its parental gene HIF1α and thus the proliferation, migration and invasion of renal cancer cells.
Assuntos
Carcinoma de Células Renais , Subunidade alfa do Fator 1 Induzível por Hipóxia , Neoplasias Renais , MicroRNAs , RNA Circular , Humanos , Carcinoma de Células Renais/patologia , Proliferação de Células , Hipóxia , MicroRNAs/genética , Invasividade Neoplásica/genética , RNA Circular/genética , RNA Circular/metabolismo , RNA Interferente Pequeno , Subunidade alfa do Fator 1 Induzível por Hipóxia/genéticaRESUMO
To explore the clinical diagnostic efficacy of antineutrophil cytoplasmic antibody associated vasculitis (AAV) by comparing the consistency and coincidence rate of serum anti-myeloperoxidase (MPO) antibody and anti-protease 3 (PR3) antibody detected by digital liquid chip method (DLCM) and enzyme-linked immunosorbent assay (ELISA). To provide reference for the selection of detection methods of anti-MPO antibody and anti-PR3 antibody in clinical laboratory. This study is a cross-sectional study, a total of 307 cases of antineutrophil cytoplasmic antibodies were detected in the Department of Clinical Immunology, West China Hospital of Sichuan University from January to March 2021. The serum samples and related clinical information were collected. At the same time, the levels of anti-MPO antibody and anti-PR3 antibody in serum samples were detected by ELISA and DLCM, indirect immunofluorescence (IIF) was used to re-test the differential samples between the two methods. SPSS 26.0 was used to analyze the test results, Cohen's kappa coefficient analysis was used to compare the consistency of the two methods, and paired chi-square test was used to compare the sensitivity and specificity of the two methods to AAV. The results showed that the positive cases of anti-MPO antibody detected by ELISA and DLCM were 63 and 44, and the negative cases were 244 and 263; the positive cases of anti-PR3 antibody detected by ELISA and DLCM were 34 and 28, and the negative cases were 273 and 279. The results of anti-MPO antibody and anti-PR3 antibody detected by the two methods had good consistency and coincidence rate, in which the total coincidence rate of anti-MPO antibody was 92.51%, the positive coincidence rate was 66.67%, and the negative coincidence rate was 99.18%. The results of consistency analysis showed that kappa=0.741 had well consistency. The total coincidence rate of anti-PR3 antibody is 96.74%, the positive coincidence rate is 76.47%, and the negative coincidence rate is 99.27%. The consistency analysis results show that kappa=0.821 had strong consistency. The results of IIF re-test of differential samples showed that the coincidence rate between DLCM and IIF was higher. The results of comparative analysis of anti-MPO antibody and anti-PR3 antibody showed that the specificity of DLCM was better than that of ELISA, and its sensitivity was lower than that of ELISA. In conclusion, the results of anti-MPO antibody and anti-PR3 antibody detected by DLCM were consistent with those of ELISA. In the combined detection of anti-MPO antibody and anti-PR3 antibody, the specificity of DLCM is better than that of ELISA.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos , Humanos , Anticorpos Anticitoplasma de Neutrófilos/análise , Mieloblastina , Estudos Transversais , Sensibilidade e Especificidade , Técnica Indireta de Fluorescência para Anticorpo , Ensaio de Imunoadsorção Enzimática/métodosRESUMO
Objective: To examine the prevalence of multiple antiphospholipid antibodies (aPL) subtypes in healthy people and antiphospholipid syndrome (APS) patients, and to assess the value of IgA-aPL in the diagnosis of APS. Methods: According to the 2006 Sydney International APS Classification Criteria, a total of 218 APS patients who were admitted to Peking Union Medical College Hospital or West China Hospital of Sichuan University from July to December 2019 were enrolled. Among them, 66 were males, and 152 were females, aged (44.5±15.4) years, including 148 primary APS patients and 70 secondary APS patients. Age-and gender-matched controls were collected at the same period at the ratio of 1â¶1 with the APS cases. IgA/IgG/IgM anticardiolipin antibodies (aCL) and anti-ß2 glycoprotein I antibodies (aß2GPI) were detected by chemiluminescent immunoassay. The differences of indicators between groups were analyzed, and the receiver operating characteristic (ROC) curve was used to evaluate the diagnostic value of IgA-aPL for APS. Results: The positivity of IgA-aCL and IgA-aß2GPI was 20.6% and 15.6% in the APS patients, while in the IgG/IgM-aCL or IgG/IgM-aß2GPI negative individuals, the isolated positivity of IgA-aCL and IgA-aß2GPI was only 2.3% and 0.9%, respectively. Accordingly, IgA-aCL and IgA-aß2GPI isolated positivity could be used to diagnose APS (P=0.216, 1, respectively). The area under the ROC curve (AUC) of IgG/IgM-aCL for APS diagnosis was 0.833, which was significantly better than that of IgG-aCL alone (AUC=0.776, P<0.001); while the AUC of IgA/IgG/IgM-aCL was 0.833, which could not further increase the diagnostic value for APS (P=0.287). As for aß2GPI, the diagnostic efficacy of combined IgG/IgM (AUC=0.875) or IgA/IgG/IgM (AUC=0.875) antibodies was not superior to IgG-aß2GPI used alone (AUC=0.869, both P>0.05). Besides, patients with IgA-aPL were more likely to have heart valve lesions and thrombocytopenia (both P<0.05). Conclusion: Based on the existing serological markers, such as lupus anticoagulant, IgG/IgM subtype of aCL and aß2GPI, testing IgA-aCL and IgA-aß2GPI cannot further improve the predictive value of APS. However, IgA-aPL is associated with clinical manifestations of APS, including heart valve lesions and thrombocytopenia.
Assuntos
Síndrome Antifosfolipídica , Anticorpos Anticardiolipina , Anticorpos Antifosfolipídeos , Síndrome Antifosfolipídica/diagnóstico , Feminino , Humanos , Imunoglobulina A , Inibidor de Coagulação do Lúpus , MasculinoRESUMO
OBJECTIVE: Non-small cell lung cancer (NSCLC) is a common type of lung cancer. Long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was reported to play a tumor-promoting role in NSCLC; however, the regulatory mechanism of MALAT1 in NSCLC progression remains largely unknown. MATERIALS AND METHODS: The expression levels of MALAT1, miR-374b-5p and SRSF7 were measured by quantitative real-time polymerase chain reaction (qRT-PCR), and the protein level of SRSF7 was detected by Western blot analysis. Cell proliferation and apoptosis were determined by cell counting kit-8 (CCK-8) assay and flow cytometry, respectively. Cell migration and invasion were assessed by transwell assay. In addition, starBase3.0 software and dual-luciferase reporter assay were used to identify the correlations between miR-374b-5p and MALAT1 or SRSF7. Nude mouse xenograft assay was performed to explore the effects of MALAT1 on NSCLC in vivo. RESULTS: We first observed that the levels of MALAT1 and SRSF7 were upregulated while miR-374b-5p was downregulated in NSCLC tissues; meanwhile, the expression level of MALAT1 was negatively correlated with miR-374b-5p and positively correlated with SRSF7. Both knockdown of MALAT1 and miR-374b-5p overexpression inhibited proliferation, migration and invasion and induced apoptosis in NSCLC cells. Then, we identified that miR-374b-5p was a target of MALAT1 and SRSF7 was the downstream of miR-374b-5p. In addition, overexpression of SRSF7 reversed the effects of MALAT1 knockdown on proliferation, apoptosis, migration and invasion in NSCLC cells. Finally, overexpression of MALAT1 suppressed NSCLC tumor growth in vivo. CONCLUSIONS: Our results demonstrated that MALAT1 contributed to NSCLC progression through the MALAT1/miR-374b-5p/SRSF7 axis.
Assuntos
Apoptose , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Movimento Celular , Proliferação de Células , Humanos , Neoplasias Pulmonares/patologia , MicroRNAs/genética , RNA Longo não Codificante/genética , Fatores de Processamento de Serina-Arginina/genéticaRESUMO
This study aimed to investigate the effects of bovine serum albumin (BSA) on boar sperm quality during liquid storage at 17°C. Boar semen samples were collected and diluted with Modena containing different concentrations (0, 1, 2, 3, 4, 5 and 6 g/l) of BSA, and sperm motility, plasma membrane integrity, acrosome integrity, total antioxidative capacity (T-AOC) activity and malondialdehyde (MDA) content were measured and analysed. The results showed that Modena supplemented with 3, 4 and 5 g/l BSA could improve boar sperm motility, effective survival time and plasma membrane integrity (p < 0.05), decrease MDA content (p < 0.05), while no statistical difference was observed for sperm acrosome integrity and T-AOC activity among these three groups (p > 0.05). The semen sample diluted with Modena containing 4 g/l BSA could achieve optimum effect, and sperm survival time was 7.5 days. After 7 days preservation, sperm motility, plasma membrane integrity and acrosome integrity were 54%, 49% and 78%, respectively. T-AOC activity and MDA content were 1.03 U/ml and 17.5 nmol/ml, respectively. In conclusion, Modena supplemented with BSA reduced the oxidative stress and improved the sperm quality of boar semen during liquid storage at 17°C, and 4 g/l BSA was the optimum concentration. Further studies are required to obtain more concrete results on the determination of antioxidant capacities of BSA in liquid preserved boar semen.
Assuntos
Preservação do Sêmen/veterinária , Soroalbumina Bovina/farmacologia , Espermatozoides/efeitos dos fármacos , Suínos/fisiologia , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Masculino , Preservação do Sêmen/métodos , Espermatozoides/citologia , Espermatozoides/fisiologia , TemperaturaRESUMO
Pancreatic cancer remains one of the deadliest of all cancers despite aggressive surgical treatment combined with adjuvant radiotherapy and chemotherapy. Chemoresistance and radioresistance are the principal causes of failure of pancreatic cancer patients to respond to therapy. Conditionally replication competent adenovirus (CRCA)-based cancer gene therapy is an innovative strategy for treating cancers displaying inherent resistance to treatment. Limitations of current adenovirus (Ad)-based gene therapies for malignant tumors include lack of cancer-specificity, and effective and targeted delivery. To remedy this situation, CRCAs have been designed that express E1A, necessary for Ad replication, under the control of a cancer-specific progression elevated gene-3 promoter (PEG-Prom) with concomitant expression of an immunomodulatory cytokine, such as mda-7/IL-24 or interferon-γ (IFN-γ), under the control of a ubiquitous and strong cytomegalovirus promoter (CMV-Prom) from the E3 region. These bipartite CRCAs, when armed with a transgene, are called cancer terminator viruses (CTVs), i.e., Ad.PEG-E1A-CMV-mda-7 (CTV-M7) and Ad.PEG-E1A-CMV-IFN-γ (CTV-γ), because of their universal effectiveness in cancer treatment irrespective of p53/pRb/p16 or other genetic alterations in tumor cells. In addition to their selective oncolytic effects in tumor cells, the potent 'bystander antitumor' properties of MDA-7/IL-24 and IFN-γ embody the CTVs with expanded treatment properties for both primary and distant cancers. Pancreatic cancer cells display a "translational block" of mda-7/IL-24 mRNA, limiting production of MDA-7/IL-24 protein and cancer-specific apoptosis. Specific chemopreventive agents abrogate this "translational block" resulting in pancreatic cancer-specific killing. This novel chemoprevention gene therapy (CGT) strategy holds promise for both prevention and treatment of pancreatic cancers where all other strategies have proven ineffective.
Assuntos
Quimioprevenção , Terapia Genética , Neoplasias Pancreáticas/tratamento farmacológico , Adenoviridae , Animais , Apoptose/genética , Linhagem Celular Tumoral , Humanos , Camundongos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Regiões Promotoras Genéticas , Neoplasias PancreáticasRESUMO
Enhanced expression of the CCN family of secretory integrin-binding proteins correlates with many essential components of the cancerous state, including tumor cell adhesion, proliferation, invasion and migration. Consequently, CCN1 expression is elevated in various cancers, including breast cancer, and its expression directly correlates with poor patient prognosis. Using subtraction-hybridization, combined with induction of cancer cell terminal differentiation, we cloned SARI (suppressor of activator protein (AP)-1, regulated by interferon (IFN)), an IFN-beta-inducible, potent tumor suppressor gene that exerts cancer-selective growth inhibitory effects. Forced expression of SARI using an adenovirus (Ad.SARI) inhibits AP-1 function and downregulates CCN1 expression in multiple cancer lineages, resulting in a profound inhibition in anchorage-independent cell growth and tumor cell invasion. Overexpression of SARI reduces CCN1-promoter activity through inhibition of AP-1 binding. Accordingly, SARI selectively blocks expression of the transformed state in rat embryo fibroblast cells that stably overexpress c-Jun. These results illustrate that SARI inhibits AP-1 transactivating factor binding to the cis-element of the CCN1 promoter, possibly through its interaction with c-Jun. Overall, SARI can directly inhibit CCN1-induced transformation by inhibiting the transcription of CCN1, as well as indirectly by inhibiting the expression of c-Jun (and hence blocking AP-1 activity). In these contexts, transformed cells 'addicted' to AP-1 activity are rendered susceptible to SARI-mediated inhibition of expression of the transformed phenotype.
Assuntos
Transformação Celular Neoplásica/genética , Proteína Rica em Cisteína 61/fisiologia , Genes Supressores de Tumor/fisiologia , Fator de Transcrição AP-1/antagonistas & inibidores , Proteínas Supressoras de Tumor/fisiologia , Animais , Adesão Celular/genética , Proliferação de Células , Progressão da Doença , Humanos , Camundongos , Camundongos Nus , Invasividade Neoplásica , Ratos , Transfecção , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/genética , Estudos de Validação como Assunto , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The scaffolding postsynaptic density-95/disks large/zonula occludens-1 (PDZ) domain-containing protein melanoma differentiation associated gene-9 (MDA-9)/syntenin is a tandem PDZ protein overexpressed in human melanoma, and breast and gastric cancer cells. MDA-9/syntenin affects cancer cell motility and invasion through distinct biochemical and signaling pathways, including focal adhesion kinase and p38 mitogen-activated protein kinase (MAPK), resulting in activation of the nuclear factor (NF)-kappaB pathway. MDA-9/syntenin also promotes melanoma metastasis by activating c-Src, but how c-Src regulates NF-kappaB activation is unclear. Using a human melanoma model, we document that MDA-9/syntenin-c-Src interactions are positive regulators of NF-kappaB activation. Inhibition of c-Src by PP2 treatment, by blocking c-Src or mda-9/syntenin expression with small interfering RNA, or in c-Src (-/-) knockout cell lines, reduces NF-kappaB activation following overexpression of mda-9/syntenin or c-Src. Deletion or point mutations of the PDZ binding motif preventing MDA-9/syntenin association with c-Src reveals that both PDZ domains, with PDZ2 being the dominant module, are required for activating downstream signaling pathways, including p38 MAPK and NF-kappaB. We also document that MDA-9/syntenin-c-Src complexes functionally cooperate with NF-kappaB to promote anchorage-independent growth, motility and invasion of melanoma cells. These findings underscore PDZ domains of MDA-9/syntenin as promising potential therapeutic targets for intervening in a decisive component of cancer progression, namely, metastatic tumor spread.
Assuntos
NF-kappa B/metabolismo , Fosfoproteínas/metabolismo , Sinteninas/metabolismo , Técnicas de Inativação de Genes , Inativação Gênica , Genes src/genética , Humanos , Melanoma/genética , Melanoma/prevenção & controle , Domínios PDZ/genética , Fosfoproteínas/genética , Mutação Puntual , RNA Interferente Pequeno/genética , Deleção de Sequência , Sinteninas/genética , Transcrição Gênica , Ativação Transcricional/genéticaRESUMO
Gene therapy is being examined as a potential strategy for treating prostate cancer. Serotype 5 adenovirus (Ad.5) is routinely used as a vector for transgene delivery. However, the infectivity of Ad.5 is dependent on Coxsackie-adenovirus receptors (CARs); many tumor types show a reduction in this receptor in vivo, thereby limiting therapeutic gene transduction. Serotype chimerism is one approach to circumvent CAR deficiency; this strategy is used to generate an Ad.5/3-recombinant Ad that infects cancer cells through Ad.3 receptors in a CAR-independent manner. In this report, the enhanced transgene delivery and efficacy of Ad.5/3-recombinant virus was evaluated using an effective wide-spectrum anticancer therapeutic melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24). Our data show that in low CAR human prostate cancer cells (PC-3), a recombinant Ad.5/3 virus delivering mda-7/IL-24 (Ad.5/3-mda-7) is more efficacious than an Ad.5 virus encoding mda-7/IL-24 (Ad.5-mda-7) in infecting tumor cells, expressing MDA-7/IL-24 protein, inducing cancer-specific apoptosis, inhibiting in vivo tumor growth and exerting an antitumor 'bystander' effect in a nude mouse xenograft model. Considering the fact that Ad.5-mda-7 has shown significant objective responses in a phase I clinical trial for solid tumors, Ad.5/3-mda-7 is predicted to exert enhanced therapeutic benefit in patients with prostate cancer.
Assuntos
Adenoviridae/genética , Terapia Genética/métodos , Interleucinas/genética , Neoplasias da Próstata/terapia , Receptores Virais/metabolismo , Animais , Linhagem Celular Tumoral , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Humanos , Imuno-Histoquímica , Interleucinas/biossíntese , Masculino , Camundongos , Camundongos Nus , Neoplasias da Próstata/genética , Transgenes , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Neuroblastoma, derived from neural crest progenitor cells, is the most common extracranial solid tumor of childhood. Astrocyte elevated gene-1 (AEG-1) is a primary mediator of tumor progression and metastasis in several human cancers. In this study, we investigated the potential contribution of AEG-1 in human neuroblastoma pathogenesis. AEG-1 expression was significantly elevated in neuroblastoma patient-derived samples and neuroblastoma cell lines as compared with normal peripheral nerve tissues, normal astrocytes and immortalized melanocytes. Knockdown of AEG-1 by small interfering RNA reduced the tumorigenic properties of highly aggressive neuroblastoma cells. Conversely, over-expression of AEG-1 enhanced proliferation and expression of the transformed state in less aggressive neuroblastoma cells through activation of the phosphatidylinositol 3-kinase-Akt-signaling pathway and stabilization of MYCN. These provocative results indicate that AEG-1 may play a crucial role in the pathogenesis of neuroblastoma and could represent a potential target for therapeutic intervention.
Assuntos
Moléculas de Adesão Celular/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Animais , Astrócitos/metabolismo , Moléculas de Adesão Celular/deficiência , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica , Feto/citologia , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Melanócitos/metabolismo , Proteínas de Membrana , Camundongos , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/genética , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/metabolismo , Nervos Periféricos/citologia , Nervos Periféricos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Ligação a RNA , Transdução de SinaisRESUMO
The prognosis and response to conventional therapies of malignant melanoma inversely correlate with disease progression. With increasing thickness, melanomas acquire metastatic potential and become inherently resistant to radiotherapy and chemotherapy. These harsh realities mandate the design of improved therapeutic modalities, especially those targeting metastases. To develop an approach to effectively treat this aggressive disease, we constructed a conditionally replication-competent adenovirus in which expression of the adenoviral E1A gene, necessary for replication, is driven by the cancer-specific promoter of progression-elevated gene-3 (PEG-3) and which simultaneously expresses mda-7/IL-24 in the E3 region of the adenovirus (Ad.PEG-E1A-mda-7), a cancer terminator virus (CTV). This CTV produces large quantities of MDA-7/IL-24 protein as a function of adenovirus replication uniquely in cancer cells. Infection of Ad.PEG-E1A-mda-7 (CTV) in normal human immortal melanocytes and human melanoma cells demonstrates cancer cell-selective adenoviral replication, mda-7/IL-24 expression, growth inhibition and apoptosis induction. Injecting Ad.PEG-E1A-mda-7 CTV into xenografts derived from MeWo human metastatic melanoma cells in athymic nude mice completely eliminated not only primary treated tumors but also distant non-treated tumors (established in the opposite flank), thereby implementing a cure. These provocative findings advocate potential therapeutic applications of this novel virus for treating patients with advanced melanomas with metastases.
Assuntos
Adenoviridae/genética , Adenoviridae/isolamento & purificação , Melanoma/genética , Melanoma/virologia , Regiões Terminadoras Genéticas/genética , Adenoviridae/fisiologia , Proteínas E1A de Adenovirus/genética , Animais , Progressão da Doença , Terapia Genética/métodos , Humanos , Melanoma/patologia , Camundongos , Camundongos Nus , Metástase Neoplásica/genética , Metástase Neoplásica/prevenção & controle , Transplante de Neoplasias , Transplante Heterólogo , Replicação ViralRESUMO
Astrocyte elevated gene-1 (AEG-1) displays oncogenic properties. Its expression is elevated in diverse neoplastic states and it cooperates with Ha-ras to promote cellular transformation. Overexpression of AEG-1 augments invasion and anchorage-independent growth of transformed cells, while AEG-1 siRNA inhibits Ha-ras-mediated colony formation, supporting a potential functional role in tumorigenesis. Additionally, oncogenic Ha-ras induces AEG-1 expression through the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway. In the present study, we investigated whether AEG-1 could induce serum-independent cell growth, another property of oncogenes. Overexpression of AEG-1 inhibited serum starvation-induced apoptosis through activation of PI3K-Akt signaling, one of the effector pathways induced by activated Ras. AEG-1 also affected the phosphorylation state of Akt substrates that are implicated in apoptosis suppression, including glycogen synthase kinase 3beta, c-Myc, murine double minute 2, p53, p21/mda-6 and Bad. Additionally, AEG-1 blocked the activity of serum starvation-induced caspases. Taken together, these observations provide evidence that AEG-1 is an oncogene cooperating with Ha-ras as well as functioning as a downstream target gene of Ha-ras and may perform a central role in Ha-ras-mediated carcinogenesis. Activation of survival pathways may be one mechanism by which AEG-1 exerts its oncogenic properties.
Assuntos
Astrócitos/enzimologia , Moléculas de Adesão Celular/fisiologia , Proteínas de Membrana/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Transdução de Sinais/fisiologia , Animais , Morte Celular/fisiologia , Linhagem Celular Transformada , Sobrevivência Celular/fisiologia , Marcação de Genes , Genes ras/fisiologia , Humanos , Camundongos , Proteínas de Ligação a RNA , Ratos , Proteínas ras/fisiologiaRESUMO
Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) is a cancer-specific, growth-suppressing and apoptosis-inducing gene with broad-spectrum antitumor activity. However, when administered by means of a replication-incompetent adenovirus, Ad.mda-7, several colorectal carcinoma cell lines are resistant to its antiproliferative and antisurvival effects. We have presently endeavored to determine if K-ras mutations, present in approximately 40-50% of colorectal cancers and which may mediate resistance to chemotherapy and radiotherapy, represent a predisposing genetic factor mitigating reduced sensitivity to Ad.mda-7. To suppress ras expression, three structurally different replication-incompetent adenoviral vectors were engineered that express (1) an intracellular, neutralizing single-chain antibody (scAb) to p21 ras (Ad.K-ras scAb), (2) an antisense (AS) K-ras gene (Ad.K-ras AS) or (3) both mda-7/IL-24 and a K-ras AS gene in a single bipartite virus (Ad.m7.KAS). Simultaneous inhibition of K-ras and expression of mda-7/IL-24 enhanced killing of colorectal carcinoma cells with mutated K-ras, but not with wild-type K-ras. The extent of killing depended on the degree of K-ras downregulation, with Ad.K-ras AS being generally more efficient than Ad.K-ras scAb in combination with Ad.mda-7. These findings support an effective dual-combinatorial approach for the therapy of colorectal cancers that employs a unique cancer-specific suppressor gene (mda-7/IL-24) with targeted inhibition of oncogene (ras) expression.
Assuntos
Apoptose , Neoplasias Colorretais/patologia , Genes ras/fisiologia , Interleucinas/metabolismo , Mutação/genética , Adenoviridae , Adjuvantes Imunológicos , Northern Blotting , Western Blotting , Ciclo Celular , Diferenciação Celular , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Terapia Genética , Vetores Genéticos , Humanos , Interleucinas/genética , Necrose , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução Genética , Células Tumorais Cultivadas , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/metabolismoRESUMO
Subtraction hybridization applied to terminally differentiating human melanoma cells identified mda-7/IL-24, a cytokine belonging to the IL-10 gene superfamily. Adenoviral-mediated delivery of mda-7/IL-24 (Ad.mda-7) provokes apoptosis selectively in a wide spectrum of cancers in vitro in cell culture, in vivo in human tumor xenograft animal models and in patients with advanced carcinomas and melanomas. In human prostate cancer cells, a role for mitochondrial dysfunction and induction of reactive oxygen species in the apoptotic process has been established. Ectopic overexpression of bcl-xL and bcl-2 prevents these changes including apoptosis induction in prostate tumor cells by Ad.mda-7. We now document that this resistance to apoptosis can be reversed by treating bcl-2 family overexpressing prostate tumor cells with ionizing radiation in combination with Ad.mda-7 or purified GST-MDA-7 protein. Additionally, radiation augments apoptosis induction by mda-7/IL-24 in parental and neomycin-resistant prostate tumor cells. Radiosensitization to mda-7/IL-24 is dependent on JNK signaling, as treatment with the JNK 1/2/3 inhibitor SP600125 abolishes this effect. Considering that elevated expression of bcl-xL and bcl-2 are frequent events in prostate cancer development and progression, the present studies support the use of ionizing radiation in combination with mda-7/IL-24 as a means of augmenting the therapeutic benefit of this gene in prostate cancer, particularly in the context of tumors displaying resistance to radiation therapy owing to bcl-2 family member overexpression.
Assuntos
Terapia Genética , Interleucinas/genética , Neoplasias da Próstata/terapia , Proteínas Proto-Oncogênicas c-bcl-2/análise , Tolerância a Radiação , Proteína bcl-X/análise , Apoptose , Linhagem Celular Tumoral , Terapia Combinada , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , Sistema de Sinalização das MAP Quinases , Masculino , Fosforilação , Neoplasias da Próstata/química , Neoplasias da Próstata/patologiaRESUMO
We investigated the role of radiation-induced mitogen activated protein kinase (MAPK) pathway activity in the regulation of proliferation, cell survival and vascular endothelial growth factor (VEGF) production in primary astrocytes and in T9 and RT2 glioblastoma cells derived from Fisher 344 rats. In these cells, ionizing radiation (2 Gy) caused activation of the MAPK pathway which was blocked by specific inhibitor drugs. Blunting of radiation-induced MAPK activity weakly enhanced radiation-induced apoptosis 24 h after exposure in RT2 cells. Furthermore, blunting of MAPK activation weakly enhanced the ability of radiation to reduce RT2 cell growth in clonogenic growth assays. These findings argue that inhibition of MAPK signaling reduces proliferation and enhances cell killing by ionizing radiation in transformed astrocytes. Proliferation and survival of cancer cells has been linked in vivo to enhanced expression of angiogenic growth factors. Recently we demonstrated that the gene product of a novel rodent radiation-responsive gene, progression elevated gene 3 (PEG-3), could enhance vascular endothelial growth factor (VEGF) promoter activity in rodent fibroblasts, leading to increased VEGF protein levels and tumorigenic behavior in vivo. Thus PEG-3 and VEGF expression could be expected to directly correlate with the oncogenic potential of transformed cells. RT2 cells expressed more PEG-3 and VEGF protein than T9 cells, and were more tumorigenic in vivo than T9 cells. Radiation activated the PEG-3 promoter via MAPK signaling and ectopic over-expression of PEG-3 enhanced both basal MAPK activity and basal VEGF promoter activity. Basal MAPK activity partially correlated with basal VEGF promoter activity and VEGF protein levels in primary astrocytes, T9 and RT2 cells. Radiation increased the activity of the VEGF promoter and VEGF protein levels in primary astrocytes, T9 and RT2 cells which were dependent upon MAPK function. Furthermore, inhibition of AP-1 transcription factor signaling by dominant negative c-Jun (TAM67) also significantly reduced basal, and to a lesser extent radiation-induced, VEGF promoter function in RT2 cells. Collectively, our data demonstrate that radiation-induced MAPK signaling can both protect cells from radiation-induced cell death as well as enhance protein levels of pro-angiogenic factors such as VEGF. Enhanced VEGF expression in RT2 cells may be mediated via MAPK and JNK pathway signaling which converges upon the AP-1 transcription factor complex.
Assuntos
Antígenos de Diferenciação , Astrócitos/efeitos da radiação , Fatores de Crescimento Endotelial/metabolismo , Glioma/radioterapia , Linfocinas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Apoptose , Sobrevivência Celular , Células Clonais , Fatores de Crescimento Endotelial/genética , Flavonoides/farmacologia , Glioblastoma , Linfocinas/genética , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas , Proteínas Proto-Oncogênicas c-jun , Radiação Ionizante , Ratos , Ratos Endogâmicos F344 , Transdução de Sinais , Fator de Transcrição AP-1/antagonistas & inibidores , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularAssuntos
Genes Supressores de Tumor , Terapia Genética , Substâncias de Crescimento/genética , Interleucinas , Melanoma/terapia , Adenovírus Humanos , Sequência de Aminoácidos , Animais , Apoptose , Sequência de Bases , Diferenciação Celular , Previsões , Terapia Genética/métodos , Vetores Genéticos , Humanos , Dados de Sequência MolecularRESUMO
Cancer is a progressive disease culminating in acquisition of metastatic potential by a subset of evolving tumor cells. Generation of an adequate blood supply in tumors by production of new blood vessels, angiogenesis, is a defining element in this process. Although extensively investigated, the precise molecular events underlying tumor development, cancer progression, and angiogenesis remain unclear. Subtraction hybridization identified a genetic element, progression elevated gene-3 (PEG-3), whose expression directly correlates with cancer progression and acquisition of oncogenic potential by transformed rodent cells. We presently demonstrate that forced expression of PEG-3 in tumorigenic rodent cells, and in human cancer cells, increases their oncogenic potential in nude mice as reflected by a shorter tumor latency time and the production of larger tumors with increased vascularization. Moreover, inhibiting endogenous PEG-3 expression in progressed rodent cancer cells by stable expression of an antisense expression vector extinguishes the progressed cancer phenotype. Cancer aggressiveness of PEG-3 expressing rodent cells correlates directly with increased RNA transcription, elevated mRNA levels, and augmented secretion of vascular endothelial growth factor (VEGF). Furthermore, transient ectopic expression of PEG-3 transcriptionally activates VEGF in transformed rodent and human cancer cells. Taken together these data demonstrate that PEG-3 is a positive regulator of cancer aggressiveness, a process regulated by augmented VEGF production. These studies also support an association between expression of a single nontransforming cancer progression-inducing gene, PEG-3, and the processes of cancer aggressiveness and angiogenesis. In these contexts, PEG-3 may represent an important target molecule for developing cancer therapeutics and inhibitors of angiogenesis.
Assuntos
Antígenos de Diferenciação , Proteínas de Neoplasias/genética , Neoplasias/genética , Neovascularização Patológica/genética , Animais , Proteínas de Ciclo Celular , Transformação Celular Neoplásica , Progressão da Doença , Fatores de Crescimento Endotelial/biossíntese , Predisposição Genética para Doença , Humanos , Linfocinas/biossíntese , Camundongos , Camundongos Nus , Fenótipo , Proteína Fosfatase 1 , Proteínas Proto-Oncogênicas , Ratos , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Several components of the eukaryotic protein synthesis apparatus have been associated with oncogenic transformation of cells. Altered expression of translation elongation factor 1 alpha (EF-1 alpha), a core component of protein synthesis and closely related sequences have been linked with transformed phenotypes by several independent studies, in diverse systems. A dominant acting oncogene, prostate tumor inducing gene-1 (PTI-1) has provided further evidence for this link. PTI-1 appears to be a hybrid molecule with components derived from both prokaryotic and eukaryotic origins. The predicted protein coding moiety represents an EF-1 alpha molecule, truncated N-terminal to amino acid residue 68 and having six additional point mutations. This coding sequence is fused to a 5' untranslated region (UTR) showing strongest homology to ribosomal RNA derived from Mycoplasma hyopneumoniae. Expression studies using the cloned cDNA in nude mouse tumor formation assays have confirmed the oncogenic nature of the molecule. A broad spectrum of tumor derived cell lines, from varied tissue sources and blood samples from patients having confirmed prostate carcinoma, all scored positive for expression of PTI-1, while corresponding normal tissues or blood samples were negative. Based on its near identity to EF-1 alpha, it is proposed that PTI-1 represents a new class of oncogene whose transforming capacity probably arises through mechanisms including: (i) protein translational infidelity, resulting in the synthesis of mutant polypeptides due to loss of proofreading function during peptide chain elongation, (ii) by its association with and alteration of the cytoskeleton, (iii) by impinging on one particular or several different signal transduction pathways through its properties as a G-protein.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias/genética , Proteínas Oncogênicas/genética , Biossíntese de Proteínas , Sequência de Aminoácidos , Animais , Elementos Antissenso (Genética)/genética , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/metabolismo , Clonagem Molecular , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas Oncogênicas/sangue , Proteínas Oncogênicas/metabolismo , Fator 1 de Elongação de Peptídeos , Neoplasias da Próstata/genética , Neoplasias da Próstata/secundário , Células Tumorais CultivadasRESUMO
DNA structure and expression of p53 gene in human hepatoma cell lines SMMC-7721, YY-8103 and a spontaneously transformed liver cell line L-02 were analysed using the following method: analysis of allelic losses on chromosome 17p, PCR/SSCP, Northern blot and immunoprecipitation. There was no point mutation found in the exons 4-9 of the p53 gene, and a low level of expression of p53 gene was detected in the three cell lines. These observations were in agreement to the reported results of the relevant experiment using the human hepatoma cell line QGY-7703. Sensitivities of these cell lines and other eight human hepatoma cell lines (QGY-7703, PLC/PRF/5, Tong/HCC, Huh-7, FOCUS, Hep3B, SK-Hep-1, HepG2) with known p53 backgrounds to parvovirus H-1 was assayed using MTT method. Abnormality in the structure and/or function was observed in all of the cell lines examined except HepG2. The cell line HepG2 with normal structure and function of the p53 gene was found to be the least sensitive to H-1 in comparison to all the cell lines which have defeated structure and/or function of the p53 gene. The present study serves as a preliminary evidence that enhancement of the sensitivity of human hepatoma cell lines to H-1 is correlated to the abnormality of the structure and/or function of the p53 gene.
Assuntos
Carcinoma Hepatocelular/genética , Genes p53/genética , Neoplasias Hepáticas/genética , Parvoviridae/genética , Expressão Gênica , Humanos , Mutação , Sensibilidade e Especificidade , Células Tumorais CultivadasRESUMO
A reciprocal subtraction differential RNA display (RSDD) approach has been developed that permits the rapid and efficient identification and cloning of both abundant and rare differentially expressed genes. RSDD comprises reciprocal subtraction of cDNA libraries followed by differential RNA display. The RSDD strategy was applied to analyze the gene expression alterations resulting during cancer progression as adenovirus-transformed rodent cells developed an aggressive transformed state, as documented by elevated anchorage-independence and enhanced in vivo oncogenesis in nude mice. This approach resulted in the identification and cloning of both known and a high proportion (>65%) of unknown sequences, including cDNAs displaying elevated expression as a function of progression (progression-elevated gene) and cDNAs displaying suppressed expression as a function of progression (progression-suppressed gene). Sixteen differentially expressed genes, including five unknown progression-elevated genes and six unknown progression-suppressed genes, have been characterized. The RSDD scheme should find wide application for the effective detection and isolation of differentially expressed genes.