RESUMO
Inflammatory microenvironment signalling plays a crucial role in tumour progression (i.e. cancer cell proliferation, survival, angiogenesis and metastasis) in many types of human malignancies. However, the role of inflammation in brain tumour pathology remains poorly understood. Here, we report that interferon regulatory factor 7 is a crucial regulator of brain tumour progression and heterogeneity. Ectopic expression of interferon regulatory factor 7 in glioma cells promotes tumorigenicity, angiogenesis, microglia recruitment and cancer stemness in vivo and in vitro through induction of interleukin 6, C-X-C motif chemokine 1 and C-C motif chemokine 2. In particular, interferon regulatory factor 7-driven interleukin 6 plays a pivotal role in maintaining glioma stem cell properties via Janus kinase/signal transducer and activator of transcription-mediated activation of Jagged-Notch signalling in glioma cells and glioma stem cells derived from glioma patients. Accordingly, the short hairpin RNA-mediated depletion of interferon regulatory factor 7 in glioma stem cells markedly suppressed interleukin 6-Janus kinase/signal transducer and activator of transcription-mediated Jagged-Notch-signalling pathway, leading to decreases in glioma stem cell marker expression, tumoursphere-forming ability, and tumorigenicity. Furthermore, in a mouse model of wound healing, depletion of interferon regulatory factor 7 suppressed tumour progression and decreased cellular heterogeneity. Finally, interferon regulatory factor 7 was overexpressed in patients with high-grade gliomas, suggesting its potential as an independent prognostic marker for glioma progression. Taken together, our findings indicate that interferon regulatory factor 7-mediated inflammatory signalling acts as a major driver of brain tumour progression and cellular heterogeneity via induction of glioma stem cell genesis and angiogenesis.
Assuntos
Glioma/patologia , Fator Regulador 7 de Interferon/metabolismo , Interleucina-6/metabolismo , Células-Tronco Neoplásicas/fisiologia , Receptor Notch1/metabolismo , Transdução de Sinais/fisiologia , Antígeno AC133 , Antígenos CD/metabolismo , Astrócitos/metabolismo , Encéfalo/citologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células , Células Cultivadas , Quimiocina CCL2/metabolismo , Quimiocina CXCL1/metabolismo , Imunoprecipitação da Cromatina , Biologia Computacional , Células Endoteliais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Glicoproteínas/metabolismo , Humanos , Fator Regulador 7 de Interferon/genética , Neovascularização Patológica/induzido quimicamente , Neovascularização Patológica/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/fisiologia , Peptídeos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução Genética/métodos , Ensaio Tumoral de Célula-TroncoRESUMO
Telomerase reverse transcriptase (TERT), the catalytic subunit of the enzyme telomerase, is robustly expressed in cancer cells. TERT enables cells to avoid chromosome shortening during repeated replication by maintaining telomere length. However, several lines of evidence indicate that many cancer cells exhibit shorter telomere length than normal tissues, implying an additional function of TERT in tumor formation and progression. Here, we report a telomerase activity-independent function of TERT that induces cancer stemness in glioma cells. Overexpression of TERT712, a telomerase activity-deficient form of TERT, in U87MG cells promoted cell self-renewal in vitro, and induced EGFR expression and formation of gliomas exhibiting cellular heterogeneity in vivo. In patients with glioblastoma multiforme, TERT expression showed a high correlation with EGFR expression, which is closely linked to the stemness gene signature. Induction of differentiation and TERT-knockdown in glioma stem cells led to a marked reduction in EGFR expression, cancer stemness, and anticancer drug resistance. Together, our findings indicate that TERT plays a crucial role in tumor progression by promoting cancer stemness through expression of EGFR.
Assuntos
Neoplasias do Sistema Nervoso Central/patologia , Receptores ErbB/metabolismo , Glioma/patologia , Proteínas Mutantes/metabolismo , Células-Tronco Neoplásicas/citologia , Telomerase/metabolismo , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Neoplasias do Sistema Nervoso Central/metabolismo , Receptores ErbB/genética , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Glioma/metabolismo , Humanos , Proteínas de Filamentos Intermediários/metabolismo , Antígeno Ki-67/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Mutantes/genética , Proteínas do Tecido Nervoso/metabolismo , Nestina , Interferência de RNA , Fatores de Transcrição SOXB1/metabolismo , Telomerase/genética , Transcrição Gênica , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Although human telomerase catalytic subunit (TERT) has several cellular functions including telomere homeostasis, genomic stability, cell proliferation, and tumorigenesis, the molecular mechanism underlying anti-apoptosis regulated by TERT remains to be elucidated. Here, we show that ectopic expression of TERT in spontaneously immortalized human fetal fibroblast (HFFS) cells, which are a telomerase- and p53-positive, leads to increases of cell proliferation and transformation, as well as a resistance to DNA damage response and inactivation of p53 function. We found that TERT and a mutant TERT (no telomerase activity) induce expression of basic fibroblast growth factor (bFGF), and ectopic expression of bFGF also allows cells to be resistant to DNA-damaging response and to suppress activation of p53 function under DNA-damaging induction. Furthermore, loss of TERT or bFGF markedly increases a p53 activity and DNA-damage sensitivity in HFFS, HeLa and U87MG cells. Therefore, our findings indicate that a novel TERT-bFGF axis accelerates the inactivation of p53 and consequent increase of resistance to DNA-damage response.
Assuntos
Apoptose , Domínio Catalítico , Fator 2 de Crescimento de Fibroblastos/genética , Telomerase/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Transformada , Proliferação de Células , Dano ao DNA , Feto/citologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Telomerase/deficiênciaRESUMO
The basic helix-loop-helix myogenic regulatory factors play critical roles in skeletal myogenesis. Among the myogenic regulatory factors (MRFs), MRF4 shows a biphasic expression pattern during the formation of myotomes, although its function remains unclear. In this study, we used BEF (spontaneously immortalized bovine embryonic fibroblast that shows myogenic differentiation by overexpression of MyoD) and C2C12 cells to investigate the function of MRF4. Ectopic expressions of MRF4 did not stimulate myogenic differentiation in the BEF and C2C12 cells, but did show a marked increase of cell proliferation, upregulation of cyclin E, and downregulation of p21WAF1. Furthermore, MRF4 was found to induce degradation of the MyoD protein, which acts as a transcriptional activator for p21WAF1, and thus indicates that MRF4 accelerates cell proliferation by suppressing MyoD-dependent p21WAF1 expression. However, forced expression of MyoD in the MRF4-overexpressing cells inhibited cell proliferation and partially induced myogenic differentiation, which suggests that MyoD is a potential negative intercessor of MRF4 in the regulation of the cell cycle. Taken together, these results indicate that MRF4 and MyoD play competitive roles in myogenesis by stimulating cell proliferation and differentiation, respectively.
Assuntos
Células Musculares/citologia , Células Musculares/fisiologia , Desenvolvimento Muscular/fisiologia , Proteína MyoD/metabolismo , Mioblastos/citologia , Mioblastos/fisiologia , Fatores de Regulação Miogênica/metabolismo , Transdução de Sinais/fisiologia , Animais , Diferenciação Celular , Linhagem Celular , Proliferação de Células , CamundongosRESUMO
BACKGROUND: The pig, Sus scrofa domestica includes both the miniature and commercial domestic breed. These animals have influenced the human life and economies and have been studied throughout history. Although the miniature breeds are more recent and have increasingly been used in a variety of biomedical studies, their cell lines have rarely been established. Therefore, we sought to establish primary and immortal cell lines derived from both the miniature and domestic pig to better enable insight into possible in vivo growth differences. RESULTS: The in vitro lifespan of primary domestic pig fibroblast (PF) and miniature pig fibroblast (MPF) cells using a standard 3T3 protocol was determined. Both of the primary PF and MPF cells were shown to have a two-step replicative senescence barrier. Primary MPF cells exhibited a relatively shorter lifespan and slower proliferation rate compared to those of primary PF cells. Beyond senescence barriers, lifespan-extended PF and MPF cells were eventually established and indicated spontaneous cellular immortalization. In contrast to the immortalized PF cells, immortal MPF cells showed a transformed phenotype and possessed more frequent chromosomal abnormalities and loss of p53 regulatory function. The lifespan of primary MPF and PF cells was extended by inactivation of the p53 function using transduction by SV40LT without any detectable senescent phenotype. CONCLUSION: These results suggest that p53 signaling might be a major determinant for the replicative senescence in the MPF cells that have the shorter lifespan and slower growth rate compared to PF cells in vitro.
Assuntos
Linhagem Celular , Proliferação de Células , Senescência Celular/fisiologia , Fibroblastos/citologia , Animais , Antígenos Transformantes de Poliomavirus/genética , Sobrevivência Celular/fisiologia , Células Cultivadas , Aberrações Cromossômicas , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/fisiologia , Feminino , Fibroblastos/fisiologia , Fenótipo , Transdução de Sinais , Sus scrofa , Suínos , Porco Miniatura , Telomerase/genética , Transdução Genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Using normal swine kidney epithelial (SKE) cells that were shown to be senescent at passages 12 to 14, we have established one lifespan-extended cell line and two lifespan-extended cell lines by exogenous introduction of the human catalytic subunit of telomerase (hTERT) and simian virus 40 large T-antigen (SV40LT), all of which maintain epithelial morphology and express cytokeratin, a marker of epithelial cells. SV40LT- and hTERT-transduced immortal cell lines appeared to be smaller and exhibited more uniform morphology relative to primary and spontaneously immortalized SKE cells. We determined the in vitro lifespan of primary SKE cells using a standard 3T6 protocol. There were two steps of the proliferation barrier at 12 and 20, in which a majority of primary SKE cells appeared enlarged, flattened, vacuolated, and ss-galactosidase-positive, all phenotypical characteristics of senescent cells. Lifespan-extended SKE cells were eventually established from most of the cellular foci, which is indicative of spontaneous cellular conversion at passage 23. Beyond passage 25, the rate of population doubling of the established cells gradually increased. At passage 30, immortal cell lines grew faster than primary counterpart cells in 10% FBS-DMEM culture conditions, and only SV40LT-transduced immortal cells grew faster than primary and other SKE immortal cells in 0.5% FBS-DMEM. These lifespan-extended SKE cell lines failed to grow in an anchorage-independent manner in soft-agar dishes. Hence, three immortalized swine kidney epithelial cells that are not transformed would be valuable biological tools for virus propagation and basic kidney epithelial cell research.
Assuntos
Linhagem Celular Transformada , Células Epiteliais/citologia , Rim/citologia , Suínos , Animais , Técnicas de Cultura de Células , Proteínas de Ligação a DNA/genética , Células Epiteliais/enzimologia , Rim/enzimologia , Vírus 40 dos Símios/genética , Telomerase/genética , Transformação GenéticaRESUMO
We have established three immortal bovine muscular epithelial (BME) cell lines, one spontaneously immortalized (BMES), the second SV40LT-mediated (BMEV) and the third hTERT-mediated (BMET). The morphology of the three immortal cell lines was similar to that of early passage primary BME cells. Each of the immortal cell lines made cytokeratin, a typical epithelial marker. BMET grew faster than the other immortal lines and the BME cells, in 10% FBS-DMEM medium, whereas neither the primary cells nor the three immortal cell lines grew in 0.5% FBS-DMEM. The primary BME cells and the immortal cell lines, with the exception of BMES, made increasing amounts of p53 protein when treated with doxorubicin, a DNA damaging agent. On the other hand, almost half of the cells in populations of the three immortal cell lines may lack p16(INK4a) regulatory function, compared to primary BME cells that were growth arrested by enforced expression of p16(INK4a). In soft-agar assays, the primary cells and immortal cell lines proved to be less transformed in phenotype than HeLa cells. The three immortal epithelial-type cell lines reported here are the first cell lines established from muscle tissue of bovine or other species.
Assuntos
Células Epiteliais/citologia , Células Epiteliais/metabolismo , Músculos/citologia , Animais , Bovinos , Adesão Celular , Linhagem Celular , Linhagem Celular Transformada , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Cinética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Genomic instability and apoptosis evasion are hallmarks of cancer, but the molecular mechanisms governing these processes remain elusive. Here, we found that survivin, a member of the apoptosis-inhibiting gene family, and aurora B kinase, a chromosomal passenger protein, were co-overexpressed in the various glioblastoma cell lines and tumors. Notably, exogenous introduction of the aurora B in human BJ cells was shown to decrease cell growth and increase the senescence-associated beta-galactosidase activity by activation of p53 tumor suppressor. However, aurora B overexpression failed to inhibit cell proliferation in BJ and U87MG cells transduced with dominant-negative p53 as well as in p53(-/-) mouse astrocytes. Aurora B was shown to increase centrosome amplification in the p53(-/-) astrocytes. Survivin was shown to induce anchorage-independent growth and inhibit anti-proliferation and drug-sensitive apoptosis caused by aurora B. Overexpression of both survivin and aurora B further accelerated the proliferation of BJ cells. Taken together, the present study indicates that survivin should accelerate tumorigenesis by inhibiting the anti-proliferative effect of p53 tumor suppressor that is activated by aurora B in normal and glioblastoma cells containing intact p53.