RESUMEN
The mouse submandibular gland (SMG) is important organ for embryonic development, and branching morphogenesis is regulated by many molecules containing transcription factors. Real-time reverse transcriptase polymerase chain reaction revealed that the expression of Brachyury increased in the SMG and peaked between E12.5-E13.5, concomitant with the early stage of branching morphogenesis. The expression of Brachyury in SMG rudiments between E12.5-E13.5 was confirmed by western blotting. In addition, fibronectin and Btbd7 (regulated by fibronectin), which are both essential for cleft formation, were expressed strongly during the same period. The Sox2 and Wnt3a, which regulate cell growth, were also expressed strongly during E12.5-E13.5. On the other hand, cleft formation and branching morphogenesis was suppressed by knockdown of Brachyury gene, suggesting that Brachyury plays a central role in regulating cell growth and cleft formation in early-stage embryonic mouse salivary gland development.
Asunto(s)
Proteínas Fetales/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Morfogénesis/fisiología , Factores de Transcripción SOXB1/metabolismo , Glándulas Salivales/embriología , Glándulas Salivales/metabolismo , Proteínas de Dominio T Box/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Fibronectinas/metabolismo , Ratones , Ratones Endogámicos ICR , Proteínas Nucleares/metabolismo , Glándulas Salivales/citología , Distribución TisularRESUMEN
Salivary gland (SG) defects have a wide range of health implications, including xerostomia, bacterial infections, and oral health issues. Branching morphogenesis is critical for SG development. A clear understanding of the mechanisms underlying this process will accelerate SG regeneration studies. Fibroblast growth factor receptor 2 (FGFR2) interacts with multiple fibroblast growth factors (FGFs), which promote development. FGFR2 consists of two isoforms, FGFR2b and FGFR2c. FGFR2b is critical for SG development, but little is known about the expression and function of FGFR2c. We investigated the expression of all FGFR family members in fetal SGs between embryonic day 12.5 (E12.5) and E18.5. Based on RT-PCR, we observed an increase in the expression of not only Fgfr2b, but also Fgfr2c in early-stage embryonic mouse SGs, suggesting that FGFR2c is related to SG development. The branch number decreased in response to exogenous FGF2 stimulation, and this effect was suppressed by a mouse anti-FGFR2c neutralizing antibody (NA) and siRNA targeting FGFR2c, whereas FGFR2b signaling was not inhibited. Moreover, the expression of marker genes related to EMT was induced by FGF2, and this expression was suppressed by the NA. These results suggested that branching morphogenesis in SGs is regulated by FGFR2c, in addition to FGFR2b. Interestingly, FGFR2c signaling also led to increased fgf10 expression, and this increase was suppressed by the NA. FGFR2c signaling regulates branching morphogenesis through the activation of FGFR2b signaling via increased FGF10 autocrine. These results provide new insight into the mechanisms by which crosstalk between FGFR2b and FGFR2c results in efficient branching morphogenesis.
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Desarrollo Embrionario/genética , Factor 10 de Crecimiento de Fibroblastos/genética , Morfogénesis/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Glándulas Salivales/crecimiento & desarrollo , Animales , Embrión de Mamíferos , Células Epiteliales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Ratones , Isoformas de Proteínas/genética , Glándulas Salivales/metabolismo , Transducción de Señal/genéticaRESUMEN
We have recently unravelled a novel function for CD82 in E-cadherin-mediated cellular adhesion. CD82 inhibits ß-catenin tyrosine phosphorylation and stabilizes E-cadherin-ß-catenin complexes at the cell membrane. This function inhibits cancer cell dissociation from the primary cancer nest and limits metastasis. In this study, we focused on the effect of CD82 on the Wnt/ß-catenin (canonical) pathway, which controls the cellular distribution of ß-catenin. CD82 had no effect on the expression of Wnt proteins but led to significant downregulation of Frizzled (Fzd) 2, 3, 5, 7 and 9, suggesting downregulation of the Wnt/ß-catenin pathway. CD82 also inhibited phosphorylation of ß-catenin at Ser45, Ser33, Ser37 and Thr41 by downregulation of glycogen synthase kinase-3ß (GSK-3ß) and kinase casein kinase 1α (CK1α). Downregulation of GSK-3ß and CK1α also led to accumulation of ß-catenin in the cytoplasm or at the cell membrane. CD82 translocated ß-catenin to the cell membrane, suggesting that CD82 strengthens the interaction between E-cadherin and ß-catenin. We concluded that CD82 attenuates Wnt signalling by controlling ß-catenin cellular distribution at multiple levels: i) inhibition of ß-catenin nuclear translocation by downregulation of Fzd receptor proteins; ii) accumulation of ß-catenin at the cell membrane by downregulation of GSK-3ß and CK1α; and iii) stabilization of the E-cadherin-ß-catenin complex.
Asunto(s)
Carcinoma/metabolismo , Proteína Kangai-1/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismo , Carcinoma/genética , Línea Celular Tumoral , Receptores Frizzled/metabolismo , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Fosforilación , Estabilidad Proteica , Transporte de Proteínas , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMEN
BACKGROUND: The high frequencies of recurrence and distant metastasis of adenoid cystic carcinoma (AdCC) emphasize the need to better understand the biological factors associated with these outcomes. To analyze the mechanisms of AdCC metastasis, we established the green fluorescence protein (GFP)-transfected subline ACCS-GFP from the AdCC parental cell line and the metastatic ACCS-M GFP line from an in vivo metastasis model. METHODS: Using these cell lines, we investigated the involvement of the epithelial-mesenchymal transition (EMT) and cancer stem cell (CSCs) in AdCC metastasis by real-time RT-PCR for EMT related genes and stem cell markers. Characteristics of CSCs were also analyzed by sphere-forming ability and tumorigenicity. Short hairpin RNA (shRNA) silencing of target gene was also performed. RESULTS: ACCS-M GFP demonstrated characteristics of EMT and additionally displayed sphere-forming ability and high expression of EMT-related genes (Snail, Twist1, Twist2, Slug, zinc finger E-box binding homeobox 1 and 2 [Zeb1 and Zeb2], glycogen synthase kinase 3 beta [Gsk3ß and transforming growth factor beta 2 [Tgf-ß2]), stem cell markers (Nodal, Lefty, Oct-4, Pax6, Rex1, and Nanog), and differentiation markers (sex determining region Y [Sox2], Brachyury, and alpha fetoprotein [Afp]). These observations suggest that ACCS-M GFP shows the characteristics of CSCs and CSCs may be involved in the EMT of AdCC. Surprisingly, shRNA silencing of the T-box transcription factor Brachyury (also a differentiation marker) resulted in downregulation of the EMT and stem cell markers. In addition, sphere-forming ability, EMT characteristics, and tumorigenicity were simultaneously lost. Brachyury expression in clinical samples of AdCC was extremely high and closely related to EMT. This finding suggests that regulation of EMT by Brachyury in clinical AdCC may parallel that observed in vitro in this study. CONCLUSIONS: The use of a single cell line is a limitation of this study. However, parallel data from in vitro and clinical samples suggest the possibility that EMT is directly linked to CSCs and that Brachyury is a regulator of EMT and CSCs.
Asunto(s)
Carcinoma Adenoide Quístico/patología , Transición Epitelial-Mesenquimal , Proteínas Fetales/metabolismo , Neoplasias de la Boca/patología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Proteínas de Dominio T Box/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Carcinoma Adenoide Quístico/genética , Carcinoma Adenoide Quístico/metabolismo , Línea Celular Tumoral , Femenino , Proteínas Fetales/genética , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Neoplasias de la Boca/genética , Neoplasias de la Boca/metabolismo , Metástasis de la Neoplasia , Fenotipo , Esferoides Celulares , Proteínas de Dominio T Box/genéticaRESUMEN
Objective. To evaluate the efficacy of selective submandibular neck dissection (SMND) in patients with oral squamous cell carcinoma (OSCC) with or without nodal metastasis. Patients. From a total of 384 patients with untreated OSCC who underwent radical excision, we identified 229 with clinically N0 necks and 68 with clinically N1 necks in level I. Main Outcome Measures. The Kaplan-Meier 5-year regional control and 5-year disease specific survival (DSS) were compared for SMND, radical neck dissection (RND), and modified radical neck dissection (MRND). Results. In clinically node-negative necks, the regional control rates were 85.2% with SMND and 83.3% with MRND (P = 0.89), and 5-year DSS rates were 86.5% and 87.0%, respectively, (P = 0.94). In clinically N1 necks, the regional control rates were 81.3% with SMND and 83.0% with RND (P = 0.72), and the DSS rates were 81.3% and 80.0%, respectively, (P = 0.94). Type of neck dissection was not significantly associated with regional control or DSS on either univariate or multivariate analysis using Cox's proportional hazard model. Conclusions. SMND can be effectively applied in elective and therapeutic management to patients with OSCC that are clinically assessed as N0 or N1 to level I of the neck.
RESUMEN
The high frequencies of recurrence and distant metastasis of adenoid cystic carcinoma (AdCC) are significant obstacles for the long-term cure of patients with AdCC and emphasize the need for better understanding of the biological factors associated with these outcomes. To identify proteins that mediate AdCC metastasis, we established three AdCC cell lines expressing green fluorescent protein (GFP) from the ACCS cell line using orthotopic transplantation and in vivo selection in nude mice: Parental ACCS-GFP, highly tumorigenic ACCS-T GFP and metastatic ACCS-M GFP. ACCS-GFP and ACCS-M GFP were subjected to DNA microarray analysis and the results were used for data mining studies. DNA microarray analysis revealed significantly altered biological processes in the ACC-M GFP cells, including events related to cell adhesion (three categories) and signaling (three categories). In particular, a significant down-regulation of cell adhesion molecules, such as cadherins and integrin subunits was observed. The loss of E-cadherin and integrins and the gain of vimentin in ACCS-M GFP cells were confirmed by immunoblotting. These results suggest that epithelial-mesenchymal transition (EMT) is a putative event in AdCC metastasis that induces tumor cell dissemination from the primary tumor site. In summary, in this study we established a useful nude mouse metastasis model which will enable further AdCC metastasis research and clinical treatment trials and we also provide evidence that EMT is significantly involved in the AdCC metastatic process.
Asunto(s)
Carcinoma Adenoide Quístico/secundario , Transición Epitelial-Mesenquimal , Animales , Carcinoma Adenoide Quístico/metabolismo , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Línea Celular Tumoral , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundario , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Metástasis Linfática , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica , Trasplante de Neoplasias , Transcripción GenéticaRESUMEN
Numerous past studies have suggested a critical role of the paracrine effect between tumor vascular endothelial growth factor (VEGF)-C and lymphatic FLT-4 in solid tumor-associated lymphangiogenesis. In contrast, the pathophysiological role of tumor cell-associated FLT-4 in tumor progression remains to be elucidated. Here, we investigated this role using a tumor implantation model. SAS cells, an oral squamous carcinoma cell line expressing both VEGF-C and FLT-4 but neither FLK-1/KDR nor VEGF-D were adopted for experiments. Stable transformants of dominant-negative (dn) SAS cells were established in which the cytoplasmic domain-deleted FLT-4 was exogenously overexpressed, which can lead to inactivation of endogenous FLT-4 through competitive antagonism and is associated with down-activation of endogenous FLT-4-related intracellular signals. In vitro and in vivo proliferation assays showed lower proliferative activity of dn-SAS cells. An immunohistochemical study revealed that the tumor lymphangiogenesis was significantly suppressed, and the level of human VEGF-C mRNA was significantly lower in dn-SAS cell-derived tumor tissues. Moreover, in vitro studies demonstrated that the significant suppression of VEGF-C and VEGF-A expression was evident in dn-SAS cells or wild-type SAS cells treated with either the FLT-4 kinase inhibitor MAZ51 or the inhibitor of FLT-4-related signals. These findings together suggested that the VEGF-C/FLT-4 autocrine loop in tumor cells was a potential enhancer system to promote cancer progression, and FLT-4 in tumor tissue might become an effective target for cancer therapy.
Asunto(s)
Comunicación Autocrina , Carcinoma de Células Escamosas/enzimología , Linfangiogénesis , Neoplasias de la Boca/enzimología , Factor C de Crecimiento Endotelial Vascular/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Comunicación Autocrina/efectos de los fármacos , Carcinoma de Células Escamosas/genética , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Indoles/farmacología , Linfangiogénesis/efectos de los fármacos , Masculino , Ratones , Neoplasias de la Boca/genética , Naftalenos/farmacología , Trasplante de Neoplasias , Transducción de Señal/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor C de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 3 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
OBJECTIVE: A tooth with a dentigerous cyst (DC) does not always erupt by marsupialization. The eruption duration and conditions of DC-associated premolars were examined to predict such eruption following marsupialization. STUDY DESIGN: The eruption and conditions including depth, root formation, inclination, and eruption space were examined retrospectively in 21 DC-associated mandibular premolars using dental and panoramic radiograms. RESULTS: Fifteen of 21 premolars erupted half within 3 months and all 15 erupted completely within 10 months after marsupialization, without orthodontic traction. The age of the patients, tooth depth, and inclination were significantly different between the erupted and non-erupted groups, whereas there was no significant difference in the root formation or the eruption space between the 2 groups. CONCLUSIONS: The successful eruption of a DC-associated premolar can be predicted within 3 months after marsupialization. Furthermore, the eruption may be affected by the patient's age, tooth depth, and tooth inclination.
Asunto(s)
Diente Premolar/fisiopatología , Quiste Dentígero/cirugía , Enfermedades Mandibulares/cirugía , Erupción Dental/fisiología , Adolescente , Factores de Edad , Diente Premolar/diagnóstico por imagen , Niño , Quiste Dentígero/fisiopatología , Femenino , Estudios de Seguimiento , Predicción , Humanos , Masculino , Enfermedades Mandibulares/fisiopatología , Odontogénesis/fisiología , Radiografía Panorámica , Estudios Retrospectivos , Ápice del Diente/diagnóstico por imagen , Cuello del Diente/diagnóstico por imagen , Corona del Diente/diagnóstico por imagen , Raíz del Diente/diagnóstico por imagen , Raíz del Diente/fisiopatología , Diente Impactado/diagnóstico por imagen , Diente Impactado/fisiopatología , Diente Impactado/cirugía , Diente no Erupcionado/fisiopatologíaRESUMEN
BACKGROUND AND OBJECTIVE: Matrix metalloproteinase (MMP)-13 has wide substrate specificity compared with other MMPs and appears to be involved in periodontitis. Previously, we reported that roxithromycin (RXM) inhibits vascular endothelial growth factor expression induced by tumour necrosis factor-alpha in human periodontal ligament cells, but little is known about the effect of RXM on MMP-13 expression in human gingival epithelial cells. We therefore examined the effect of RXM on MMP-13 mRNA expression and production in cultured human gingival epithelial cells. MATERIAL AND METHODS: Human epithelial cell lines (Ca9-22, TU4, SCCTF and HSC-3) were plated in tissue culture dishes. Then, the culture supernatants and sediments were collected and the production of MMP-13 was analysed using enzyme-linked immunosorbent assay; the expression of MMP-13 mRNA and runt-related gene 2 mRNA was assessed using reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time RT-PCR. We also studied the effect of Runx2 short interfering RNA (siRNA) on the induction of MMP-13. RESULTS: Roxithromycin downregulated the induction of MMP-13 in Ca9-22 cells. Roxithromycin suppressed the expression of MMP-13 mRNA not only in Ca9-22 cells, but also in other human epithelial cell lines. Roxithromycin strongly inhibited the expression of Runx2 mRNA. Furthermore, Runx2 siRNA inhibited the induction of MMP-13 in Ca9-22 cells. CONCLUSION: These results indicate that RXM suppresses MMP-13 via the downregulation of Runx2 in human gingival epithelial cell cultures.
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Antibacterianos/farmacología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Inducción Enzimática/efectos de los fármacos , Encía/enzimología , Inhibidores de la Metaloproteinasa de la Matriz , Roxitromicina/farmacología , Línea Celular Tumoral , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/fisiología , Regulación hacia Abajo , Células Epiteliales/enzimología , Encía/citología , Humanos , Metaloproteinasa 13 de la Matriz/biosíntesis , Metaloproteinasa 13 de la Matriz/genética , Interferencia de ARN , ARN Mensajero/biosíntesis , ARN Interferente Pequeño/biosíntesis , ARN Interferente Pequeño/fisiologíaRESUMEN
The prognosis of patients with oral squamous cell carcinoma (SCC) is influenced by the presence of lymph node metastasis. In this study, we analyzed the relationship between lymphangiogenesis and the expression of VEGF-C and VEGF-D in association with lymph node metastasis in oral SCC. Oral SCC biopsy specimens (160 cases) were examined for lymphatic vessel density (LVD) and the expression of VEGF-C and VEGF-D immunohistochemically. The levels of VEGF-C and VEGF-D expression and LVD were significantly associated with lymph node metastasis (p<0.001). The expression of VEGF-C and VEGF-D increased the LVD significantly (p<0.001). Multivariate analysis showed that VEGF-C expression and LVD were significantly associated with lymph node metastasis (p<0.001). This study presents clinical evidence for the important roles of VEGF-C and VEGF-D in lymphangiogenesis and lymphatic metastasis of oral SCC, and suggests that VEGF-C or LVD can effectively predict lymphatic metastasis of oral SCC.
Asunto(s)
Ganglios Linfáticos/metabolismo , Neoplasias de la Boca/irrigación sanguínea , Neoplasias de la Boca/metabolismo , Neoplasias de Células Escamosas/irrigación sanguínea , Neoplasias de Células Escamosas/metabolismo , Factor C de Crecimiento Endotelial Vascular/biosíntesis , Factor D de Crecimiento Endotelial Vascular/biosíntesis , Adulto , Anciano , Biomarcadores de Tumor/biosíntesis , Estudios de Cohortes , Femenino , Humanos , Inmunohistoquímica , Ganglios Linfáticos/patología , Metástasis Linfática , Masculino , Persona de Mediana Edad , Neoplasias de la Boca/patología , Neoplasias de Células Escamosas/patología , Pronóstico , Análisis de Regresión , Estudios Retrospectivos , Tasa de SupervivenciaRESUMEN
BACKGROUND: Interleukin-1 alpha (IL-1 alpha) is thought to play a crucial role in the growth of keratocystic odontogenic tumors (KCOTs) in the jaw. The function of IL-1 alpha is regulated by the local levels of IL-1 alpha, its receptor and receptor antagonist (IL-1Ra) in tissues. In this study, the expression of these proteins was investigated both before and after marsupialization in KCOTs. METHODS: The expression of IL-1 alpha, IL-1 receptor type I (IL-1RI) and IL-1Ra was detected immunohistochemically in 10 specimens of KCOTs. RESULTS: IL-1 alpha was intensively expressed throughout the epithelium in all cases, while mild expression of IL-1 alpha was detected in the subepithelial layer endothelial cells and fibroblasts. Mild or intensive immunoreactivity for IL-1RI was also observed in the epithelial cells in all cases, and in the endothelial cells and fibroblasts in five cases respectively. The expression of IL-1Ra was detected in the epithelial cells in five cases, and in the endothelial cells and fibroblasts in three cases. After marsupialization, the immunoreactivity for IL-1 alpha and IL-1RI in the epithelial cells decreased, while the immunoreactivity for IL-1Ra in the epithelial cells increased. However, the immunoreactivity for IL-1RI and IL-1Ra in endothelial cells and fibroblasts did not change significantly. CONCLUSION: The effects of IL-1 alpha on the epithelial cells might be downregulated after marsupialization by changing the expression levels of IL-1 alpha, IL-1RI and IL-1Ra in the epithelium of KCOTs.
Asunto(s)
Proteína Antagonista del Receptor de Interleucina 1/metabolismo , Interleucina-1alfa/metabolismo , Quistes Odontogénicos/metabolismo , Tumores Odontogénicos/metabolismo , Receptores de Interleucina-1/metabolismo , Adulto , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Neoplasias Maxilomandibulares/complicaciones , Neoplasias Maxilomandibulares/metabolismo , Masculino , Persona de Mediana Edad , Quistes Odontogénicos/complicaciones , Tumores Odontogénicos/complicacionesRESUMEN
BACKGROUND: Antisense (AS) induced down-regulation of uPAR in ACCS adenoid-cyctic carcinoma cells decreased the cellular adhesion and invasion on various extracellular matrices. Additionally, ACCS-AS cells showed an increased EGFR expression and other behavioral similarities to NA-SCC, a typical highly proliferative but less invasive squamous cell carcinoma (SCC) cell line of the head and neck. ACCS, ACCS-AS and NA-SCC cells were used to elucidate the relationships between uPAR down-regulation and EGFR inhibition. RESULTS: Tyrosine kinase inhibitor Gefitinib (IRESSA, ZD 1839) significantly reduced the chemotactic cell migration and adhesion. This was associated with reduced EGFR and ERK activation. In addition, anti-proliferative effect of gefitinib in uPAR down-regulated ACCS-AS was significantly higher than parental ACCS, to levels comparable to gefitinib-sensitive NA-SCC cells. This was evidenced by both reduced dosage and duration of treatment. Furthermore, time-lapse videography showed that treatment with gefitinib was also associated with cell rounding and loss of pseudopodia, mostly in ACCS-AS rather than parental ACCS cells. There were also evidences of formation and exocytosis of vacuole-like structures in ACCS-AS, as well as NA-SCC, but not in parental ACCS cells. Interestingly, immunocytochemistry showed that the exocytotic vacuoles actually contained de-activated EGFR. CONCLUSION: Our results suggested that down-regulation of uPAR affected the fate of EGFR in high EGFR expressing cells. Furthermore, combining the uPAR down-regulation with EGFR inhibition showed a synergistic anti-tumor effect and might provide an alternative method to increase anti-proliferative effect of tyrosine kinase inhibitors with lower doses and duration to reduce their side effects during cancer control.
Asunto(s)
Regulación hacia Abajo , Receptores ErbB/metabolismo , Adhesiones Focales/efectos de los fármacos , Adhesiones Focales/metabolismo , Neoplasias de Cabeza y Cuello/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quimiotaxis/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Receptores ErbB/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/patología , Humanos , ARN Mensajero/genética , Activador de Plasminógeno de Tipo Uroquinasa/genéticaRESUMEN
In this study, we analyzed the effect of the metastasis suppressor CD82/KAI-1, a member of the tetraspanin superfamily, on intercellular adhesion on cancer cells. The newly established invasion assay and the cell aggregation assay revealed that CD82 strengthens E-cadherin-mediated intercellular adhesion. Interestingly, ectopic expression of CD82 stabilized E-cadherin/beta-catenin complex formation. Furthermore, CD82 reduced tyrosine phosphorylation of beta-catenin on HGF stimulation. Taken together, CD82 may stabilize or strengthen E-cadherin-dependent intercellular adhesion by regulating beta-catenin-mediated signal transduction on cancer cells, and consequently, prevent cancer cells from seceding from the primary tumor site.
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Cadherinas/metabolismo , Proteína Kangai-1/fisiología , Neoplasias/metabolismo , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Humanos , Neoplasias/patología , Fosforilación , Tirosina/metabolismo , beta Catenina/química , beta Catenina/metabolismoRESUMEN
It has been proposed that the metastasis suppressor CD82/KAI-1, which is a member of the tetraspanin superfamily, regulates biological activity by associating with cell surface receptors or proteins. We show a novel association between CD82 and the hepatocyte growth factor (HGF) receptor c-Met. Although ectopic expression of CD82 in nonsmall cell lung carcinoma cells did not affect the tyrosine phosphorylation of c-Met, these cells showed significant suppression of HGF-induced lamellipodial protrusion and cell migration. CD82 selectively attenuated c-Met signaling via the Ras-Cdc42/Rac and the phosphatidylinositol 3-kinase/Cdc42/Rac pathways. In contrast, another c-Met signaling pathway that involves phosphatidylinositol 3-kinase/Akt and phosphatidylinositol 3-kinase/mitogen activated protein kinase was not affected by CD82. Signaling adapter proteins for c-Met, such as Grb2 and p85, exhibited reduced association with c-Met in cells that ectopically expressed CD82. These results indicate that the CD82-c-Met complex inhibits HGF-induced cancer cell migration by the inactivation of small GTP-binding proteins of the Rho family via c-Met adapter proteins.
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Movimiento Celular , Proteína Kangai-1/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Proto-Oncogénicas c-met/metabolismo , Transducción de Señal , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Anticuerpos/inmunología , Línea Celular Tumoral , Proliferación Celular , Activación Enzimática , Regulación Neoplásica de la Expresión Génica , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Proteína Kangai-1/genética , Proteína Kangai-1/inmunología , Neoplasias/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Unión Proteica , Proteínas ras/metabolismo , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Adenoid cystic carcinoma (ACC) is one of the most common malignancies to arise in the salivary glands, yet very little is known of the genetic alterations that are involved in the pathogenesis of this disease. To further examine the genetic changes that underlie ACC, we analyzed genomic DNA obtained from 22 primary ACC and two ACC-derived cell lines by high-density oligonucleotide single-nucleotide polymorphism genotyping arrays (Affymetrix GeneChip Human Mapping 100K Set). Allelotype calls were analyzed by the Haplotype Correction version of the Linkage Disequilibrium Hidden Markov Model to determine loss of heterozygosity using information derived only from tumor samples. Comparison of data obtained from matched tumor-normal samples suggested that only deletion calls of >3 Mb were reliable. Within these parameters, ACC samples revealed a mean of three deletions per tumor, and no consensus areas of deletion were observed across the majority of tumors. Similarly, copy number analysis of primary hybridization data revealed no consensus areas of gene amplification. This is in contrast to a much higher rate of genomic alterations detected in a cohort of squamous carcinomas analyzed by the same methods. Our data show that most ACC have predominantly stable genomes, which is consistent with the theory that telomere crisis does not play a significant role in early stages of ACC tumor progression. Our data suggest that gene mutation and/or epigenetic events that cannot be detected by assay of gross alteration of chromosomal structure are likely to underlie the malignant transformation events of this tumor type.
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Carcinoma Adenoide Quístico/genética , Análisis Mutacional de ADN/métodos , Dosificación de Gen , Pérdida de Heterocigocidad , Polimorfismo de Nucleótido Simple/genética , Neoplasias de las Glándulas Salivales/genética , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma Adenoide Quístico/secundario , Línea Celular Tumoral , Deleción Cromosómica , ADN de Neoplasias/análisis , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Masculino , Repeticiones de Microsatélite , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias de las Glándulas Salivales/patologíaRESUMEN
Fibroblasts isolated from jaw cysts expressed calcium-sensing receptor (CasR). In the fibroblasts elevated extracellular Ca(2+) ([Ca(2+)](o)) increased fluo-3 fluorescence intensity, and the production of inositol(1,4,5)trisphosphate and active protein kinase C. Phospholipase C inhibitor U-73122 attenuated the Ca(2+)-induced increase in fluo-3 fluorescence intensity. Elevated [Ca(2+)](o) enhanced the expression of cyclooxygenase-2 (COX-2) mRNA and protein, and the secretion of prostaglandin E(2) in the fibroblasts. CasR activator neomycin also increased the expression of COX-2 mRNA, and U-73122 attenuated the Ca(2+)-induced expression of COX-2 mRNA. Elevated [Ca(2+)](o)-induced phosphorylation of extracellular signal-regulated protein kinase-1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK), and U-73122 inhibited the Ca(2+)-induced phosphorylation. The inhibitors for each kinase, PD98059, SB203580, and SP600125, attenuated the Ca(2+)-induced expression of COX-2 mRNA. These results suggest that in jaw cyst fibroblasts elevated extracellular Ca(2+) may enhance COX-2 expression via the activation of ERK1/2, p38 MAPK, and JNK through CasR.
Asunto(s)
Huesos/citología , Calcio/metabolismo , Ciclooxigenasa 2/metabolismo , Fibroblastos/enzimología , Receptores Sensibles al Calcio/metabolismo , Antracenos/farmacología , Huesos/efectos de los fármacos , Huesos/enzimología , Señalización del Calcio , Cationes Bivalentes/metabolismo , Ciclooxigenasa 2/genética , Dinoprostona/metabolismo , Estrenos/farmacología , Fibroblastos/efectos de los fármacos , Flavonoides/farmacología , Humanos , Imidazoles/farmacología , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Pirrolidinonas/farmacología , ARN Mensajero/metabolismo , Receptores Sensibles al Calcio/genética , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismoRESUMEN
Some modification on the surgical technique for bone grafting to the alveolar clefts has been required. Secondary bone grafting was performed with and without free-periosteum on 34 (mean age, 9.8 +/- 1.1 years) and 44 (mean age, 10.7 +/- 1.2 years) alveolar clefts, respectively. Vertical bone formation which scored 3 or 4 was obtained in 97.1% of free-periosteum grafted clefts, while it was obtained in 79.5% of the control group. The rate of postoperative wound dehiscence in free-periosteum grafted clefts (20.6%) was not significantly different from the control group (11.4%). In the wound dehiscence cases, however, the grafted periosteum covered the grafted bone and prevented bone exposure. The score of bone formation in the free-periosteum grafted clefts (3.57 +/- 0.79) was significantly higher than that of the control group (2.60 +/- 1.34). Thus, free-periosteum grafting in secondary bone grafting is useful for bone formation in alveolar clefts.
Asunto(s)
Proceso Alveolar/anomalías , Proceso Alveolar/cirugía , Trasplante Óseo/métodos , Periostio/trasplante , Niño , Humanos , Ilion/cirugía , Maxilar/cirugía , Dehiscencia de la Herida Operatoria/epidemiologíaRESUMEN
Differentiation-inducing factors (DIFs) are morphogens which induce cell differentiation in Dictyostelium. We reported that DIF-1 and DIF-3 inhibit proliferation and induce differentiation in mammalian cells. In this study, we investigated the effect of DIF-1 on oral squamous cell carcinoma cell lines NA and SAS, well differentiated and poorly differentiated cell lines, respectively. Although DIF-1 did not induce the expression of cell differentiation makers in these cell lines, it inhibited the proliferation of NA and SAS in a dose-dependent manner by restricting the cell cycle in the G0/G1 phase. DIF-1 induced cyclin D1 degradation, but this effect was prevented by treatment with lithium chloride and SB216763, the inhibitors of glycogen synthase kinase-3beta (GSK-3beta). Depletion of endogenous GSK-3beta by RNA interference also attenuated the effect of DIF-1 on cyclin D1 degradation. Therefore, we investigated the effect of DIF-1 on GSK-3beta and found that DIF-1 dephosphorylated GSK-3beta on Ser9 and induced the nuclear translocation of GSK-3beta, suggesting that DIF-1 activated GSK-3beta. Then, we examined the effect of DIF-1 on cyclin D1 mutants (Thr286Ala, Thr288Ala, and Thr286/288Ala). We revealed that Thr286Ala and Thr286/288Ala mutants were highly resistant to DIF-1-induced degradation compared with wild-type cyclin D1, indicating that the phosphorylation of Thr286 was critical for cyclin D1 degradation induced by DIF-1. These results suggest that DIF-1 induces degradation of cyclin D1 through the GSK-3beta-mediated phosphorylation of Thr286.
Asunto(s)
Antineoplásicos/uso terapéutico , Carcinoma de Células Escamosas/tratamiento farmacológico , Ciclina D1/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Hexanonas/uso terapéutico , Hidrocarburos Clorados/uso terapéutico , Neoplasias de la Boca/tratamiento farmacológico , Antineoplásicos/farmacología , Carcinoma de Células Escamosas/metabolismo , Ciclo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Ciclina D1/genética , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta , Hexanonas/farmacología , Humanos , Hidrocarburos Clorados/farmacología , Neoplasias de la Boca/metabolismo , Mutación , Fosforilación , Transporte de Proteínas , Interferencia de ARN , Treonina/metabolismoRESUMEN
IkappaB kinase (IKK) alpha and beta share the function to phosphorylate IkappaB to activate a transcription factor NF-kappaB. Recent reports, however, revealed differences in the functions of the two kinases. The present study was designed to determine a unique function of IKKalpha on the differentiation of squamous cell carcinoma (SCC). Transfection with IKKalpha gene, but neither IKKbeta nor NF-kappaB gene, inhibited the constitutive expressions as well as extracellular calcium-induced expressions of involucrin and filaggrin, epithelial differentiation markers, in cultured SCC cells. Morphological changes from polygonal to fibroblastic shape were seen in the SCC cells stably expressing green-fluorescent protein (GFP)-fused IKKalpha while intracellular localization of GFP-IKKalpha differed from that of GFP-IKKbeta. Interestingly, phorbol myristate acetate together with IKKalpha gene transfection strongly inhibited the expression of involucrin in SCC cells and induced the phosphorylation of serine residue in IKKalpha, suggesting that protein kinase C is involved in the effect of IKKalpha on the differentiation of SCC cells. In conclusion, high expression of IKKalpha may serve as an intracellular signal to halt the epithelial differentiation of SCC cells.
Asunto(s)
Carcinoma de Células Escamosas/enzimología , Quinasa I-kappa B/fisiología , Neoplasias de la Boca/enzimología , Proteínas Serina-Treonina Quinasas/genética , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Transformación Celular Neoplásica , Proteínas Filagrina , Humanos , Quinasa I-kappa B/genética , Proteínas de Filamentos Intermediarios/antagonistas & inhibidores , Neoplasias de la Boca/patología , Precursores de Proteínas/antagonistas & inhibidoresRESUMEN
Adenoid cystic carcinoma (AdCC) cell lines (ACCS and ACCT) showed higher migration responses and adhesion to the extracellular matrix (ECM), especially types I and IV collagen, than did the oral squamous cell carcinoma (SCC) lines (NA and TF). The response to collagens was largely and exclusively inhibited by anti-alpha(2) integrin antibody. Moreover, AdCC cell lines expressed higher surface levels of urokinase-type plasminogen activator receptor (uPAR) than did SCC cell lines. When AdCC cells were plated on collagen, the surface level of uPAR was increased, and numerous focal adhesions consisting of uPAR, vinculin, and paxillin were assembled; whereas collagen-stimulated SCC cell counterparts or AdCC cells plated on other types of ECM, such as fibronectin, failed to assemble such definite focal adhesions. In order to elucidate the association of uPAR with collagen-induced events, an ACCS-AS cell line transfected with a vector expressing antisense uPAR RNA was established and shown to have reduced uPAR (about 10% that of parental ACCS at both the protein and mRNA levels). ACCS-AS showed a strong reduction of collagen-stimulated migration and focal adhesion assembly of alpha(2) integrin, vinculin, and paxillin. These findings suggest that AdCC has a proclivity for migrating to types I and IV collagens due to the overexpression of uPAR, which plays a key role in focal adhesion assembly and migration.