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1.
Oncogene ; 34(32): 4219-28, 2015 Aug 06.
Artículo en Inglés | MEDLINE | ID: mdl-25347745

RESUMEN

Zinc-finger, MYND-type containing 10 (ZMYND10), or more commonly called BLU, expression is frequently downregulated in nasopharyngeal carcinoma (NPC) and many other tumors due to promoter hypermethylation. Functional evidence shows that the BLU gene inhibits tumor growth in animal assays, but the detailed molecular mechanism responsible for this is still not well understood. In current studies, we find that 93.5% of early-stage primary NPC tumors show downregulated BLU expression. Using a PCR array, overexpression of the BLU gene was correlated to the angiogenesis network in NPC cells. Moreover, expression changes of the MMP family, VEGF and TSP1, were often detected in different stages of NPC, suggesting the possibility that BLU may be directly involved in the microenvironment and anti-angiogenic activity in NPC development. Compared with vector-alone control cells, BLU stable transfectants, derived from poorly-differentiated NPC HONE1 cells, suppress VEGF165, VEGF189 and TSP1 expression at both the RNA and protein levels, and significantly reduce the secreted VEGF protein in these cells, reflecting an unknown regulatory mechanism mediated by the BLU gene in NPC. Cells expressing BLU inhibited cellular invasion, migration and tube formation. These in vitro results were further confirmed by in vivo tumor suppression and a matrigel plug angiogenesis assay in nude mice. Tube-forming ability was clearly inhibited, when the BLU gene is expressed in these cells. Up to 70-90% of injected tumor cells expressing increased exogenous BLU underwent cell death in animal assays. Overexpressed BLU only inhibited VEGF165 expression in differentiated squamous NPC HK1 cells, but also showed an anti-angiogenic effect in the animal assay, revealing a complicated mechanism regulating angiogenesis and the microenvironment in different NPC cell lines. Results of these studies indicate that alteration of BLU gene expression influences anti-angiogenesis pathways and is important for the development of NPC.


Asunto(s)
Cromosomas Humanos Par 3/genética , Neoplasias Nasofaríngeas/genética , Neovascularización Patológica/genética , Transducción de Señal/genética , Proteínas Supresoras de Tumor/genética , Animales , Western Blotting , Carcinoma , Línea Celular Tumoral , Movimiento Celular/genética , Células Cultivadas , Mapeo Cromosómico , Proteínas del Citoesqueleto , Regulación hacia Abajo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/metabolismo , Ratones Desnudos , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trombospondina 1/genética , Trombospondina 1/metabolismo , Trasplante Heterólogo , Microambiente Tumoral/genética , Proteínas Supresoras de Tumor/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo
2.
Br J Cancer ; 108(3): 613-20, 2013 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-23299542

RESUMEN

BACKGROUND: Lobular endocervical glandular hyperplasia (LEGH) is a rare lesion of the uterine cervix. It has been proposed that LEGH may represent a precursor lesion to a group of mucinous adenocarcinoma with gastric phenotype (GA) that is independent of high-risk human papillomavirus (H-HPV) infection. Carbonic anhydrase-IX (CA-IX) is highly expressed in conventional glandular lesions (CGLs). However, expression of CA-IX in LEGH or GA has not been studied. METHODS: In all, 12 CGLs, 7 LEGHs, 6 LEGHs with coexisting adenocarcinoma in situ (AIS, 3) and GA (3) were identified from Japanese women with a cytological diagnosis of atypical glandular cells of undetermined significance. Immunostaining was used to detect CA-IX and p16(INK)4(a) (hereafter termed p16) protein expression in the tissues and CA-IX protein expression in the Papanicolaou smears (PSs). Polymerase chain reaction was used to detect H-HPV DNA in liquid-based cytology. RESULTS: Out of 12 (83%) CGLs, 10 were positive with H-HPV and high levels of CA-IX expression were seen in all (100%) cases. P16 protein expression was observed in 11 out of 12 (92%) cases. None of the LEGHs, LEGHs with AIS or GA were positive for H-HPV and only 8 out of 13 (62%) showed focal weak (1+) p16 expression. In contrast, all cases (100%) exhibited strong CA-IX protein expression. CONCLUSION: Our study suggests that there are different molecular mechanisms of carcinogenesis resulting in CGLs vs LEGHs associated with AIS or GA. There is also a possible link between LEGHs and GAs. Furthermore, CA-IX expression may serve as a useful biomarker for the detection of GAs in the absence of H-HPV infection.


Asunto(s)
Antígenos de Neoplasias/metabolismo , Biomarcadores de Tumor/metabolismo , Anhidrasas Carbónicas/metabolismo , Hiperplasia/patología , Neoplasias Glandulares y Epiteliales/patología , Infecciones por Papillomavirus/patología , Neoplasias del Cuello Uterino/patología , Adenocarcinoma Mucinoso/enzimología , Adenocarcinoma Mucinoso/patología , Adenocarcinoma Mucinoso/virología , Adulto , Anciano , Anciano de 80 o más Años , Anhidrasa Carbónica IX , Carcinoma Lobular/enzimología , Carcinoma Lobular/patología , Carcinoma Lobular/virología , ADN Viral/genética , Femenino , Humanos , Hiperplasia/enzimología , Hiperplasia/virología , Técnicas para Inmunoenzimas , Masculino , Persona de Mediana Edad , Neoplasias Glandulares y Epiteliales/enzimología , Neoplasias Glandulares y Epiteliales/virología , Papillomaviridae/genética , Infecciones por Papillomavirus/enzimología , Infecciones por Papillomavirus/virología , Reacción en Cadena de la Polimerasa , Pronóstico , Neoplasias Gástricas/enzimología , Neoplasias Gástricas/patología , Neoplasias Gástricas/virología , Neoplasias del Cuello Uterino/enzimología , Neoplasias del Cuello Uterino/virología
3.
Oncogenesis ; 1: e21, 2012 Jul 09.
Artículo en Inglés | MEDLINE | ID: mdl-23552737

RESUMEN

Carcinogenesis and cancer progression, driven by mutations in oncogenes and tumor-suppressor genes, result in biological differences between normal and cancer cells in various cellular processes. Specific genes and signaling molecules involved in such cellular processes may be potential therapeutic targets of agents that specifically interact with the key factors in cancer cells. Increased glucose uptake is fundamental to many solid tumors and well associated with increases in glycolysis and the overexpression of glucose transporters (GLUTs) such as GLUT1 and GLUT3 at the plasma membrane. Here, we used cell-based screening to identify glycogen synthase kinase-3ß (GSK-3ß) inhibitors that selectively target GLUT3-expressing tumorigenic HeLa cell hybrids as compared with non-tumorigenic hybrids that express GLUT1 alone. The GSK-3 inhibitors as well as GSK-3ß RNAi suppressed GLUT3 expression at the level of transcription, leading to apoptosis. This suppression was associated with NF-κB in a p53-independent manner. Furthermore, GSK-3 inhibitors exhibited a synergistic effect with anticancer agents such as adriamycin and camptothecin in GULT3-overexpressing colon cancer cells, but little effect in non-producing A431 cells. These results suggest a potential use of GSK-3 inhibitors to selectively kill cancer cells that overexpress GLUT3.

4.
Oncogene ; 31(32): 3709-20, 2012 Aug 09.
Artículo en Inglés | MEDLINE | ID: mdl-22158051

RESUMEN

Alpha B-crystallin (CRYAB) maps within the nasopharyngeal carcinoma (NPC) tumor-suppressive critical region 11q22-23 and its downregulation is significantly associated with the progression of NPC. However, little is known about the functional impact of CRYAB on NPC progression. In this study we evaluated the NPC tumor-suppressive and progression-associated functions of CRYAB. Activation of CRYAB suppressed NPC tumor formation in nude mice. Overexpression of CRYAB affected NPC progression-associated phenotypes such as loss of cell adhesion, invasion, interaction with the tumor microenvironment, invasive protrusion formation in three dimensional Matrigel culture, as well as expression of epithelial-mesenchymal transition-associated markers. CRYAB mediates this ability to suppress cancer progression by inhibition of E-cadherin cytoplasmic internalization and maintenance of ß-catenin in the membrane that subsequently reduces the levels of expression of critical downstream targets such as cyclin-D1 and c-myc. Both ectopically expressed and recombinant CRYAB proteins were associated with endogenous E-cadherin and ß-catenin, and, thus, the cadherin/catenin adherens junction. The CRYAB α-crystallin core domain is responsible for the interaction of CRYAB with both E-cadherin and ß-catenin. Taken together, these results indicate that CRYAB functions to suppress NPC progression by associating with the cadherin/catenin adherens junction and modulating the ß-catenin function.


Asunto(s)
Uniones Adherentes/metabolismo , Cadherinas/metabolismo , Carcinoma/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Cadena B de alfa-Cristalina/metabolismo , beta Catenina/metabolismo , Animales , Carcinoma/patología , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Progresión de la Enfermedad , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/patología , Invasividad Neoplásica , Trasplante de Neoplasias , Transporte de Proteínas , Carga Tumoral
5.
Oncogene ; 31(6): 728-38, 2012 Feb 09.
Artículo en Inglés | MEDLINE | ID: mdl-21743496

RESUMEN

Fibulin-2 (FBLN2) has been identified as a candidate tumor-suppressor gene in nasopharyngeal carcinoma (NPC). Originally identified through a chromosome 3 NotI genomic microarray screen, it shows frequent deletion or methylation in NPC. FBLN2 is located on chromosome 3p25.1 and is associated with tumor development through its important interactions with the extracellular matrix (ECM) proteins. FBLN2 encodes two isoforms. The short isoform (FBLN2S) is expressed abundantly in normal tissues, but is dramatically downregulated in NPC, while the long isoform (FBLN2L) is either not detectable or is expressed only at low levels in both normal and tumor tissues. Reintroduction of this FBLN2S inhibited cell proliferation, migration, invasion and angiogenesis in vitro. Furthermore, in vivo studies in nude mice show its expression is associated with tumor and angiogenesis suppression. FBLN2-associated angiogenesis occurs via concomitant downregulation of vascular endothelial growth factor and matrix metalloproteinase 2. This study provides compelling evidence that FBLN2S has an important tumor-suppressive and anti-angiogenic role in NPC.


Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Neovascularización Patológica/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Secuencia de Bases , Western Blotting , Proteínas de Unión al Calcio/genética , Carcinoma , Línea Celular , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Metilación de ADN , Proteínas de la Matriz Extracelular/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/patología , Invasividad Neoplásica , Neovascularización Patológica/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante Heterólogo , Carga Tumoral , Proteínas Supresoras de Tumor/genética , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo
6.
Br J Cancer ; 104(5): 841-9, 2011 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-21326238

RESUMEN

BACKGROUND: Oesophageal squamous cell carcinoma (SCC) causes the highest number of cancer deaths in some regions of Northern China. Previously, we narrowed down a critical region at 9q33-34, identified to be associated with tumour-suppressive function of deleted in oesophageal cancer 1 (DEC1) in oesophageal SCC. METHODS: We generated DEC1 antibody and constructed tissue microarrays (TMAs) utilising tissue specimens from Henan, a high-risk region for oesophageal SCC, to investigate the importance of DEC1 expression in this cancer. RESULTS: Tissue microarray immunohistochemical staining reveals significant loss of DEC1 from hyperplasia, to tumour, and to lymph node metastasis. In addition, the loss of DEC1 in tumour is age-dependent. Interestingly, there is significant abrogation of DEC1 expression in patients with a family history of oesophageal SCC. Deleted in oesophageal cancer 1 localises to both the cytoplasm and nucleus. The vesicular pattern of DEC1 in the cytoplasm appears to localise at the Golgi and Golgi-endoplasmic reticulum intermediate compartment. CONCLUSION: This is the first TMA study to suggest a clinical association of DEC1 in lymph node metastatic oesophageal SCC, early onset oesophageal SCC and familial oesophageal SCC development. Subcellular localisation of DEC1 and its expression in oesophageal SCC tissues provide important insight for further deciphering the molecular mechanism of DEC1 in oesophageal SCC development.


Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Neoplasias Esofágicas/metabolismo , Salud de la Familia , Metástasis Linfática , Proteínas Supresoras de Tumor/metabolismo , Adulto , Anciano , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Citoplasma/metabolismo , Progresión de la Enfermedad , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Análisis de Matrices Tisulares
7.
Br J Cancer ; 104(2): 353-60, 2011 Jan 18.
Artículo en Inglés | MEDLINE | ID: mdl-21157448

RESUMEN

BACKGROUND: High-risk human papillomavirus (H-HPV) infection is linked to cervical neoplasia but its role in detecting cervical glandular lesions (GLs) is unclear. Carbonic anhydrase IX (CA-IX) is a hypoxic biomarker that is highly expressed in neoplastic cervical GLs. The diagnostic utility of these biomarkers was evaluated by the Gynecologic Oncology Group in Japanese women with a cytological diagnosis of atypical glandular cells. METHODS: Immunostaining was used to detect CA-IX in a conventional Pap smear. Immunoreactivity of CA-IX was interpreted by a panel of pathologists blinded to the histological diagnosis. Polymerase chain reaction was used to detect H-HPV in a liquid-based cytology specimen. RESULTS: Significant cervical lesions (SCLs), defined as cervical intraepithelial neoplasia (CIN2, CIN3), adenocarcinoma in situ or invasive carcinoma, were observed in 37/88 (42%) of women. CA-IX testing alone (n=88) had a sensitivity of 89, 100 or 73% for SCLs, GLs or significant squamous lesions (SLs), respectively, with a false negative rate (FNR) of 14%. Testing for H-HPV (n=84) had a sensitivity of 65, 53 or 80% for SCLs, GLs or SLs, respectively, with a FNR of 22%. The combination of CA-IX and H-HPV testing had a sensitivity of 97, 100 or 93% for SCLs, GLs or SLs, respectively, with a FNR of 5%. Among eight H-HPV-negative GLs, six (75%) had a diagnosis of lobular endocervical glandular hyperplasia (LEGH). CONCLUSION: The combination of CA-IX and HPV testing improved the diagnostic accuracy. The low rate of H-HPV positivity in the GLs was associated with coexisting LEGH independent of H-HPV.


Asunto(s)
Alphapapillomavirus/patogenicidad , Anhidrasas Carbónicas/metabolismo , Displasia del Cuello del Útero/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Alphapapillomavirus/genética , Femenino , Genotipo , Humanos , Japón , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Displasia del Cuello del Útero/enzimología , Displasia del Cuello del Útero/virología
8.
Oncogene ; 26(1): 148-57, 2007 Jan 04.
Artículo en Inglés | MEDLINE | ID: mdl-16799631

RESUMEN

A gene critical to esophageal cancer has been identified. Functional studies using microcell-mediated chromosome transfer of intact and truncated donor chromosomes 3 into an esophageal cancer cell line and nude mouse tumorigenicity assays were used to identify a 1.61 Mb tumor suppressive critical region (CR) mapping to chromosome 3p14.2. This CR is bounded by D3S1600 and D3S1285 microsatellite markers. One candidate tumor suppressor gene, ADAMTS9, maps to this CR. Further studies showed normal expression levels of this gene in tumor-suppressed microcell hybrids, levels that were much higher than observed in the recipient cells. Complete loss or downregulation of ADAMTS9 gene expression was found in 15 out of 16 esophageal carcinoma cell lines. Promoter hypermethylation was detected in the cell lines that do not express this gene. Re-expression of ADAMTS9 was observed after demethylation drug treatment, confirming that hypermethylation is involved in gene downregulation. Downregulation of ADAMTS9 was also found in 43.5 and 47.6% of primary esophageal tumor tissues from Hong Kong and from the high-risk region of Henan, respectively. Thus, this study identifies and provides functional evidence for a CR associated with tumor suppression on 3p14.2 and provides the first evidence that ADAMTS9, mapping to this region, may contribute to esophageal cancer development.


Asunto(s)
Proteínas ADAM/genética , Carcinoma de Células Escamosas/genética , Cromosomas Humanos Par 3 , Neoplasias Esofágicas/genética , Genes Supresores de Tumor , Proteína ADAMTS9 , Secuencia de Bases , Mapeo Cromosómico , ADN , Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular
9.
Cytometry B Clin Cytom ; 70(2): 45-55, 2006 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-16456867

RESUMEN

BACKGROUND: Although tumor hypoxia has been associated with a more aggressive phenotype and lower cure rate, there is no consensus as to the method best suited for routine measurement. Binding of the chemical hypoxia marker, pimonidazole, and expression of the endogenous hypoxia markers HIF-1alpha and CAIX were compared for their ability to detect hypoxia in tumor biopsies from 67 patients with advanced carcinoma of the cervix. METHODS: Two biopsies were taken one day after administration of pimonidazole and were analyzed for pimonidazole binding using flow cytometry or immunohistochemistry. CAIX and HIF-1alpha expression and degree of colocalization were measured in sequential antibody-stained sections. Patient subsets were examined for tumor oxygen tension using an Eppendorf electrode, S phase DNA content, or change in HIF-1alpha expression over the course of treatment. RESULTS: Approximately 6% of the tumor area stained positive for pimonidazole, HIF-1alpha, or CAIX. The CAIX positive fraction correlated with the pimonidazole positive fraction (r = 0.60). Weaker but significant correlations were observed between pimonidazole and HIF-1alpha (r = 0.31) and CAIX and HIF-1alpha (r = 0.41). Taking the extent of marker colocalization into consideration increased the confidence that all markers were identifying hypoxic regions. Over 65% of stained areas showed a high degree of colocalization with the other markers. Oxygen microelectrode measurements and S phase fraction were not correlated with the hypoxic fraction measured using the three hypoxia markers. HIF-1alpha levels tended to decrease with time after the start of therapy. CONCLUSIONS: Endogenous hypoxia marker binding shows reasonable agreement, in extent and location, with binding of pimonidazole. CAIX staining pattern is a better match to the pimonidazole staining pattern than is HIF-1alpha, and high CAIX expression in the absence (or low levels) of HIF-1alpha may indicate a different biology.


Asunto(s)
Antígenos de Neoplasias/metabolismo , Biomarcadores de Tumor/metabolismo , Anhidrasas Carbónicas/metabolismo , Hipoxia de la Célula/fisiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Nitroimidazoles/metabolismo , Oxígeno/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Biopsia , Anhidrasa Carbónica IX , Cuello del Útero/metabolismo , Cuello del Útero/patología , ADN de Neoplasias/análisis , Electrodos , Femenino , Citometría de Flujo , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Fenotipo , Valor Predictivo de las Pruebas , Pronóstico , Fase S , Factores de Tiempo , Neoplasias del Cuello Uterino/patología
10.
J Med Genet ; 42(7): 565-76, 2005 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-15994878

RESUMEN

BACKGROUND: STRA13 is a bHLH transcription factor that plays a crucial role in cell differentiation, proliferation, apoptosis, and response to hypoxia. OBJECTIVE: To assess STRA13 involvement in carcinogenesis and evaluate its diagnostic value. METHODS: A comprehensive analysis was undertaken of the endogenous protein expression in 389 normal and corresponding malignant specimens, using newly generated polyclonal antibodies. RESULTS: STRA13 was commonly expressed in epithelial cells of normal and neoplastic tissues where it was confined mostly to the nucleus. Intense cytoplasmic STRA13 immunoreactivity was characteristic of myoepithelial and differentiated squamous epithelial cells of all organ sites and their neoplastic counterparts, suggesting application of STRA13 as a myoepithelial cell marker. A distinctive apical granular cytoplasmic staining pattern observed in the pancreas and large intestine was retained in corresponding metastatic carcinomas, providing for identification of the primary sites of these disseminating tumours. In less differentiated tumours there was a tendency to lose the cytoplasmic staining or to switch to nuclear STRA13 staining. Analysis of STRA13, HIF-1alpha, and CAIX expression patterns in a large set of various tumours substantiated the association of STRA13 with HIF-1alpha expression and hypoxia in vivo. Investigation of the molecular mechanisms of STRA13 nucleo-cytoplasmic shuttling suggested that STRA13 employs nuclear import/export that utilises the NLS/NES motifs situated within the N-terminus and in the middle of the protein. CONCLUSIONS: STRA13 may serve as a marker for myoepithelial cells, for the degree of tumour differentiation, and for identification of the primary site of certain metastatic tumours. In combination with CAIX and CAXII markers, it may lead to a more accurate classification of all renal carcinomas.


Asunto(s)
Antígenos de Diferenciación/biosíntesis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/biosíntesis , Biomarcadores de Tumor/biosíntesis , Células Epiteliales/metabolismo , Proteínas de Homeodominio/biosíntesis , Neoplasias/diagnóstico , Neoplasias/metabolismo , Especificidad de Anticuerpos , Antígenos de Neoplasias/biosíntesis , Anhidrasa Carbónica IX , Anhidrasas Carbónicas/biosíntesis , Diferenciación Celular , Línea Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Células Epiteliales/patología , Humanos , Inmunohistoquímica , Neoplasias Renales/clasificación , Neoplasias Renales/diagnóstico , Neoplasias Renales/metabolismo , Neoplasias/clasificación
11.
Eur J Gynaecol Oncol ; 26(1): 31-5, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-15754996

RESUMEN

INTRODUCTION: Tumour hypoxia has been found to be associated with tumour aggressiveness. Our primary aim was to explore the relationship between pretreatment tumour oxygenation in primary vulvar carcinoma and nodal status. Our secondary objective was to assess if there was a relationship between the clinical and biological variables. METHODS: 20 women with ISCC of the vulva were assessed with pretreatment primary tumour oxygenation with an Eppendorf pO2 probe. Patients underwent standard surgical management. Pathological assessment of the primary and nodal tissues was then performed. Primary tumour specimens were also stained for microvessel density and carbonic anhydrase IX. The relationship between smoking, preoperative Hgb, tumour CAIX expression, MVD, and Eppendorf pO2 measurements vs nodal metastasis and between these clinical and biological variables was assessed. RESULTS: Seven patients had positive lymph nodes, 13 had negative nodes. While neither current smoking status, tumour size, tumour oxygen measurements, MVD and CAIX expression correlated with metastatic nodal disease, a low preoperative Hgb correlated with pathological nodal status (p < 0.027). CONCLUSIONS: Although this analysis failed to demonstrate a strong correlation between various measures of tumour oxygenation with nodal metastasis, it may be due to the small number of patients. Only preoperative anaemia is correlated with nodal metastasis in early ISCC of the vulva.


Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Oxígeno , Neoplasias de la Vulva/metabolismo , Anciano , Carcinoma de Células Escamosas/secundario , Femenino , Humanos , Ganglios Linfáticos/metabolismo , Metástasis Linfática , Invasividad Neoplásica , Consumo de Oxígeno , Valor Predictivo de las Pruebas , Neoplasias de la Vulva/patología
12.
J Med Genet ; 40(4): 257-61, 2003 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-12676895

RESUMEN

PURPOSE: Carbonic anhydrase enzymes (CAs) are universally involved in many fundamental physiological processes, including acid base regulation and fluid formation and movement. In glaucoma patients, CA inhibitors are very effective in lowering intraocular pressure by reducing the rate of aqueous humour secretion mediated by the CAs in the ciliary epithelium. In this work, we investigated the expression and tissue distribution of two recently discovered CA genes CA9 (CAIX) and CA12 (CAXII) in fetal, neonatal, and adult human eyes with and without glaucoma. METHODS: CAIX and CAXII expression in 16 normal and 10 glaucomatous eyes, and in cultured non-pigmented ciliary epithelial cells (NPE) from normal and glaucoma eye donors was assessed by immunostaining. In addition, northern blot hybridisation was performed to assess expression of CA4, CA9, and CA12 mRNA in cultured NPE cells from normal and glaucoma donors. RESULTS: CAXII was localised primarily to the NPE with its expression prominent during embryonic eye development but which decreased significantly in adults. CAIX expression in the NPE was very low. The epithelium of cornea and lens occasionally expressed both enzymes at low levels during development and in adult eye, and no expression was detected in the retina. The NPE from glaucoma eyes expressed higher levels of CAXII, but not CAIX, in comparison with normal eyes. This expression pattern was retained in cultured NPE cell lines. NPE cells from a glaucoma patient showed a five-fold increase in the CA12 mRNA level with no detectable expression of CA9 mRNA. Also, no expression of the CA4 gene encoding a GPI anchored plasma membrane protein was detected on these northern blots. CONCLUSIONS: Transmembrane CAIX and CAXII enzymes are expressed in the ciliary cells and, thus, may be involved in aqueous humour production. CA12 may be a targeted gene in glaucoma.


Asunto(s)
Anhidrasas Carbónicas/genética , Membrana Celular/enzimología , Glaucoma/genética , Northern Blotting , Anhidrasas Carbónicas/metabolismo , Células Cultivadas , Cuerpo Ciliar/citología , Cuerpo Ciliar/enzimología , Células Epiteliales/enzimología , Regulación Enzimológica de la Expresión Génica , Glaucoma/enzimología , Glaucoma/patología , Humanos , Inmunohistoquímica , Isoenzimas/genética , Isoenzimas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo
13.
Cancer Res ; 61(24): 8924-9, 2001 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-11751418

RESUMEN

The presence of radiation-resistant hypoxic cells in some solid tumors is known to predict for relapse after radiotherapy. Use of an endogenous marker of hypoxia would be a convenient alternative to current methods that measure tumor oxygenation, provided the marker could be shown to reliably identify viable, radiation-resistant, hypoxic cells. Carbonic anhydrase 9 (CA9) is a transmembrane protein overexpressed in a wide variety of tumor types and induced by hypoxia. Using a monoclonal antibody and cell sorting, CA9-positive cells in SiHa cervical carcinoma xenografts growing in immunodeficient mice were found to be clonogenic, resistant to killing by ionizing radiation, and preferentially able to bind the hypoxia marker pimonidazole. CA9 and pimonidazole immunostaining were compared in formalin-fixed sections from tumors of 18 patients undergoing treatment for cancer of the cervix. Excellent colocalization was observed, although the area of the tumor section that bound anti-CA9 antibodies represented double the number of cells that bound anti-pimonidazole antibodies. Occasional regions staining with pimonidazole but not CA9 could be indicative of transient changes in tumor perfusion. Results support the hypothesis that CA9 is a useful endogenous marker of tumor hypoxia.


Asunto(s)
Antígenos de Neoplasias , Biomarcadores de Tumor/metabolismo , Anhidrasas Carbónicas , Proteínas de Neoplasias/metabolismo , Oxígeno/metabolismo , Neoplasias del Cuello Uterino/enzimología , Animales , Anhidrasa Carbónica IX , Hipoxia de la Célula , Femenino , Glioma/enzimología , Glioma/metabolismo , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas de Neoplasias/biosíntesis , Trasplante de Neoplasias , Nitroimidazoles/metabolismo , Nitroimidazoles/farmacología , Tolerancia a Radiación , Fármacos Sensibilizantes a Radiaciones/metabolismo , Fármacos Sensibilizantes a Radiaciones/farmacología , Trasplante Heterólogo , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/radioterapia
14.
Cancer Res ; 61(19): 7118-21, 2001 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-11585743

RESUMEN

High-risk human papillomavirus (HPV) types 16 and 18 are involved in the multistep process of cervical cancer. Transfection of normal keratinocytes with high-risk HPV-DNA generally gives rise to immortal cultures. This may be explained by the loss of senescence genes as a consequence of HPV-induced genetic instability. On the basis of the dominance of cellular senescence over immortality, fusion of normal keratinocytes with HPV-immortalized cells results in complementation of these putative gene defects. In a previous study, we showed that underrepresentation of chromosome 10 is a characteristic phenomenon during the early phase of immortalization. Here we show that introduction of a normal copy of chromosome 10 into HPV16-immortalized cells (HPKII) by Microcell-mediated chromosome transfer resulted in senescence of a significant number of hybrids. By using several derivatives of chromosome 10 for further fusion experiments, the chromosomal region responsible for senescence could be assigned to 10p14-p15. The potential significance of loss of gene function in this region is underlined by the high frequency (38.7%) of loss of heterozygosity in cervical cancers including early stage tumors.


Asunto(s)
Senescencia Celular/genética , Cromosomas Humanos Par 10/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/virología , Línea Celular Transformada , Transformación Celular Viral , Mapeo Cromosómico , ADN Viral/genética , Femenino , Eliminación de Gen , Técnicas de Transferencia de Gen , Humanos , Queratinocitos/citología , Queratinocitos/fisiología , Queratinocitos/virología , Pérdida de Heterocigocidad , Papillomaviridae/genética , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/virología , Displasia del Cuello del Útero/genética , Displasia del Cuello del Útero/patología , Displasia del Cuello del Útero/virología
15.
Biochem J ; 359(Pt 3): 669-77, 2001 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-11672442

RESUMEN

MN/CA IX (MN) is a tumour-associated isoenzyme of the carbonic anhydrase family. Previous deletion analysis of the MN promoter established that protected regions (PRs) 1 and 2 are crucial for its transcriptional activity. Computer-assisted searching indicated putative binding sites for activator protein (AP) 2 and specificity protein (SP) 1 transcription factors, plus a CACCC box in PR1 and an AP1 site in PR2. PR1 produced four complexes in electrophoretic mobility-shift assay (EMSA) with HeLa nuclear extracts. Of these, three were completely competed with the SP1 and transforming growth factor-beta retinoblastoma control-element CACCC box (RCE) probes, whereas the AP2 probe competed against the same three complexes partially. Supershift EMSA identified SP1 in the complex 1 and SP3 in the complexes 2 and 4. Point mutations in the SP1 site abrogated the PR1 function, while mutations affecting the overlapping CACCC box/AP2 site in PR1 had minor effect on MN promoter activity. Block-replaced MN promoter mutants that had a consensus binding site (SP1 or AP2) or the RCE in place of PR1 demonstrated the stringent selectivity of the PR1 position as only the SP1 mutant reconstituted the MN promoter activity. The consensus SP1 probe generated the same SP1 and SP3 complexes as PR1 in EMSA; therefore we conclude that SP activity is both necessary and sufficient in the PR1 position. The critical role of AP1 in the PR2 position was confirmed by supershift of the PR2 complex with c-Fos antibody and markedly decreased activity of the construct with a mutated AP1 site. Detailed deletion analysis proved that PR1+PR2 account for 90% of the MN promoter activity, while neither PR1 nor PR2 on their own are sufficient for transactivation. Thus, synergistic co-operation between SP and AP1 factors bound to the adjacent PR1 and PR2, respectively, is necessary for MN transcriptional activity. The PR1+PR2 module also stimulated transcription from a heterologous promoter. The modulation of AP1 activity with PMA stimulated MN expression and activated the MN promoter, whereas inhibition of protein kinase C activity had no effect on MN expression in HeLa cells.


Asunto(s)
Antígenos de Neoplasias , Anhidrasas Carbónicas , Proteínas de Unión al ADN/metabolismo , Proteínas de Neoplasias/genética , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/metabolismo , Biomarcadores de Tumor , Western Blotting , Anhidrasa Carbónica IX , Proteínas de Unión al ADN/genética , Ensayo de Cambio de Movilidad Electroforética , Elementos de Facilitación Genéticos , Células HeLa , Humanos , Proteínas de Neoplasias/metabolismo , Mutación Puntual , Unión Proteica , Proteína Quinasa C/metabolismo , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp3 , Acetato de Tetradecanoilforbol/farmacología , Factor de Transcripción AP-1/genética , Factores de Transcripción/genética
16.
Mol Cell Biol ; 21(17): 5846-56, 2001 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-11486024

RESUMEN

We have developed a model system of human fibrosarcoma cell lines that do or do not possess and express an oncogenic mutant allele of N-ras. HT1080 cells contain an endogenous mutant allele of N-ras, whereas the derivative MCH603 cell line contains only wild-type N-ras. In an earlier study (S. Gupta et al., Mol. Cell. Biol. 20:9294-9306, 2000), we had shown that HT1080 cells produce rapidly growing, aggressive tumors in athymic nude mice, whereas MCH603 cells produced more slowly growing tumors and was termed weakly tumorigenic. An extensive analysis of the Ras signaling pathways (Raf, Rac1, and RhoA) provided evidence for a potential novel pathway that was critical for the aggressive tumorigenic phenotype and could be activated by elevated levels of constitutively active MEK. In this study we examined the role of phosphoinositide 3-kinase (PI 3-kinase) in the regulation of the transformed and aggressive tumorigenic phenotypes expressed in HT1080 cells. Both HT1080 (mutant N-ras) and MCH603 (wild-type N-ras) have similar levels of constitutively active Akt, a downstream target of activated PI 3-kinase. We find that both cell lines constitutively express platelet-derived growth factor (PDGF) and PDGF receptors. Transfection with tumor suppressor PTEN cDNA into HT1080 and constitutively active PI 3-kinase-CAAX cDNA into MCH603 cells, respectively, resulted in several interesting and novel observations. Activation of the PI 3-kinase/Akt pathway, including NF-kappaB, is not required for the aggressive tumorigenic phenotype in HT1080 cells. Activation of NF-kappaB is complex: in MCH603 cells it is mediated by Akt, whereas in HT1080 cells activation also involves other pathway(s) that are activated by mutant Ras. A threshold level of activation of PI 3-kinase is required in MCH603 cells before stimulatory cross talk to the RhoA, Rac1, and Raf pathways occurs, without a corresponding activation of Ras. The increased levels of activation seen were similar to those observed in HT1080 cells, except for Raf and MEK, which were more active than HT1080 levels. This cross talk results in conversion to the aggressive tumorigenic phenotype. This latter observation is consistent with our previous observation that overstimulation of the activity of endogenous members of Ras signaling pathways, activated MEK in particular, is a prerequisite for aggressive tumorigenic growth.


Asunto(s)
Fibrosarcoma/fisiopatología , Proteínas I-kappa B , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Serina-Treonina Quinasas , Actinas/metabolismo , Animales , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Ratones , Ratones Desnudos , Inhibidor NF-kappaB alfa , FN-kappa B/metabolismo , Fosforilación , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-sis/metabolismo , Células Tumorales Cultivadas , Proteína Letal Asociada a bcl
17.
Oncogene ; 20(30): 3929-36, 2001 Jul 05.
Artículo en Inglés | MEDLINE | ID: mdl-11494121

RESUMEN

Although apoptosis plays an essential role in the embryogenesis and homeostasis of multicellular organisms, this mechanism has not yet been fully clarified. We isolated a novel human apoptosis-inducing gene, ASY, which encodes an endoplasmic reticulum-targeting protein without any known apoptosis-related motifs. This gene is identical to the Nogo-B, a splice variant of the Nogo-A which has recently been shown to be an inhibitor of neuronal regeneration in the central nervous system. Ectopic expression of the ASY gene led to extensive apoptosis, particularly in cancer cells. Furthermore, transcription of the ASY gene was suppressed in small cell lung cancer. These results suggest that a new type of apoptosis-inducing gene, namely, ASY, may be involved in the development of certain types of cancer.


Asunto(s)
Apoptosis/genética , Genes Reguladores , Proteínas de la Mielina/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Carcinoma de Células Pequeñas/genética , Carcinoma de Células Pequeñas/metabolismo , ADN Complementario/genética , Retículo Endoplásmico/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosarcoma/metabolismo , Fibrosarcoma/patología , Células HeLa/metabolismo , Células HeLa/patología , Humanos , Células Híbridas/metabolismo , Células Híbridas/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Melanoma/metabolismo , Melanoma/patología , Datos de Secuencia Molecular , Proteínas de la Mielina/biosíntesis , Proteínas de Neoplasias/biosíntesis , Proteínas Nogo , Osteosarcoma/metabolismo , Osteosarcoma/patología , Empalme del ARN , ARN Mensajero/genética , ARN Neoplásico/genética , Transfección , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/patología
18.
J Natl Cancer Inst ; 93(15): 1159-65, 2001 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-11481388

RESUMEN

BACKGROUND: Antibodies isolated from cancer patients have been used to identify genes encoding tumor-associated antigen epitopes relevant to immune responses in cancer patients. In this report, we used an immunoglobulin G (IgG) purified from serum of a patient with breast cancer to identify its corresponding epitope, gene, and protein-retinoblastoma-binding protein-1-like protein-1 (RBP1L1)-and determined whether it is a potential molecular marker for various cancers. METHODS: IgG purified from the serum of a patient with breast cancer was used to screen an MCF-7 breast cancer cell complementary DNA (cDNA) expression library for immunoreactive clones. The cDNAs identified were cloned and sequenced. Immunoreactivity of specific amino acids in the epitope was determined by western blot analysis and enzyme-linked immunosorbent assay. The cellular location of the antigen was determined by immunoperoxidase staining with purified RBP1L1-specific IgG. Gene expression in various human carcinomas and normal tissues was examined by northern blot analysis and the reverse transcription-polymerase chain reaction. RESULTS: Our purified IgG recognized just one epitope on RBP1L1. The complete 5802-base-pair RBP1L1 cDNA encodes a 1226-amino acid protein containing the antigenic epitope IKPSLGSKK. The derived protein sequence of RBP1L1 shares 74% and 37% amino acid identity, respectively, with a partial sequence of the retinoblastoma-binding protein and the complete sequence of retinoblastoma-binding protein-1. The RBP1L1 epitope was localized to the cytoplasm of MCF-7 cells but was not detected in peripheral blood mononuclear cells. High expression of RBP1L1 messenger RNA was found in human breast, lung, colon, pancreatic, and ovarian cancers and in normal testis, but expression was limited in other normal tissues. CONCLUSIONS: RBP1L1 appears to be a molecular marker associated with a broad range of human malignancies.


Asunto(s)
Biomarcadores de Tumor/análisis , Neoplasias de la Mama/inmunología , Proteínas de Unión al ADN/análisis , Epítopos/genética , Inmunoglobulina G/aislamiento & purificación , Neoplasias/química , Testículo/química , Northern Blotting , Western Blotting , ADN Complementario/análisis , Ensayo de Inmunoadsorción Enzimática , Femenino , Regulación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glutatión Transferasa/metabolismo , Humanos , Técnicas para Inmunoenzimas , Inmunoglobulina G/inmunología , Masculino , Neoplasias/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Terminología como Asunto , Regulación hacia Arriba
19.
Cancer Res ; 61(11): 4536-40, 2001 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-11389086

RESUMEN

To understand genetic differences and similarities between tumorigenic and nontumorigenic HeLa x fibroblast hybrid cells, subtractive suppression hybridization (SSH), based on suppression PCR and a combination of normalization and subtraction in a single procedure, was used. Using the nontumorigenic CGL1 and tumorigenic CGL3, forward (CGL1-CGL3) and reverse (CGL3-CGL1) subtracted libraries were constructed. Among 192 clones, seven were identified as differentially expressed genes specific for either CGL1 or CGL3. All seven were not reported previously as differentially expressed genes in this hybrid system. In the forward subtraction, p16 was isolated, indicating the involvement of the loss of tumorigenic phenotype. Subsequent transfection of wild-type p16 to the tumorigenic CGL3 showed growth suppression in colony formation assay; however, no tumor suppression was observed when the transfectant was inoculated into nude mice. These results indicate that: (a) SSH is a suitable method to identify differentially expressed genes in two types of cells; and (b) although p16 plays some roles in growth suppression, the p16-transfected CGL3 is still capable to proliferate in vivo.


Asunto(s)
Fibroblastos/fisiología , Regulación Neoplásica de la Expresión Génica , Genes p16/fisiología , Células HeLa/fisiología , Animales , Femenino , Fibroblastos/patología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Células HeLa/patología , Humanos , Células Híbridas/patología , Células Híbridas/fisiología , Ratones , Ratones Desnudos , Hibridación de Ácido Nucleico/métodos , Reacción en Cadena de la Polimerasa , Transfección
20.
Proc Natl Acad Sci U S A ; 98(13): 7504-9, 2001 Jun 19.
Artículo en Inglés | MEDLINE | ID: mdl-11390984

RESUMEN

Clear cell-type renal cell carcinomas (clear RCC) are characterized almost universally by loss of heterozygosity on chromosome 3p, which usually involves any combination of three regions: 3p25-p26 (harboring the VHL gene), 3p12-p14.2 (containing the FHIT gene), and 3p21-p22, implying inactivation of the resident tumor-suppressor genes (TSGs). For the 3p21-p22 region, the affected TSGs remain, at present, unknown. Recently, the RAS association family 1 gene (isoform RASSF1A), located at 3p21.3, has been identified as a candidate lung and breast TSG. In this report, we demonstrate aberrant silencing by hypermethylation of RASSF1A in both VHL-caused clear RCC tumors and clear RCC without VHL inactivation. We found hypermethylation of RASSF1A's GC-rich putative promoter region in most of analyzed samples, including 39 of 43 primary tumors (91%). The promoter was methylated partially or completely in all 18 RCC cell lines analyzed. Methylation of the GC-rich putative RASSF1A promoter region and loss of transcription of the corresponding mRNA were related causally. RASSF1A expression was reactivated after treatment with 5-aza-2'-deoxycytidine. Forced expression of RASSF1A transcripts in KRC/Y, a renal carcinoma cell line containing a normal and expressed VHL gene, suppressed growth on plastic dishes and anchorage-independent colony formation in soft agar. Mutant RASSF1A had reduced growth suppression activity significantly. These data suggest that RASSF1A is the candidate renal TSG gene for the 3p21.3 region.


Asunto(s)
Carcinoma de Células Renales/genética , Cromosomas Humanos Par 3 , Genes Supresores de Tumor , Neoplasias Renales/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Supresoras de Tumor , Azacitidina/farmacología , Adhesión Celular , División Celular/efectos de los fármacos , Mapeo Cromosómico , Metilación de ADN , Cartilla de ADN , ADN de Neoplasias/química , ADN de Neoplasias/metabolismo , Doxiciclina/toxicidad , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Regiones Promotoras Genéticas , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas
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