RESUMEN

BACKGROUND: We have previously shown that transducin-like enhancer of split 3 (TLE3) is associated with outcome specifically in patients with taxane-treated breast cancer and not in patients treated with anthracycline-based regimens without a taxane. The purpose of this study was to assess the association between TLE3 expression and recurrence in patients with ovarian carcinoma treated with a taxane containing regimen as opposed to those treated with a platinum-based agent alone. METHODS: We carried out immunohistochemical staining of TLE3 in two series of ovarian cancer specimens from the University of Alabama at Birmingham, Birmingham, AL and the Royal Hospital for Women, Sydney, Australia. Local and distant recurrences within the first five years of follow-up were analyzed using Kaplan-Meier, Cox proportional hazard, and multivariate analysis to assess an association between TLE3 expression and response to therapy. RESULTS: TLE3 was expressed in approximately 30% of tumors and expression was associated with a favorable outcome only in patients who had received taxane as part of their treatment regimen (n = 173, HR = 0.62, P = 0.012; P(interaction) = 0.024). Further analysis revealed that the predictive association between TLE3 expression and outcome was strongest in patients with nonserous histology. CONCLUSION: High TLE3 expression predicts a favorable response to taxane containing chemotherapy regimens in ovarian carcinoma. IMPACT: Our findings warrant an independent evaluation of TLE3 as a potential therapeutic response marker for taxane-based chemotherapy in ovarian cancer.

Asunto(s)

Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Hidrocarburos Aromáticos con Puentes/uso terapéutico , Proteínas Co-Represoras/biosíntesis , Neoplasias Glandulares y Epiteliales/tratamiento farmacológico , Neoplasias Glandulares y Epiteliales/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Taxoides/uso terapéutico , Biomarcadores de Tumor/biosíntesis , Hidrocarburos Aromáticos con Puentes/administración & dosificación , Carcinoma Epitelial de Ovario , Estudios de Cohortes , Femenino , Humanos , Estimación de Kaplan-Meier , Análisis Multivariante , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/patología , Modelos de Riesgos Proporcionales , Taxoides/administración & dosificación , Resultado del Tratamiento

RESUMEN

To identify epigenetic-based biomarkers for diagnosis of ovarian cancer we performed MeDIP-Chip in A2780 and CaOV3 ovarian cancer cell lines. Validation by Sequenom massARRAY methylation analysis confirmed a panel of six gene promoters (ARMCX1, ICAM4, LOC134466, PEG3, PYCARD & SGNE1) where hypermethylation discriminated 27 serous ovarian cancer clinical samples versus 12 normal ovarian surface epithelial cells (OSE) (ROC of 0.98). Notably, CpG sites across the transcription start site of a potential long-intergenic non-coding RNA (lincRNA) gene (LOC134466), was shown to be hypermethylated in 81% of serous EOC and could differentiate tumours from OSE (p<0.05). We propose that this potential biomarker panel holds great promise as a diagnostic test for high-grade (Type II) serous ovarian cancer.

Asunto(s)

Biomarcadores/análisis , Metilación de ADN , Epigenómica , Perfilación de la Expresión Génica , Genoma Humano , Neoplasias Ováricas/genética , Regiones Promotoras Genéticas/genética , Islas de CpG/genética , Cistadenocarcinoma Seroso/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Ovario/metabolismo , Ovario/patología , Células Tumorales Cultivadas

RESUMEN

OBJECTIVE: Altered DNA methylation patterns hold promise as cancer biomarkers. In this study we selected a panel of genes which are commonly methylated in a variety of cancers to evaluate their potential application as biomarkers for prognosis and diagnosis in high grade serous ovarian carcinoma (HGSOC); the most common and lethal subtype of ovarian cancer. METHODS: The methylation patterns of 10 genes (BRCA1, EN1, DLEC1, HOXA9, RASSF1A, GATA4, GATA5, HSULF1, CDH1, SFN) were examined and compared in a cohort of 80 primary HGSOC and 12 benign ovarian surface epithelium (OSE) samples using methylation-specific headloop suppression PCR. RESULTS: The genes were variably methylated in primary HGSOC, with HOXA9 methylation observed in 95% of cases. Most genes were rarely methylated in benign OSE, with the exception of SFN which was methylated in all HGSOC and benign OSE samples examined. Methylation of DLEC1 was associated with disease recurrence, independent of tumor stage and suboptimal surgical debulking (HR 3.5 (95% CI:1.10-11.07), p=0.033). A combination of the methylation status of HOXA9 and EN1 could discriminate HGSOC from benign OSE with a sensitivity of 98.8% and a specificity of 91.7%, which increased to 100% sensitivity with no loss of specificity when pre-operative CA125 levels were also incorporated. CONCLUSIONS: This study provides further evidence to support the feasibility of detecting altered DNA methylation patterns as a potential diagnostic and prognostic approach for HGSOC.

Asunto(s)

Cistadenocarcinoma Seroso/genética , Metilación de ADN , Neoplasias Ováricas/genética , Estudios de Cohortes , Cistadenocarcinoma Seroso/patología , Femenino , Proteínas de Homeodominio/genética , Humanos , Persona de Mediana Edad , Clasificación del Tumor , Neoplasias Ováricas/patología , Reacción en Cadena de la Polimerasa/métodos , Tasa de Supervivencia , Proteínas Supresoras de Tumor/genética

RESUMEN

The purpose of this study was to investigate the in vitro effect of anti-MUC1 monoclonal antibody (MAb) C595 alone and in combination with docetaxel, on the growth and survival of different epithelial ovarian cancer (EOC) cell lines. MUC1 expression was assessed on EOC cell lines (OVCAR-3, IGROV-1, A2780, CAOV-3, TOV-21G, TOV-112D, SKOV-3 and OV-90) using immunofluorescence labeling and flow cytometry. The effect of MAb C595 alone or in combination with docetaxel on the cell lines was studied by proliferation, colony and TUNEL assays. Our results indicate that all primary and metastatic EOC cell lines tested were positive to MAb C595 (MUC1); MAb C595 inhibited EOC cell proliferation in a MUC1- and dose-dependent manner; low-dose MAb C595 (1/2 of IC50) combined with docetaxel greatly improved efficiency of cell killing in EOC cells and induced apoptosis; the additive effect of MAb C595 was further confirmed in colony forming assays; and cell death following single or combined treatments was associated with the release of cytochrome c and increased caspase-3 activity. These results suggest that MAb C595 used either alone, or combined with docetaxel, is an attractive strategy for targeting human EOC.

Asunto(s)

Anticuerpos Monoclonales/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Mucina-1/metabolismo , Neoplasias Glandulares y Epiteliales , Neoplasias Ováricas , Taxoides/uso terapéutico , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Separación Celular , Docetaxel , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Etiquetado Corte-Fin in Situ , Microscopía Confocal , Mucina-1/efectos de los fármacos , Neoplasias Glandulares y Epiteliales/metabolismo , Neoplasias Ováricas/metabolismo

RESUMEN

BACKGROUND: The four-transmembrane MAL2 protein is frequently overexpressed in breast carcinoma, and MAL2 overexpression is associated with gain of the corresponding locus at chromosome 8q24.12. Independent expression microarray studies predict MAL2 overexpression in ovarian carcinoma, but these had remained unconfirmed. MAL2 binds tumor protein D52 (TPD52), which is frequently overexpressed in ovarian carcinoma, but the clinical significance of MAL2 and TPD52 overexpression was unknown. METHODS: Immunohistochemical analyses of MAL2 and TPD52 expression were performed using tissue microarray sections including benign, borderline and malignant epithelial ovarian tumours. Inmmunohistochemical staining intensity and distribution was assessed both visually and digitally. RESULTS: MAL2 and TPD52 were significantly overexpressed in high-grade serous carcinomas compared with serous borderline tumours. MAL2 expression was highest in serous carcinomas relative to other histological subtypes, whereas TPD52 expression was highest in clear cell carcinomas. MAL2 expression was not related to patient survival, however high-level TPD52 staining was significantly associated with improved overall survival in patients with stage III serous ovarian carcinoma (log-rank test, p < 0.001; n = 124) and was an independent predictor of survival in the overall carcinoma cohort (hazard ratio (HR), 0.498; 95% confidence interval (CI), 0.34-0.728; p < 0.001; n = 221), and in serous carcinomas (HR, 0.440; 95% CI, 0.294-0.658; p < 0.001; n = 182). CONCLUSIONS: MAL2 is frequently overexpressed in ovarian carcinoma, and TPD52 overexpression is a favourable independent prognostic marker of potential value in the management of ovarian carcinoma patients.

Asunto(s)

Adenocarcinoma de Células Claras/metabolismo , Adenocarcinoma Mucinoso/metabolismo , Cistadenocarcinoma Seroso/metabolismo , Neoplasias Endometriales/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Ováricas/metabolismo , Proteolípidos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Adenocarcinoma de Células Claras/patología , Adenocarcinoma Mucinoso/patología , Biomarcadores de Tumor/metabolismo , Estudios de Cohortes , Cistadenocarcinoma Seroso/patología , Neoplasias Endometriales/patología , Femenino , Humanos , Técnicas para Inmunoenzimas , Persona de Mediana Edad , Proteínas Proteolipídicas Asociadas a Mielina y Linfocito , Estadificación de Neoplasias , Neoplasia Residual/metabolismo , Neoplasia Residual/patología , Neoplasias Ováricas/patología , Pronóstico , Tasa de Supervivencia , Análisis de Matrices Tisulares

RESUMEN

Dual specificity phosphatases are characterised by their ability to dephosphorylate both phosphotyrosine and phosphoserine/threonine residues within the one substrate. The aim of this study was to characterise the phosphatase activity of the atypical dual specificity phosphatase, DUSP26 on MAP kinases, and to determine its expression, regulation and function in cancer cells. Overexpression and knockdown of DUSP26 in epithelial cells and in vitro phosphatase assays were used to demonstrate that, contrary to several published reports, DUSP26 does not act as a dual specificity phosphatase on ERK, JNK or p38 MAPKs. However, overexpression of DUSP26 in MCF10A epithelial cells suppressed colony formation and acinar growth in 3D culture, effects dependent on its phosphatase activity, while knockdown of DUSP26 in HOSE17.1 cells enhanced colony formation and cellular proliferation. DUSP26 mRNA expression was reduced in neuroblastoma, brain and ovarian cancer cell lines. Consistent with epigenetic silencing of DUSP26, expression was enhanced by treatment of cells with 5-aza-2-deoxycitidine and trichostatin A, and a CpG island upstream of the DUSP26 transcriptional start site was variably methylated in cancer cell lines. Together, these results help to clarify confusion in the literature relating to DUSP26 substrate specificity and support recent reports that substrates other than MAPKs are the primary substrates of this phosphatase. In addition, they indicate that DUSP26 may function as a tumour suppressor in particular cancers.

Asunto(s)

Proliferación Celular , Fosfatasas de Especificidad Dual/fisiología , Células Epiteliales/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/fisiología , Animales , Células CHO , Células COS , Células Cultivadas , Chlorocebus aethiops , Islas de CpG/genética , Cricetinae , Cricetulus , Fosfatasas de Especificidad Dual/antagonistas & inhibidores , Fosfatasas de Especificidad Dual/genética , Fosfatasas de Especificidad Dual/metabolismo , Células Epiteliales/metabolismo , Técnicas de Silenciamiento del Gen , Genes Supresores de Tumor/fisiología , Humanos , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/antagonistas & inhibidores , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/genética , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/metabolismo , Fosforilación/fisiología

RESUMEN

BACKGROUND: The urokinase plasminogen activator (uPA) system has been implicated in progression and poor prognosis in epithelial ovarian cancer (EOC) patients. The present study investigated the distribution of uPA and its receptor (uPAR) in EOC cell lines, primary and metastatic tumors, and the relationship between uPA/uPAR and matrix metalloproteinase (MMP) expression using immunohistochemistry. We also studied the association between uPA/uPAR expression and clinical and pathological parameters including disease progression free survival (PFS). METHODS: The expression of uPA/uPAR was examined on paraffin-embedded tissue sections from primary EOC (n=100), and matched metastatic lesions (n=30) of untreated patients, normal ovarian tissues (n=20) as well as 8 primary and metastatic EOC cell lines by immunohistochemistry. Co-immunolabeling of uPA and MMP-1, -2, -9 or MT1-MMP was examined using confocal microscopy. RESULTS: The expression of uPA/uPAR was found in most primary (92% and 88% positive, respectively), metastatic ovarian tumors (93% and 90% positive, respectively), and all of examined EOC cell lines. The majority of specimens showed moderate to strong immunostaining of tumor and stromal cells; for primary specimens, this was significantly associated with tumor stage, grade and time to relapse (P<0.01). Overexpression of uPA/uPAR was found to be associated with an unfavorable prognosis with significantly reduced median disease PFS of 16 vs. 33 months for uPA (P<0.001), and 15 vs. 28 months for uPAR (P<0.001). Co-localization of uPA with MMP-1, -2, -9 or MT1-MMP was also seen in primary tumors and metastatic lesions. CONCLUSIONS: The expression of uPA/uPAR was associated with EOC progression. uPA/uPAR are useful markers for EOC prognosis and could be promising therapeutic targets for treating incurable, recurrent EOC.

Asunto(s)

Neoplasias Ováricas/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa/biosíntesis , Activador de Plasminógeno de Tipo Uroquinasa/biosíntesis , Adulto , Anciano , Línea Celular Tumoral , Supervivencia sin Enfermedad , Femenino , Humanos , Inmunohistoquímica , Isoenzimas , Metaloproteinasa 1 de la Matriz/biosíntesis , Metaloproteinasa 14 de la Matriz/biosíntesis , Persona de Mediana Edad , Metástasis de la Neoplasia , Neoplasias Ováricas/enzimología , Neoplasias Ováricas/patología , Adhesión en Parafina

RESUMEN

DUSPs (dual-specificity phosphatases) are a heterogeneous group of protein phosphatases that can dephosphorylate both phosphotyrosine and phosphoserine/phosphothreonine residues within the one substrate. DUSPs have been implicated as major modulators of critical signalling pathways that are dysregulated in various diseases. DUSPs can be divided into six subgroups on the basis of sequence similarity that include slingshots, PRLs (phosphatases of regenerating liver), Cdc14 phosphatases (Cdc is cell division cycle), PTENs (phosphatase and tensin homologues deleted on chromosome 10), myotubularins, MKPs (mitogen-activated protein kinase phosphatases) and atypical DUSPs. Of these subgroups, a great deal of research has focused on the characterization of the MKPs. As their name suggests, MKPs dephosphorylate MAPK (mitogen-activated protein kinase) proteins ERK (extracellular-signal-regulated kinase), JNK (c-Jun N-terminal kinase) and p38 with specificity distinct from that of individual MKP proteins. Atypical DUSPs are mostly of low-molecular-mass and lack the N-terminal CH2 (Cdc25 homology 2) domain common to MKPs. The discovery of most atypical DUSPs has occurred in the last 6 years, which has initiated a large amount of interest in their role and regulation. In the past, atypical DUSPs have generally been grouped together with the MKPs and characterized for their role in MAPK signalling cascades. Indeed, some have been shown to dephosphorylate MAPKs. The current literature hints at the potential of the atypical DUSPs as important signalling regulators, but is crowded with conflicting reports. The present review provides an overview of the DUSP family before focusing on atypical DUSPs, emerging as a group of proteins with vastly diverse substrate specificity and function.

Asunto(s)

Fosfatasas de Especificidad Dual/fisiología , Animales , Proteínas de Ciclo Celular/metabolismo , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/metabolismo , Fosfatasa 1 de Especificidad Dual/metabolismo , Fosfatasa 3 de Especificidad Dual/metabolismo , Fosfatasas de Especificidad Dual/clasificación , Fosfatasas de Especificidad Dual/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/metabolismo , Fosfohidrolasa PTEN/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo

Asunto(s)

Biomarcadores de Tumor/genética , Epigénesis Genética , Neoplasias Ováricas/genética , Femenino , Humanos

RESUMEN

OBJECTIVE: To review epigenetic changes identified in ovarian cancer, focusing on their potential as clinical markers for detection, monitoring of disease progression and as markers of therapeutic response. METHODS: A comprehensive review of English language scientific literature on the topics of methylation and ovarian cancer was conducted. RESULTS: Genome-wide demethylation of normally methylated and silenced chromosomal regions, and hypermethylation and silencing of genes including tumor suppressors are common features of cancer cells. Epigenetic alterations, including CpG island DNA methylation, occur in ovarian cancer and the identification of specific genes that are altered by epigenetic events is an area of intense research. Aberrant DNA methylation in ovarian cancer is observed in early cancer development, can be detected in DNA circulating in the blood and hence provides the promise of a non-invasive cancer detection test. In addition, identification of ovarian cancer-specific epigenetic changes has promise in molecular classification and disease stratification. CONCLUSIONS: The detection of cancer-specific DNA methylation changes heralds an exciting new era in cancer diagnosis as well as evaluation of prognosis and therapeutic responsiveness and warrants further investigation.

Asunto(s)

Metilación de ADN , Neoplasias Ováricas/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Ováricas/diagnóstico , Neoplasias Ováricas/terapia , Pronóstico

RESUMEN

BACKGROUND: Currently, no specific immunohistochemical markers are available to differentiate primary mucinous epithelial ovarian cancer (MOC) from adenocarcinomas originating at other sites that have metastasised to the ovary, which may have an impact on patient management and prognosis. AIM: To investigate the expression of two intestinal markers, galectin 4 and meprin alpha, in mucinous carcinomas of the ovary and gastrointestinal tract. METHODS: Using immunohistochemical analysis, the expression of galectin 4 and meprin alpha was investigated in 10 MOCs and in 38 mucinous adenocarcinomas of colon, pancreas, stomach and appendix, the most common sites of origin of ovarian metastases. RESULTS: Total cytoplasmic galectin 4 expression was relatively consistent between the different carcinomas. Membranous meprin alpha expression was significantly lower in MOCs compared with gastrointestinal carcinomas. Moreover, meprin alpha expression showed greater discrimination between the ovarian and gastrointestinal carcinomas than the cytokeratins CK7 and CK20, the current standard immunohistochemical markers used to determine the tissue origin of mucinous carcinomas involving the ovaries. CONCLUSIONS: Meprin alpha is a useful additional marker in differentiating primary from secondary mucinous adenocarcinomas of the ovary.

Asunto(s)

Adenocarcinoma Mucinoso/diagnóstico , Biomarcadores de Tumor/metabolismo , Neoplasias Gastrointestinales/patología , Metaloendopeptidasas/metabolismo , Neoplasias Ováricas/diagnóstico , Adenocarcinoma/diagnóstico , Adenocarcinoma/metabolismo , Adenocarcinoma/secundario , Adenocarcinoma Mucinoso/metabolismo , Diagnóstico Diferencial , Femenino , Galectina 4/metabolismo , Humanos , Técnicas para Inmunoenzimas , Queratina-20/metabolismo , Queratina-7/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/secundario

RESUMEN

The isolation of fully human monoclonal antibodies (MAb) against tumor targets has to date relied largely on combinatorial library-based antibody display techniques, which generally require lengthy antigen selection procedures due to a low frequency of clones expressing compatible heavy (VH) and light chain (VL) variable genes. Here we describe a method to directly isolate immunoglobulin sequences in situ from antibody-producing cells infiltrating human tumor tissue. Single B cells and plasma cells infiltrating cervical cancer were microdissected from tissue sections using laser-assisted microscopy, and VH and VL expressed by each individual cell amplified using nested reverse transcriptase- polymerase chain reaction (RT-PCR), thus retaining the native VH and VL pairing. Sequencing analysis determined that the isolated cells expressed functional immunoglobulin variable genes, consistent with an antitumor antibody response. The immunoglobulin sequences can be reassembled as Fab or scFv fragments using conventional recombinant antibody expression plasmids. This method will allow a more direct assessment of the humoral immune response to cancer, and the potential identification of novel human therapeutic cancer antibodies.

Asunto(s)

Anticuerpos Monoclonales/metabolismo , Fragmentos de Inmunoglobulinas/metabolismo , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/patología , Microdisección/métodos , Análisis de Secuencia de Proteína/métodos , Neoplasias del Cuello Uterino/inmunología , Neoplasias del Cuello Uterino/patología , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/aislamiento & purificación , Linfocitos B/inmunología , Linfocitos B/patología , Separación Celular/métodos , Células Cultivadas , Femenino , Humanos , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/inmunología , Fragmentos de Inmunoglobulinas/aislamiento & purificación , Rayos Láser , Reacción en Cadena de la Polimerasa/métodos , Ingeniería de Proteínas/métodos , Células Tumorales Cultivadas , Neoplasias del Cuello Uterino/genética

RESUMEN

Papillomaviruses have evolved mechanisms that result in escape from host immune surveillance. The E5 protein is expressed early in papillomavirus infection in the deep layers of the infected epithelium. It is localized to the Golgi apparatus (GA) and endoplasmic reticulum. The E5 protein of bovine papillomavirus (BPV) impairs the synthesis and stability of major histocompatibility (MHC) class I complexes and prevents their transport to the cell surface due to retention in the GA. Here we show that human papillomavirus type 16 (HPV-16) E5 also causes the retention of MHC (HLA) class I complexes in the GA and impedes their transport to the cell surface, which is rescued by treatment with interferon. Unlike BPV E5, HPV-16 E5 does not affect the synthesis of HLA class I heavy chains or the expression of the transporter associated with antigen processing TAP. These results show that downregulation of surface MHC class I molecules is common to both BPV and HPV E5 proteins. Moreover, we determined that HPV-16 E5 downregulates surface expression of HLA-A and HLA-B, which present viral peptides to MHC class I-restricted cytotoxic T lymphocytes (CTLs), but not the natural killer (NK) cell inhibitory ligands HLA-C and HLA-E. Selective downregulation of cell surface HLA class I molecules may allow the virus to establish infection by avoiding immune clearance of virus-infected cells by both CTLs and NK cells.

Asunto(s)

Antígenos HLA/inmunología , Antígenos HLA-C/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Proteínas Oncogénicas Virales/farmacología , Papillomaviridae/patogenicidad , Regulación hacia Abajo , Fibroblastos , Perfilación de la Expresión Génica , Aparato de Golgi/fisiología , Humanos , Queratinocitos , Células Asesinas Naturales/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T Citotóxicos/inmunología , Células Tumorales Cultivadas , Antígenos HLA-E

RESUMEN

PURPOSE: Dysregulation of cell cycle control, in particular G(1)-S-phase transition, is implicated in the pathogenesis of most human cancers, including epithelial ovarian cancer (EOC). However, the prognostic significance of aberrant cell cycle gene expression in EOC remains unclear. EXPERIMENTAL DESIGN: The expression of selected genes from the pRb pathway that regulates G(1)-S-phase progression, including cyclin D1, p16(Ink4a), cyclin E, p27(Kip1), p21(Waf1/Cip1), and p53, was examined in a consecutive series of 134 serous EOC using immunohistochemistry and the results correlated to disease outcome. RESULTS: Molecular markers predictive of reduced overall survival in univariate analysis were overexpression of cyclin D1 (P = 0.03) and p53 (P = 0.03) and reduced expression of p27(Kip1) (P = 0.05) and p21(Waf1/Cip1) (P = 0.02), with the latter three also being prognostic for a shorter progression-free interval. In addition, patients displaying overexpression of p53 with concurrent loss of p21(Waf1/Cip1) had a significantly shorter overall (P = 0.0008) and progression-free survival (P = 0.0001). On multivariate analysis, overexpression of cyclin D1 and combined loss of p21(Waf1/Cip1) in the presence of p53 overexpression were independent predictors of overall survival. Similarly, the combination of p21(Waf1/Cip1) loss and p53 overexpression was independently predictive of a shorter progression-free interval. Overexpression of p53 and cyclin E and reduced expression of p27(Kip1) and p21(Waf1/Cip1) were significantly associated with increasing tumor grade. CONCLUSIONS: This study confirms that dysregulation of cell cycle genes is common in EOC, and that aberrant expression of critical cell cycle regulatory proteins can predict patient outcome in serous EOC.

Asunto(s)

Proteínas de Ciclo Celular/biosíntesis , Ciclina D1/biosíntesis , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/mortalidad , Regulación Neoplásica de la Expresión Génica/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/mortalidad , Proteína p53 Supresora de Tumor/biosíntesis , Biomarcadores de Tumor , Ciclo Celular , Línea Celular Tumoral , Ciclina E/biosíntesis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Femenino , Humanos , Inmunohistoquímica , Oportunidad Relativa , Modelos de Riesgos Proporcionales , Riesgo , Factores de Tiempo , Resultado del Tratamiento , Proteína p53 Supresora de Tumor/metabolismo

RESUMEN

PURPOSE: A better understanding of the molecular pathways underlying the development of epithelial ovarian cancer (EOC) is critical to identify ovarian tumor markers for use in diagnostic or therapeutic applications. The aims of this study were to integrate the results from 14 transcript profiling studies of EOC to identify novel biomarkers and to examine their expression in early and late stages of the disease. EXPERIMENTAL DESIGN: A database incorporating genes identified as being highly up-regulated in each study was constructed. Candidate tumor markers were selected from genes that overlapped between studies and by evidence of surface membrane or secreted expression. The expression patterns of three integral membrane proteins, discoidin domain receptor 1 (DDR1), claudin 3 (CLDN3), and epithelial cell adhesion molecule, all of which are involved in cell adhesion, were evaluated in a cohort of 158 primary EOC using immunohistochemistry. RESULTS: We confirmed that these genes are highly overexpressed in all histological subtypes of EOC compared with normal ovarian surface epithelium, identifying DDR1 and CLDN3 as new biomarkers of EOC. Furthermore, we determined that these genes are also expressed in ovarian epithelial inclusion cysts, a site of metaplastic changes within the normal ovary, in borderline tumors and in low-grade and stage cancer. A trend toward an association between low CLDN3 expression and poor patient outcome was also observed. CONCLUSIONS: These results suggest that up-regulation of DDR1, CLDN3, and epithelial cell adhesion molecule are early events in the development of EOC and have potential application in the early detection of disease.

Asunto(s)

Antígenos de Neoplasias/biosíntesis , Moléculas de Adhesión Celular/biosíntesis , Proteínas de la Membrana/biosíntesis , Neoplasias Ováricas/metabolismo , Ovario/metabolismo , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Biomarcadores de Tumor , Adhesión Celular , Línea Celular Tumoral , Membrana Celular/metabolismo , Claudina-3 , Estudios de Cohortes , ADN Complementario/metabolismo , Bases de Datos como Asunto , Receptor con Dominio Discoidina 1 , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Molécula de Adhesión Celular Epitelial , Epitelio/metabolismo , Femenino , Marcadores Genéticos , Humanos , Inmunohistoquímica , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismo , Factores de Tiempo , Resultado del Tratamiento , Regulación hacia Arriba

Asunto(s)

Transformación Celular Viral/inmunología , Inmunidad , Papillomaviridae/inmunología , Infecciones por Papillomavirus/inmunología , Infecciones Tumorales por Virus/inmunología , Papillomaviridae/genética , Papillomaviridae/patogenicidad , Papillomaviridae/fisiología

RESUMEN

During papillomavirus infection, the E5 protein localizes in the cell Golgi apparatus and other endomembrane compartments. Cells transformed by E5 do not express major histocompatibility class I complex (MHC I) on the cell surface, while cells transformed by the other transforming proteins E6 and E7 do. In addition, the total amount of both MHC I protein and mRNA is reduced in E5-transformed cells. Here we show that expression of bovine papillomavirus E5 causes the retention of MHC I in the Golgi apparatus, thus preventing its transport to the cell surface. We ascribe this effect to a failure of acidification of the Golgi apparatus, as similar effects are observed in control cells treated with the ionophore monensin. Treatment of E5-transformed cells with either beta- or gamma-interferon increases the synthesis of MHC I, showing that inhibition of MHC I expression by E5 is not irreversible. However, even after interferon treatment, MHC I, although increased in quantity, is not transported to the cell surface. E5 therefore affects MHC I at several levels, but prevention of MHC I transport to the cell surface appears to be the dominant effect. Lack of surface MHC I would have profound consequences for presentation of viral peptides to the immune system.

Asunto(s)

Papillomavirus Bovino 1/fisiología , Aparato de Golgi/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Proteínas Oncogénicas Virales/fisiología , Transporte de Proteínas/fisiología , Animales , Antivirales/farmacología , Papillomavirus Bovino 1/efectos de los fármacos , Bovinos , Transformación Celular Viral , Regulación de la Expresión Génica/efectos de los fármacos , Genes MHC Clase I , Genes Reporteros , Proteínas Fluorescentes Verdes , Concentración de Iones de Hidrógeno , Interferón beta/farmacología , Interferón gamma/farmacología , Ionóforos/farmacología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Monensina/farmacología , Transporte de Proteínas/efectos de los fármacos , ARN Mensajero/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Virales/fisiología

RESUMEN

The nature of the interaction between papillomaviruses (PV) and their infected host has led to the identification of ways in which the viral oncoproteins can transform the infected host cells into cancer cells. As viral persistence is required for malignancy, and persistence requires avoidance of immune attack by the host, defining the relationship between PV and the immune system is also paramount in understanding tumorigenesis. It has emerged that PV have evolved several ways in which to prevent clearance by the host immune system. The limitation of the PV replication cycle to the epithelium, together with low level expression of the virus proteins and an absence of inflammation, minimises the exposure of virus to immune cells. In addition, more recently it has been shown that, like many other viruses, PV can directly subvert the immune response, including interference with the interferon pathway, modulation of antigen presentation, inhibition of interleukin-18 activity and down-regulation of major histocompatibility class I on infected cells. Collectively these mechanisms explain how PV lesions are able to persist for long periods of time in immunocompetent hosts.

Asunto(s)

Inmunidad , Papillomaviridae/patogenicidad , Infecciones por Papillomavirus/inmunología , Infecciones Tumorales por Virus/inmunología , Proteínas Virales/metabolismo , Femenino , Humanos

Asunto(s)

Anticuerpos Monoclonales/genética , Fragmentos de Inmunoglobulinas/genética , Región Variable de Inmunoglobulina/genética , Biblioteca de Péptidos , Reacción en Cadena de la Polimerasa/métodos , Animales , Humanos , Ratones

RESUMEN

We have previously described a recombinant phagemid expression vector, pComBov, designed for the production of native sequence bovine monoclonal antibodies (mAb) generated by antibody phage display. Bovine mAb Fab fragments isolated from libraries constructed using pComBov in Escherichia coli strain XL1-Blue, which is routinely used for antibodies expressed on the surface of phage, were expressed at very low yields. Therefore, a study was undertaken to determine optimal growth conditions for maximal expression of bovine Fab fragments in E. coli. By varying the E. coli strain, and the temperature and length of the culture growth, we were able to substantially increase the yield of soluble Fab fragments. A high yield of Fab fragments was found in the culture growth medium, which enabled us to devise a rapid and simple single-step method for the purification of native (nondenatured) Fabs based on immobilized metal affinity chromatography against a six-histidine amino acid carboxyl-terminal extension of the heavy-chain constant region. Using these methods we were able to express and purify antigen-specific bovine Fab fragments from E. coli.

Asunto(s)

Clonación Molecular/métodos , Fragmentos Fab de Inmunoglobulinas/genética , Animales , Bovinos , Cromatografía de Afinidad , Escherichia coli/genética , Expresión Génica , Histidina , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/aislamiento & purificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Temperatura