RESUMEN
AIMS: To perform a population-based review of monomorphic endometrial stromal tumours and their histological mimics presenting over a 20-year period, including an evaluation of fluorescence in-situ hybridization (FISH) for the JAZF1 and YWHAE breakaparts. METHODS AND RESULTS: Forty-nine tumours were examined, comprising 13 histological mimics and 36 endometrial stromal tumours [six stromal nodules (ESNs), 25 low-grade stromal sarcomas (ESSs), and five monomorphic undifferentiated sarcomas (mUESs)]. Nine ESSs showed variant histological patterns, including smooth muscle, sex cord-like/glandular, fibrous or rhabdoid differentiation. Three ESSs were initially misclassified as benign uterine lesions, and, conversely, three benign mimics were originally reported as ESSs. One mUES showed a prominent pseudopapillary pattern. Fluorescence in-situ hybridization demonstrated JAZF1 breakaparts in five of six ESNs and 16 of 25 ESSs; however, only three of nine ESS variants were positive. YWHAE breakaparts were present in four of five mUESs. Analysis of a subsequent metastasis in the YWHAE breakapart-negative mUES demonstrated a YWHAE deletion. None of the histological mimics was positive in FISH analysis. Diffuse cyclin D1 expression was restricted to mUESs in this series. CONCLUSIONS: Endometrial stromal neoplasms continue to present diagnostic difficulty. Fluorescence in-situ hybridization analysis is helpful in distinguishing stromal tumours from their histological mimics and in distinguishing ESS from mUES.
Asunto(s)
Neoplasias Endometriales/diagnóstico , Tumores Estromáticos Endometriales/diagnóstico , Hibridación Fluorescente in Situ , Proteínas 14-3-3/análisis , Adulto , Anciano , Australia , Proteínas Co-Represoras , Ciclina D1/aislamiento & purificación , Proteínas de Unión al ADN , Diagnóstico Diferencial , Neoplasias Endometriales/patología , Tumores Estromáticos Endometriales/patología , Femenino , Humanos , Persona de Mediana Edad , Proteínas de Neoplasias/análisis , Translocación Genética/genéticaRESUMEN
Esta revisión muestra los principales biomarcadores de cáncer oral en saliva. El aspecto clínico y el grado de displasia de las lesiones precancerosas de la cavidad bucal sugieren su capacidad de malignidad; sin embargo, éstas generalmente son diagnosticadas en estadios avanzados, disminuyendo la probabilidad de supervivencia, lo que justifica el diseño de nuevas pruebas diagnósticas que determinen el grado de alteración celular, permitan comprender el proceso degenerativo en el cáncer y establezcan diagnósticos precoz. Esta búsqueda para mejorar los métodos diagnósticos, apunta a que sean sensibles, específicos y menos invasivos, por lo cual el estudio de diferentes biomarcadores en saliva que desde una perspectiva molecular proporcionan información adicional al examen clínico e histopatológico, es considerada como una alternativa eficaz y más cómoda con respecto a los ensayos en sangre. Los biomarcadores que se han descrito en saliva algunos mostrando mayor relación con la carcinogénesis oral son: Ciclina D1, cyfra 21-1, endotelina-1, galectinas 1, 3 y 7, Ki67, lactato deshidrogenasa, metaloproteinasas 2 y 9, proteína p53, proteína de unión a calcio (S100P) y telomerasa (AU)
This review shows the main oral cancer biomarkers in saliva. The clinical appearance and the degree of dysplasia, precancerous lesions of the oral cavity suggests its ability to malignancy, but these are usually diagnosed in advanced stages, decreasing the likelihood of survival, justifying the design of new diagnostic tests to determine the degree of cell alteration as to understand the degenerative process in cancer diagnosis and establish early. This search for improved diagnostic methods, aims to be sensitive, specific and less invasive, so the study of biomarkers in saliva from a molecular perspective provide additional information to clinical and histopathological examination is considered as a more comfortable and effective to establish a diagnosis. Biomarkers that have been described in saliva some showing more related to oral carcinogenesis are cyclin D1, Cyfra 21-1, Endothelin-1, Galectins 1, 3 and 7, Ki67, Lactate dehydrogenase, Metalloproteinases 2 and 9, p53 protein, protein calcium-binding (S100P) and Telomerase (AU)
Asunto(s)
Humanos , Neoplasias de la Boca/diagnóstico , Saliva/citología , Biomarcadores de Tumor/análisis , Ciclina D1/aislamiento & purificación , Endotelina-1/aislamiento & purificación , Galectinas/aislamiento & purificación , Antígeno Ki-67/aislamiento & purificación , Lactato Deshidrogenasas/aislamiento & purificación , Proteína p53 Supresora de Tumor/aislamiento & purificación , Proteínas de Unión al Calcio/aislamiento & purificación , Telomerasa/aislamiento & purificaciónRESUMEN
CDK2 and CDK4 known promoter of cell cycling catalyze phosphorylation of RB protein. Enzyme specificity between two CDKs that work at a different cell cycle phase is not clearly understood. In order to define kinase properties of CDK2 and CDK4 in complex with cycline A or cycline D1 in relation to their respective role in cell cycling regulation, we examined enzymatic properties of both CDK4/cycline D1 and CDK2/cycline A in vitro. Association constant, Km for ATP in CDK4/cyclin D1 was found as 418 microM, a value unusually high whereas CDK2/cyclin A was 23 microM, a value close to most of other regulatory protein kinases. Turnover value for both CDK4/cyclin D1 and CDK2/cyclin A were estimated as 3.4 and 3.9 min(-1) respectively. Kinetic efficiency estimation indicates far over one order magnitude less efficiency for CDK4/cyclin D1 than the value of CDK2/cycline A (9.3 pM(-1) min(-1) and 170 pM(-1) min(-1) respectively). In addition, inhibition of cellular CDK4 caused increase of cellular levels of ATP, even though inhibition of CDK2 did not change it noticeably. These data suggest cellular CDK4/cyclin D1 activity is tightly associated with cellular ATP concentration. Also, analysis of phosphorylated serine/threonine sites on RB catalyzed by CDK4/cyclin D1 and CDK2/cyclin A showed significant differences in their preference of phosphorylation sites in RB C-terminal domain. Since RB is known to regulate various cellular proteins by binding and this binding is controlled by its phosphorylation, these data shown here clearly indicate significant difference in their biochemical properties between CDK4/cyclin D1 and CDK2/cyclin A affecting regulation of cellular RB function.
Asunto(s)
Quinasas CDC2-CDC28/metabolismo , Ciclina A/metabolismo , Ciclina D1/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Proteínas Proto-Oncogénicas , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Baculoviridae/genética , Quinasas CDC2-CDC28/genética , Quinasas CDC2-CDC28/aislamiento & purificación , Ciclina A/genética , Ciclina A/aislamiento & purificación , Ciclina D1/genética , Ciclina D1/aislamiento & purificación , Quinasa 2 Dependiente de la Ciclina , Quinasa 4 Dependiente de la Ciclina , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/aislamiento & purificación , Humanos , Cinética , Datos de Secuencia Molecular , Fosforilación , Conformación Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Transgenic mice expressing specific oncogenes usually develop tumors in a stochastic fashion suggesting that tumor progression is a multi-step process. To gain further understanding of the interactions between oncogenes and tumor suppressor genes during tumorigenesis, we have crossed a transgenic strain (TG.NK) carrying an activated c-neu oncogene driven by the MMTV enhancer/promoter with p53-deficient mice. c-neu transgenic mice have stochastic breast tumor formation and normal appearing salivary glands. However, c-neu mice heterozygous for a p53 deletion develop parotid gland tumors and loose their wild type p53 allele. c-neu mice with a homozygous p53 deletion have increased rates of parotid tumor onset suggesting that inactivation of p53 is required and sufficient for parotid gland transformation in the presence of activated c-neu. In contrast to the dramatic effect of p53 in parotid gland transformation, p53 loss has little effect on the rate or stochastic appearance of mammary tumors. In addition, p53 loss was accompanied by the down regulation of p21 in parotid gland tumors but not breast tumors. The parotid gland tumors were aneuploid and demonstrated increased levels of Cyclin D1 expression. These observations suggest that in c-neu transgenic mice, p53 alterations have differential tissue effects and may be influenced by the tissue specific expression of genes influencing p53 activity.
Asunto(s)
Genes erbB-2 , Genes p53 , Neoplasias de las Glándulas Salivales/genética , Aneuploidia , Animales , Transformación Celular Neoplásica/genética , Ciclina D1/aislamiento & purificación , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/aislamiento & purificación , Femenino , Regulación Neoplásica de la Expresión Génica , Pérdida de Heterocigocidad , Masculino , Neoplasias Mamarias Animales/etiología , Neoplasias Mamarias Animales/genética , Ratones , Ratones Transgénicos , Neoplasias de la Parótida/genética , Neoplasias de las Glándulas Salivales/etiología , Glándulas Salivales/metabolismo , Procesos Estocásticos , Distribución Tisular , Proteína p53 Supresora de Tumor/aislamiento & purificaciónRESUMEN
Using single and double transgenic mouse models, we investigated how c-Myc modulates the mammary epithelial cell cycle to induce cancer and how TGFalpha enhanced the process. In c-myc transgenic mice, c-myc expression was high in the hyperplastic mammary epithelium and in the majority of tumor areas. However, the tumors displayed focal areas of low expression of c-myc but high rates of proliferation. In contrast to E2F1 and cyclin A2, which were induced and co-localized with c-myc expression, induction of cyclins D1 and E occurred only in these tumor foci. Overexpression of cyclin D1 also occurred in the hyperplastic epithelium of tgfalpha-single and tgfalpha/c-myc-double transgenic mice. In tgfalpha/c-myc tumors, cells positive for cyclins D1 and E were randomly spread, without showing a reciprocal relationship to c-myc expression. In contrast to c-myc tumors, most tgfalpha/c-myc tumors showed undetectable levels of retinoblastoma protein (pRB), and the loss of pRB occurred in some cases at the mRNA level. These results suggest that E2F1 and cyclin A2 may be induced by c-Myc to mediate the onset of mammary cancer, whereas overexpression of cyclins D1 and E may occur later to facilitate tumor progression. TGFalpha may play its synergistic role, at least in part, by inducing cyclin D1 and facilitating the loss of pRB.
Asunto(s)
Proteínas Portadoras , Proteínas de Ciclo Celular , Transformación Celular Neoplásica/genética , Proteínas de Unión al ADN , Neoplasias Mamarias Experimentales/genética , Proteínas Proto-Oncogénicas c-myc/genética , Factor de Crecimiento Transformador alfa/genética , Animales , Apoptosis , Ciclo Celular/genética , Ciclina A/aislamiento & purificación , Ciclina D1/aislamiento & purificación , Ciclina D3 , Ciclina E/aislamiento & purificación , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Ciclinas/aislamiento & purificación , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Células Epiteliales , Femenino , Hibridación in Situ , Etiquetado Corte-Fin in Situ , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Transgénicos , Modelos Biológicos , Proteína de Retinoblastoma/aislamiento & purificación , Proteína 1 de Unión a Retinoblastoma , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción DP1 , Factores de Transcripción/aislamiento & purificaciónRESUMEN
We describe and discuss a method of protein extraction for Western blot analysis from formalin-fixed, paraffin-embedded tissue sections. From 5-mm2 50-micron-thick tissue sections, an abundance of proteins could be extracted by incubating the sections in lysis buffer containing 2% sodium dodecyl sulfate (SDS) at 100C for 20 min followed by incubation at 60C for 2 hr. Extracts yielded discernible protein bands ranging from 10 kD to 120 kD as identified by SDS-polyacrylamide gel electrophoresis (PAGE). Western blot analysis successfully detected membrane-bound protein such as E-cadherin, cytosolic protein such as beta-catenin, and nuclear proteins including proliferating cell nuclear antigen (PCNA), mutant-type p53, cyclin D1, cyclin E, and cyclin-dependent kinases (CDKs). With this technique, we could examine cyclin D1 and CDK2 expression in small adenomas compared with cancer tissues and normal mucosa. The simple method of protein extraction described here should make it possible to use large-scale archives of formalin-fixed, paraffin-embedded samples for Western blot analysis, and its application could lead to detailed analysis of protein expression. This new technique should yield valuable information for molecular biology.
Asunto(s)
Western Blotting/métodos , Quinasas CDC2-CDC28 , Formaldehído , Parafina , Proteínas/aislamiento & purificación , Adenoma/química , Neoplasias Colorrectales/química , Ciclina D1/química , Ciclina D1/aislamiento & purificación , Quinasa 2 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/química , Quinasas Ciclina-Dependientes/aislamiento & purificación , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/aislamiento & purificación , Antígeno Nuclear de Célula en Proliferación/química , Antígeno Nuclear de Célula en Proliferación/aislamiento & purificación , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/aislamiento & purificación , Proteínas/química , Fijación del TejidoRESUMEN
The association of cyclin D1 with nuclear structures was investigated in normal human fibroblasts by using hypotonic detergent extraction procedures, immunofluorescence quantitation with flow cytometry, and Western blot analysis. About 20% of the total cellular levels of cyclin D1 was found to be tightly bound to nuclear structures, being the complex formation resistant to DNase I treatment and to high salt extraction. Maximal levels of the insoluble form of the protein were found in the middle to late G1 phase of the cell cycle. Cell fractionation and immunoprecipitation techniques after in vivo 32P-labeling showed that both soluble and nuclear-bound forms of cyclin D1 were phosphorylated. Both fractions were reactive to an anti-phosphotyrosine antibody, while only the latter was detectable with an anti-phosphoserine antibody. Treatment with the protein kinase inhibitor staurosporine, which induces a cell cycle arrest in early G1 phase, strongly reduced cyclin D1 phosphorylation. Concomitantly, the ratio of nuclear-bound/total cyclin D1 levels was reduced by about 60%, compared with the control value. The protein kinase A specific inhibitor isoquinoline-sulfonamide (H-89) induced a similar reduction in the ratio, with no significant modification in the total amount of protein. In contrast, both calphostin C and bisindolylmaleimide, specific inhibitors of protein kinase C, consistently increased by 30-50% the ratio of nuclear-bound/total amount of the cyclin protein. These results suggest that, during the G1 phase, formation of an insoluble complex of cyclin D1 occurs at nuclear matrix structures and that this association is mediated by a protein kinase A-dependent pathway.