RESUMEN
To study the postulated mutant p53 (mutp53) "gain of function" effects in mammary tumor development, progression and metastasis, we crossed SV40 transgenic WAP-T mice with mutant p53 transgenic WAP-mutp53 mice. Compared to tumors in monotransgenic WAP-T mice, tumors in bitransgenic WAP-T x WAP-mutp53 mice showed higher tumor grading, enhanced vascularization, and significantly increased metastasis. Bitransgenic tumors revealed a gene signature associated with the oncogenic epithelial-mesenchymal transition pathway (EMT gene signature). In cultures of WAP-T tumor-derived G-2 cancer cells, which are comprised of subpopulations displaying "mesenchymal" and "epithelial" phenotypes, this EMT gene signature was associated with the "mesenchymal" compartment. Furthermore, ectopic expression of mutp53 in G-2 cells sufficed to induce a strong EMT phenotype. In contrast to these in vitro effects, monotransgenic and bitransgenic tumors were phenotypically similar suggesting that in vivo the tumor cell phenotype might be under control of the tumor microenvironment. In support, orthotopic transplantation of G-2 cells as well as of G-2 cells expressing ectopic mutp53 into syngeneic mice resulted in tumors with a predominantly epithelial phenotype, closely similar to that of endogenous primary tumors. We conclude that induction of an EMT gene signature by mutp53 in bitransgenic tumors primarily promotes tumor cell plasticity, that is, the probability of tumor cells to undergo EMT processes under appropriate stimuli, thereby possibly increasing their potential to disseminate and metastasize.
Asunto(s)
Transición Epitelial-Mesenquimal , Neoplasias Mamarias Experimentales/patología , Proteína p53 Supresora de Tumor/fisiología , Animales , Línea Celular Tumoral , Femenino , Humanos , Ratones , Mutación , Metástasis de la Neoplasia , Microambiente TumoralRESUMEN
Mammary carcinomas developing in SV40 transgenic WAP-T mice arise in two distinct histological phenotypes: as differentiated low-grade and undifferentiated high-grade tumors. We integrated different types of information such as histological grading, analysis of aCGH-based gene copy number and gene expression profiling to provide a comprehensive molecular description of mammary tumors in WAP-T mice. Applying a novel procedure for the correlation of gene copy number with gene expression on a global scale, we observed in tumor samples a global coherence between genotype and transcription. This coherence can be interpreted as a matched transcriptional regulation inherited from the cells of tumor origin and determined by the activity of cancer driver genes. Despite common recurrent genomic aberrations, e.g. gain of chr. 15 in most WAP-T tumors, loss of chr. 19 frequently occurs only in low-grade tumors. These tumors show features of "basal-like" epithelial differentiation, particularly expression of keratin 14. The high-grade tumors are clearly separated from the low-grade tumors by strong expression of the Met gene and by coexpression of epithelial (e.g. keratin 18) and mesenchymal (e.g. vimentin) markers. In high-grade tumors, the expression of the nonmutated Met protein is associated with Met-locus amplification and Met activity. The role of Met as a cancer driver gene is supported by the contribution of active Met signaling to motility and growth of mammary tumor-derived cells. Finally, we discuss the independent origin of low- and high-grade tumors from distinct cells of tumor origin, possibly luminal progenitors, distinguished by Met gene expression and Met signaling.
Asunto(s)
Neoplasias Mamarias Experimentales/patología , Proteínas de la Leche/genética , Proteínas Proto-Oncogénicas c-met/fisiología , Animales , Línea Celular Tumoral , Hibridación Genómica Comparativa , Femenino , Neoplasias Mamarias Experimentales/genética , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Clasificación del Tumor , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas c-met/genética , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
Telomerase is activated in the majority of invasive breast cancers, but the time point of telomerase activation during mammary carcinogenesis is not clear. We have recently presented a transgenic mouse model to study human telomerase reverse transcriptase (TERT) gene expression in vivo (hTERTp-lacZ). In the present study, hTERTp-lacZxWAP-T bitransgenic mice were generated to analyze the mechanisms responsible for human and mouse TERT upregulation during tumor progression in vivo. We found that telomerase activity and TERT expression were consistently upregulated in SV40-induced invasive mammary tumors compared to normal and hyperplastic tissues and ductal carcinoma in situ (DCIS). Human and mouse TERT genes are regulated similarly in the breast tissue, involving the CEBP transcription factors. Loss of CEBP-α and induction of CEBP-ß expression correlated well with the activation of TERT expression in mouse mammary tumors. Transfection of CEBP-α into human or murine cells resulted in TERT repression, whereas knockdown of CEBP-α in primary human mammary epithelial cells resulted in reactivation of endogenous TERT expression and telomerase activity. Conversely, ectopic expression of CEBP-ß activated endogenous TERT gene expression. Moreover, ChIP and EMSA experiments revealed binding of CEBP-α and CEBP-ß to human TERT-promoter. This is the first evidence indicating that CEBP-α and CEBP-ß are involved in TERT gene regulation during carcinogenesis.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Transformación Celular Neoplásica/patología , Neoplasias Mamarias Experimentales/patología , Proteínas de la Leche/genética , Regiones Promotoras Genéticas/genética , Telomerasa/genética , Animales , Western Blotting , Proteínas Potenciadoras de Unión a CCAAT/genética , Inmunoprecipitación de Cromatina , Ensayo de Cambio de Movilidad Electroforética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Luciferasas/metabolismo , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Telomerasa/metabolismo , Activación Transcripcional , Células Tumorales CultivadasRESUMEN
This unit describes an easy, rapid, and universal procedure to process fresh and nitrogen-frozen tissue specimens for high-resolution cell cycle and DNA ploidy analysis. Unlike other protocols, this procedure does not require treating tissues with enzymes, detergents, or other plasma membrane-lysing chemicals, but it achieves tissue dispersion by a simple two-step mechanical process that can be performed in â¼5 min. Resulting single-cell suspensions are fixed with ethanol, stained with propidium iodide, and subjected to flow cytometric DNA content analysis. The method can be applied without any alterations to all tissue types (except bones) derived from several species and results in highly reproducible cell cycle profiles of excellent resolution. The described protocol can be used to reliably and accurately detect subtle cell cycle and ploidy alterations in tissue specimens, including cell cycle arrest, aneuploidy, and apoptosis/necrosis-associated DNA fragmentation.
Asunto(s)
Ciclo Celular , Técnicas Citológicas/métodos , Especificidad de Órganos , Ploidias , Animales , Secciones por Congelación , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
Cell cycle alterations are fundamental to many physiological processes but their detection has proven difficult when cells are in the context of a tissue structure. Here we describe an easy, rapid and optimization-free procedure for obtaining high resolution cell cycle profiles from nearly all tissue types derived from mouse, human and sheep. Using a standardized and non-enzymatic procedure that is universally suitable for soft, solid and epithelial tissues alike, we reproducibly obtain cell cycle profiles of highest quality with half peak coefficients of variation below 2.0. We are able to reduce preparation-derived debris to almost zero and efficiently exclude doublets, but retain multinucleated cells and apoptotic subG1-fragments. Applying this technique, we determine DNA-indices as small as 1.09 in tumor samples containing large necrotic areas and follow ploidy changes within different sections of individual tumors. Moreover, we examine tissue-specific cell cycle arrest and apoptosis as an in vivo stress response caused by radiation of mice. This method significantly improves the quality of DNA content analysis in tissues and extends the spectrum of applications. It allows assessing changes in ploidy, cell cycle distribution and apoptosis/necrosis in vivo and should be instrumental in all research that involves experimental animal models and/or patient biopsies.
Asunto(s)
Ciclo Celular , ADN/análisis , Citometría de Flujo/métodos , Ploidias , Aneuploidia , Animales , Apoptosis , Fase G1 , Humanos , Ratones , Ratones Endogámicos C57BL , OvinosRESUMEN
BACKGROUND: In analogy to normal stem cell differentiation, the current cancer stem cell (CSC) model presumes a hierarchical organization and an irreversible differentiation in tumor tissue. Accordingly, CSCs should comprise only a small subset of the tumor cells, which feeds tumor growth. However, some recent findings raised doubts on the general applicability of the CSC model and asked for its refinement. METHODOLOGY/PRINCIPAL FINDINGS: In this study we analyzed the CSC properties of mammary carcinoma cells derived from transgenic (WAP-T) mice. We established a highly tumorigenic WAP-T cell line (G-2 cells) that displays stem-like traits. G-2 cells, as well as their clonal derivates, are closely related to primary tumors regarding histology and gene expression profiles, and reflect heterogeneity regarding their differentiation states. G-2 cultures comprise cell populations in distinct differentiation states identified by co-expression of cytoskeletal proteins (cytokeratins and vimentin), a combination of cell surface markers and a set of transcription factors. Cellular subsets sorted according to expression of CD24a, CD49f, CD61, Epcam, Sca1, and Thy1 cell surface proteins, or metabolic markers (e.g. ALDH activity) are competent to reconstitute the initial cellular composition. Repopulation efficiency greatly varies between individual subsets and is influenced by interactions with the respective complementary G-2 cellular subset. The balance between differentiation states is regulated in part by the transcription factor Sox10, as depletion of Sox10 led to up-regulation of Twist2 and increased the proportion of Thy1-expressing cells representing cells in a self-renewable, reversible, quasi-mesenchymal differentiation state. CONCLUSIONS/SIGNIFICANCE: G-2 cells constitute a self-reproducing cancer cell system, maintained by bi- and unidirectional conversion of complementary cellular subsets. Our work contributes to the current controversial discussion on the existence and nature of CSC and provides a basis for the incorporation of alternative hypotheses into the CSC model.
Asunto(s)
Homeostasis , Neoplasias Mamarias Animales/patología , Modelos Biológicos , Animales , Biomarcadores/metabolismo , Comunicación Celular , Diferenciación Celular , Línea Celular Tumoral , Células Clonales , Células Epiteliales/patología , Perfilación de la Expresión Génica , Humanos , Neoplasias Mamarias Animales/genética , Mesodermo/patología , Ratones , Ratones Transgénicos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Fenotipo , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Transgenic mouse models offer an excellent opportunity for studying the molecular basis of cancer development and progression. Here we applied flat-panel volume computed tomography (fpVCT) to monitor tumor progression as well as the development of tumor vasculature in vivo in a transgenic mouse model for oncogene-induced mammary carcinogenesis (WAP-T mice). WAP-T mice develop multiple mammary carcinomas on oncogene induction within 3 to 5 months. Following induction, 3-dimensional fpVCT data sets were obtained by serial single scans of entire mice in combination with iodine containing contrast agents and served as basis for precise measurements of tumor volumes. Thereby, we were able to depict tumors within the mammary glands at a very early stage of the development. Tumors of small sizes (0.001 cm(3)) were detected by fpVCT before being palpable or visible by inspection. The capability to determine early tumor onset combined with longitudinal noninvasive imaging identified diverse time points of tumor onset for each mammary carcinoma and different tumor growth kinetics for multiple breast carcinomas that developed in single mice. Furthermore, blood supply to the breast tumors, as well as blood vessels around and within the tumors, were clearly visible over time by fpVCT. Three-dimensional visualization of tumor vessels in high resolution was enhanced by the use of a novel blood pool contrast agent. Here, we demonstrate by longitudinal fpVCT imaging that mammary carcinomas develop at different time points in each WAP-T mouse, and thereafter show divergent growth rates and distinct vascularization patterns.
Asunto(s)
Tomografía Computarizada de Haz Cónico/métodos , Neoplasias Mamarias Experimentales/diagnóstico por imagen , Neoplasias Mamarias Experimentales/patología , Oncogenes/fisiología , Interpretación de Imagen Radiográfica Asistida por Computador/métodos , Animales , Proliferación Celular , Femenino , Cinética , Neoplasias Mamarias Experimentales/etiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de la Leche/genética , Regiones Promotoras GenéticasRESUMEN
In human breast cancer, mutations in the p53 gene are associated with poor prognosis. However, analysis of patient data so far did not clarify, whether missense point mutations in the p53 gene, in addition to causing loss of wild-type p53 function, also confer a gain of function phenotype to the encoded mutant p53. As heterogeneity of patient material and data might obscure a clear answer, we studied the effects of a coexpressed mutant p53(R270H) in transgenic mice in which SV40 early proteins initiate the development of mammary adenocarcinoma (WAP-T mice). In such tumors the endogenous wild-type p53 is functionally compromised by complex formation with SV40 T-antigen, thereby constituting a loss of wild-type p53 function situation that allowed analysis of the postulated gain of function effects of mutant p53(R270H). We found that mutant p53(R270H) in bi-transgenic mice enhanced the transition from intraepithelial neoplasia to invasive carcinoma, resulting in a higher frequency of invasive carcinoma per gland and per mouse, a more severe tumor phenotype, and more frequent pulmonary metastasis. Surprisingly, mutant p53(R270H) in this system does not increase genomic instability. Therefore, other postulated gain of function activities of mutant p53 must be responsible for the effects described here.
Asunto(s)
Adenocarcinoma/genética , Transformación Celular Neoplásica/genética , Genes p53 , Neoplasias Mamarias Experimentales/genética , Mutación Missense , Proteína p53 Supresora de Tumor/genética , Animales , Antígenos Transformadores de Poliomavirus , Arginina , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Histidina , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Oncogenes , Fenotipo , Mutación PuntualRESUMEN
Oxygen free radicals are thought to play an essential role in senescence, especially those derived from peroxisomes. Therefore, the activities of different isoforms of the peroxisomal hydrogen peroxide (H2O2)-scavenging enzyme catalase (CAT) were analysed during senescence of Arabidopsis. CAT2 activity decreased with bolting time parallel with cytosolic ascorbate peroxidase 1 (APX1) activity before loss of chlorophyll could be measured. At the same time point, the H2O2 content increased. Subsequently, the stress-inducible CAT3 isoform was activated and APX1 activity was recovered, accompanied by a decline of the H2O2 content. In very late stages, low activities of the seed-specific CAT1 became detectable in leaves, but H2O2 increased again. Further analyses of CAT expression by promoter: beta-glucuronidase (GUS) fusions in transgenic plants revealed a vasculature-specific CAT3 expression, whereas CAT2 expression turned out to be specific for photosynthetic active tissues. CAT2 expression is down-regulated during leaf senescence, while CAT3 expression is induced with age and corresponds to an accumulation of H2O2 in the vascular bundles. CAT2 down-regulation on the transcriptional level appears as the initial step in creating the H2O2 peak during bolting time, while the decrease in APX1 activity might only be a secondary and amplifying effect.
Asunto(s)
Arabidopsis/enzimología , Catalasa/metabolismo , Senescencia Celular/genética , Arabidopsis/citología , Arabidopsis/genética , Proteínas de Arabidopsis/antagonistas & inhibidores , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiología , Ascorbato Peroxidasas , Catalasa/genética , Catalasa/fisiología , Regulación de la Expresión Génica de las Plantas , Glucuronidasa/análisis , Herbicidas/farmacología , Peróxido de Hidrógeno/análisis , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacología , Isoenzimas/genética , Isoenzimas/metabolismo , Isoenzimas/fisiología , Estrés Oxidativo/genética , Paraquat/farmacología , Peroxidasas/antagonistas & inhibidores , Peroxidasas/metabolismo , Peroxisomas/enzimología , Hojas de la Planta/enzimología , Plantas Modificadas Genéticamente/metabolismo , Proteínas Recombinantes de Fusión/análisisRESUMEN
We describe the construction and phenotypic characterization of 23 whey acidic protein (WAP)-mutp53 transgenic mouse lines. The mutp53-expressing lines showed a mosaic expression pattern for the transgenes, leading to a heterogeneous yet mouse line-specific expression pattern for mutp53 upon induction. Only few lines were obtained, in which the majority of the induced mammary epithelial cells expressed the mutp53 transgene, most of the transgenic lines did not express mutp53, or expressed the transgene in less than 2% of the induced mammary epithelial cells. Hormone requirements for mutp53 transgene expression from the WAP-promoter differed in high and low expressing lines, being low in high expressing lines, and even lower in multiparous mutp53 mice, where persistent expression of the transgene occurred. Repeated induction of mutp53 expression through repeated parturition resulted in the formation of expanding mutp53-expressing foci within the mammary alveolar epithelium. The data suggest that epigenetic mechanisms play a role in modulating the expression of the mutp53 transgene. To support this idea, we crossed a nonexpressing WAP-mutp53 line with a strongly SV40 T-antigen-expressing WAP-T mouse line. In the bitransgenic mice, T-antigen-induced chromatin remodeling led to re-expression of epigenetically silenced mutp53 transgene(s). In these mice, mutp53 expression was much more variable compared to SV40 T-antigen expression, and seemed to depend on the coexpression of SV40 T-antigen. Mutp53 expression in this system thus resembles the situation in many human tumors, where one can observe a heterogeneous expression of mutp53, despite a homogeneous distribution of the p53 mutation in the tumor cells.