RESUMEN
The present work introduces a rigorous stochastic model, called the generalized stochastic microdosimetric model (GSM^{2}), to describe biological damage induced by ionizing radiation. Starting from the microdosimetric spectra of energy deposition in tissue, we derive a master equation describing the time evolution of the probability density function of lethal and potentially lethal DNA damage induced by a given radiation to a cell nucleus. The resulting probability distribution is not required to satisfy any a priori conditions. After the initial assumption of instantaneous irradiation, we generalized the master equation to consider damage induced by a continuous dose delivery. In addition, spatial features and damage movement inside the nucleus have been taken into account. In doing so, we provide a general mathematical setting to fully describe the spatiotemporal damage formation and evolution in a cell nucleus. Finally, we provide numerical solutions of the master equation exploiting Monte Carlo simulations to validate the accuracy of GSM^{2}. Development of GSM^{2} can lead to improved modeling of radiation damage to both tumor and normal tissues, and thereby impact treatment regimens for better tumor control and reduced normal tissue toxicities.
Asunto(s)
Modelos Teóricos , Radiometría , Procesos EstocásticosRESUMEN
The macrophage migration inhibitory factor (MIF) is a hypoxia regulated gene that has a variety of tumorigenic functions. In clear cell renal carcinoma (CCRC), hypoxic signaling is constitutively active because of the frequent loss of function of the von Hippel-Lindau tumor suppressor protein. We therefore sought to assess the expression of MIF in CCRC and its biological functions. We stained tumor tissue microarrays comprising sections of 128 CCRC tumors and found MIF to be moderately or highly expressed in >98%. MIF expression was further found to be dramatically elevated in blood plasma of individuals with CCRC compared with healthy controls, suggesting that measurement of MIF levels in the blood may have utility as a diagnostic marker in CCRC. At a functional level, MIF has been reported to engage the CD74 and CD44 receptors and induce signal transduction. In CCRC cell lines, depletion of MIF, CD74 or CD44 by small hairpin RNA led to a significant reduction in growth rate, and clonogenic survival, coinciding with the degree of knockdown. Interruption of the MIF pathway also decreased tumorigenic potential. Biochemically, we found that in CCRC cells MIF signaling leads to activation of the mitogen-activated protein kinase pathway and to Src phosphorylation, which is critical for regulation of p27. Together, our studies establish MIF as a protumorigenic signaling molecule that functions in an autocrine fashion to promote renal cell carcinoma and may be useful as a minimally invasive marker of disease status.
Asunto(s)
Comunicación Autocrina/efectos de los fármacos , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Factores Inhibidores de la Migración de Macrófagos/farmacología , Animales , Comunicación Autocrina/fisiología , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/metabolismo , Carcinoma de Células Renales/diagnóstico , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/metabolismo , Progresión de la Enfermedad , Humanos , Neoplasias Renales/diagnóstico , Ratones , Ratones Desnudos , Pronóstico , Trasplante Heterólogo , Células Tumorales Cultivadas , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Specific chromosomal translocations involving the ews gene and one of five members of the ets family of transcription factors create ews/ets fusion genes that are found in approximately 85% of Ewing's family of tumors. ews/ets fusion genes consistently maintain an intact and functional ets DNA binding domain (DBD) in all of these cases. We demonstrate here, however, that EWS/FLI1, the most prevalent EWS/ETS fusion, activates oncogenic pathways independent of its DBD. In in vivo tumor assays, EWS/FLI1 molecules with either point mutations or a large deletion in the ets DBD retain the ability to accelerate tumors in NIH 3T3 cells, whereas they lose the ability to bind DNA in vitro. Additionally, whereas inhibition of DBD functions of EWS/FLI1 with a dominant negative form of FLI1 is sufficient to inhibit anchorage-independent growth in NIH 3T3 cells, it is ineffective in inhibiting tumor growth in SCID mice. Usage of this dominant negative construct in a Ewing's tumor cell line, however, does reduce the rate of tumor formation, supporting the need for a functional DBD in this context. Together, these results suggest that EWS/FLI1 induces both DBD-dependent and DBD-independent oncogenic pathways.
Asunto(s)
ADN/metabolismo , Neoplasias Experimentales/etiología , Proteínas de Fusión Oncogénica/fisiología , Factores de Transcripción/fisiología , Células 3T3 , Animales , Sitios de Unión , Transformación Celular Neoplásica , Femenino , Ratones , Ratones SCID , Proteínas de Fusión Oncogénica/química , Proteína Proto-Oncogénica c-fli-1 , Proteína EWS de Unión a ARN , Sarcoma de Ewing/etiología , Factores de Transcripción/químicaRESUMEN
More than 85% of Ewing's family tumors carry a specific chromosomal translocation that fuses the NH(2) terminus of the EWS gene to the COOH terminus of the FLI1 transcription factor. It has been shown previously that both the transactivation domain encoded by EWS and the DNA binding domain of FLI1 were necessary for transforming cells to anchorage independence. We now report that a COOH-terminal domain in addition to the FLI1 DNA binding domain is necessary to promote cellular transformation. NIH 3T3 cells expressing a COOH-terminal deletion mutant (EWS/FLI1 DeltaC) have a greatly reduced capability to form colonies in soft agar and tumors in severe combined immunodeficient mice. The rate of tumor formation for NIH 3T3 that express EWS/FLI1 DeltaC is 50 days, whereas EWS/FLI1 forms tumors within 22 days. In addition, cells expressing the EWS/FLI1 DeltaC mutant failed to completely demonstrate the round-cell histology that is seen in both Ewing's tumor cell lines and NIH 3T3 cells expressing full-length EWS/FLI1. Northern and microarray analyses were performed to assess the effect of loss of the FLI1 COOH terminus on transcriptional modulation of EWS/FLI1 target genes. We found that although EWS/FLI1 DeltaC up-regulates smaller numbers of genes (21 genes) compared with EWS/FLI1 (34 genes), 41% of the EWS/FLI1 targets were also up-regulated by EWS/FLI1 DeltaC. On the other hand, EWS/FLI1 DeltaC is unable to down-regulate genes (3 genes) as efficiently as EWS/FLI1 (39 genes) with only one target gene repressed by both fusion constructs. Our study indicates that the EWS/FLI1 transcription factor has strong transcriptional activating as well as repressing properties and suggests that transcriptional activation and repression of target genes may occur through biochemically different mechanisms.
Asunto(s)
Transformación Celular Neoplásica , Proteínas de Unión al ADN/fisiología , Proteínas de Fusión Oncogénica/fisiología , Proteínas Proto-Oncogénicas , Transactivadores/fisiología , Factores de Transcripción/fisiología , Células 3T3 , Secuencia de Aminoácidos , Animales , Adhesión Celular/fisiología , División Celular/fisiología , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas de Fusión Oncogénica/genética , Estructura Terciaria de Proteína , Proteína Proto-Oncogénica c-fli-1 , Proteína EWS de Unión a ARN , Alineación de Secuencia , Relación Estructura-Actividad , Transactivadores/genética , Factores de Transcripción/genética , Activación Transcripcional/fisiologíaRESUMEN
The EWS/FLI1 fusion gene found in Ewing's sarcoma and primitive neuroectodermal tumor, is able to transform certain cell lines by acting as an aberrant transcription factor. The ability of EWS/FLI1 to modulate gene expression in cells transformed and resistant to transformation by EWS/FLI1, was assessed by Representational Difference Analysis (RDA). We found that the cyclin selective ubiquitin conjugase murine E2-C, was up regulated in NIH3T3 cells transformed by EWS/FLI1 but not in a nontransformed NIH3T3 clone expressing EWS/FLI1. We also found that mE2-C is upregulated in NIH3T3 cells transformed by other genes including activated cdc42, v-ABL and c-myc. We demonstrated that expression of mE2-C in both the EWS/FLI1 transformed and parent NIH3T3 lines varies with the cell cycle. Finally, dominant-negative mE2-C, created by changing a catalytic cysteine to serine, inhibits the in vitro ubiquitination and degradation of cyclin B in human HeLa cell extracts. These data suggest that part of the biologic effect of EWS/FLI1 could be to transcriptionally modulate genes involved in cell cycle regulation.
Asunto(s)
Proteínas Portadoras/biosíntesis , Ciclina B/metabolismo , Ligasas , Proteínas de Fusión Oncogénica/metabolismo , Factores de Transcripción/metabolismo , Enzimas Ubiquitina-Conjugadoras , Regulación hacia Arriba , Células 3T3 , Secuencia de Aminoácidos , Animales , Ciclo Celular , Línea Celular Transformada , Transformación Celular Neoplásica , Sistema Libre de Células , Expresión Génica , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas de Fusión Oncogénica/genética , Proteína Proto-Oncogénica c-fli-1 , Proteína EWS de Unión a ARN , Factores de Transcripción/genéticaRESUMEN
The identification of differential gene expressionbetween cells is a frequent goal in modern biological research. Here we demonstrate the coupling of representational difference analysis (RDA) of cDNA with microarray analysis of the output for high throughput screening. Two primary Ewing's sarcoma tissue samples with different biological behavior in vivo were compared by RDA: one which was metastatic and progressed rapidly; the other localized and successfully treated. A modified RDA protocol that minimizes the necessary starting material was employed. After a reduced number of subtractive rounds, the output of RDA was shotgun cloned into a plasmid vector. Inserts from individual colonies from the subtracted library were amplified with vector-specific primers and arrayed at high density on glass slides. The arrays were then hybridized with differentially fluorescently labeled starting amplicons from the two tissues and fluorescent signals were measured at each DNA spot. We show that the relative amounts of fluorescent signal correlate well with the abundance of fragments in the RDA amplicon and in the starting mRNA. In our system, we analyzed 192 products and 173 (90%) were appropriately detected as being >2-fold differentially expressed. Fifty unique, differentially expressed clones were identified. Therefore, the use of RDA essentially provides an enriched library of differentially expressed genes, while analysis of this library with microarrays allows rapid and reproducible screening of thousands of DNA molecules simultaneously. The coupling of these two techniques in this system resulted in a large pool of differentially expressed genes.