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1.
Blood ; 120(11): 2280-9, 2012 Sep 13.
Artículo en Inglés | MEDLINE | ID: mdl-22855598

RESUMEN

Peripheral T-cell lymphomas (PTCLs) are aggressive malignancies of mature T lymphocytes with 5-year overall survival rates of only ∼ 35%. Improvement in outcomes has been stymied by poor understanding of the genetics and molecular pathogenesis of PTCL, with a resulting paucity of molecular targets for therapy. We developed bioinformatic tools to identify chromosomal rearrangements using genome-wide, next-generation sequencing analysis of mate-pair DNA libraries and applied these tools to 16 PTCL patient tissue samples and 6 PTCL cell lines. Thirteen recurrent abnormalities were identified, of which 5 involved p53-related genes (TP53, TP63, CDKN2A, WWOX, and ANKRD11). Among these abnormalities were novel TP63 rearrangements encoding fusion proteins homologous to ΔNp63, a dominant-negative p63 isoform that inhibits the p53 pathway. TP63 rearrangements were seen in 11 (5.8%) of 190 PTCLs and were associated with inferior overall survival; they also were detected in 2 (1.2%) of 164 diffuse large B-cell lymphomas. As TP53 mutations are rare in PTCL compared with other malignancies, our findings suggest that a constellation of alternate genetic abnormalities may contribute to disruption of p53-associated tumor suppressor function in PTCL.


Asunto(s)
Reordenamiento Génico , Linfoma de Células T Periférico/genética , Mutación , Factores de Transcripción/genética , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genética , Línea Celular Tumoral , Inhibidor p16 de la Quinasa Dependiente de Ciclina/química , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Análisis Mutacional de ADN , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/mortalidad , Linfoma de Células B Grandes Difuso/patología , Linfoma de Células T Periférico/metabolismo , Linfoma de Células T Periférico/mortalidad , Linfoma de Células T Periférico/patología , Masculino , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas de Fusión Oncogénica/química , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Oxidorreductasas/química , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Homología de Secuencia de Ácido Nucleico , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/metabolismo , Estados Unidos , Oxidorreductasa que Contiene Dominios WW
2.
Nature ; 447(7143): 447-52, 2007 May 24.
Artículo en Inglés | MEDLINE | ID: mdl-17450122

RESUMEN

Although oxidative damage has long been associated with ageing and neurological disease, mechanistic connections of oxidation to these phenotypes have remained elusive. Here we show that the age-dependent somatic mutation associated with Huntington's disease occurs in the process of removing oxidized base lesions, and is remarkably dependent on a single base excision repair enzyme, 7,8-dihydro-8-oxoguanine-DNA glycosylase (OGG1). Both in vivo and in vitro results support a 'toxic oxidation' model in which OGG1 initiates an escalating oxidation-excision cycle that leads to progressive age-dependent expansion. Age-dependent CAG expansion provides a direct molecular link between oxidative damage and toxicity in post-mitotic neurons through a DNA damage response, and error-prone repair of single-strand breaks.


Asunto(s)
Envejecimiento/genética , ADN Glicosilasas/metabolismo , Neuronas/metabolismo , Expansión de Repetición de Trinucleótido/genética , Animales , Línea Celular , Roturas del ADN de Cadena Simple , Daño del ADN , ADN Glicosilasas/deficiencia , ADN Glicosilasas/genética , Reparación del ADN/genética , Femenino , Guanosina/análogos & derivados , Guanosina/metabolismo , Humanos , Enfermedad de Huntington/genética , Masculino , Ratones , Modelos Genéticos , Oxidación-Reducción
3.
Carcinogenesis ; 32(7): 1085-92, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21551128

RESUMEN

Loss of E-cadherin expression is a critical step in the development and progression of gynecological tumors. Study of the precise role of E-cadherin has been hampered by the lack of satisfactory mouse model for E-cadherin deficiency. Likewise, DNA mismatch repair (MMR) is implicated in gynecological tumorigenesis, but knockout of MMR in mice predominantly causes hematologic neoplasms. Here, we show that combined disruption of E-cadherin and DNA MMR pathways increases incidence of endometrioid tumors in mice. Twenty percent of mice knockout for Msh2 enzyme and hemizygous for E-cadherin [Msh2(-/-)/Cdh1(+/-)] developed endometrioid-like tumors in the ovary, uterus and genital area. Characteristic of these tumors was a complete loss of E-cadherin expression. Sequence analysis of E-cadherin promoter region demonstrated that the loss of E-cadherin expression is caused by inactivating mutations, implying that E-cadherin is a mutational target in Msh2-deficient mice. In addition, Msh2(-/-)/Cdh1(+/-) mice showed a reduction in overall survival as compared with their Msh2(-/-) counterparts due to the development of more aggressive lymphomas, suggesting a specific role of E-cadherin in lymphomagenesis. In conclusion, Msh2(-/-)/Cdh1(+/-) mice provide a good model of gynecological tumorigenesis and may be useful for testing molecular target-specific therapies.


Asunto(s)
Disparidad de Par Base , Cadherinas/genética , Neoplasias Endometriales/epidemiología , Regiones Promotoras Genéticas , Animales , Secuencia de Bases , Western Blotting , Cartilla de ADN , Neoplasias Endometriales/genética , Femenino , Incidencia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/fisiología
4.
J Vis Exp ; (161)2020 07 25.
Artículo en Inglés | MEDLINE | ID: mdl-32773766

RESUMEN

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic target analyses and drug response monitoring using patient derived xenografts (PDX). This strategy was validated using ovarian tumors as an example. The mate-pair next generation sequencing (MPseq) protocol was used to identify structural alterations and followed by analysis of potentially targetable alterations. Human tumors grown in immunocompromised mice were treated with drugs selected based on the genomic analyses. Results demonstrated a good correlation between the predicted and the observed responses in the PDX model. The presented approach can be used to test the efficacy of combination treatments and aid personalized treatment for patients with recurrent cancer, specifically in cases when standard therapy fails and there is a need to use drugs off label.


Asunto(s)
Análisis Mutacional de ADN , Neoplasias Ováricas/tratamiento farmacológico , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , ADN , Modelos Animales de Enfermedad , Femenino , Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Mutación , Recurrencia Local de Neoplasia
5.
Mol Oncol ; 13(2): 132-152, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30499260

RESUMEN

Ovarian cancer is the most lethal gynecologic malignancy. About 75% of ovarian cancer patients relapse and/or develop chemo-resistant disease after initial response to standard-of-care treatment with platinum-based therapies. HER2 amplifications and overexpression in ovarian cancer are reported to vary, and responses to HER2 inhibitors have been poor. Next generation sequencing technologies in conjunction with testing using patient-derived xenografts (PDX) allow validation of personalized treatments. Using a whole-genome mate-pair next generation sequencing (MPseq) protocol, we identified several high grade serous ovarian cancers (HGS-OC) with DNA alterations in genes encoding members of the ERBB2 pathway. The efficiency of anti-HER2 therapy was tested in three different PDX lines with the identified alterations and high levels of HER2 protein expression. Treatment responses to pertuzumab or pertuzumab/trastuzumab were compared in each PDX line WITH standard carboplatin and paclitaxel combination treatment. In all three PDX models, HER2-targeted therapy resulted in significant inhibition of tumor growth compared with untreated controls. However, the responses in each case were inferior to those to chemotherapy, even for chemo-resistant lines. When chemotherapy and HER2-targeted therapy were administered together, a significant regression of tumor was observed after 6 weeks of treatment compared with chemotherapy alone. Post-treatment analysis of these tissues revealed that inhibition of the ERBB2 pathway occurred at the level of phosphorylation and expression of downstream targets. In conclusion, while targeting of presumably activated ERBB2 pathway alone in HGS-OC results in a modest treatment benefit, a combination therapy including both chemotherapy drugs and HER2 inhibitors provides a far better response. Further studies are needed to address development of recurrence and sensitivity of recurrent disease to HER2-targeted therapy.


Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias Ováricas/tratamiento farmacológico , Receptor ErbB-2/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Femenino , Humanos , Ratones SCID , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Resultado del Tratamiento
6.
Mayo Clin Proc ; 94(1): 27-36, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30611450

RESUMEN

OBJECTIVE: To test the hypothesis that chromosomal rearrangements (CRs) can distinguish low risk of progression (LRP) from intermediate and high risk of progression (IHRP) to prostate cancer (PCa) and if these CRs have the potential to identify men with LRP on needle biopsy that harbor IHRP PCa in the prostate gland. PATIENTS AND METHODS: Mate pair sequencing of amplified DNA from pure populations of Gleason patterns in 154 frozen specimens from 126 patients obtained between August 14, 2001, and July 15, 2011, was used to detect CRs including abnormal junctions and copy number variations. Potential CR biomarkers with higher incidence in IHRP than in LRP to cancer and having significance in PCa biology were identified. Independent validation was performed by fluorescence in situ hybridization in 152 specimens from 124 patients obtained between February 12, 2002, and July 12, 2008. RESULTS: The number of abnormal junctions did not distinguish LRP from IHRP. Loci corresponding to genes implicated in PCa were more frequently altered in IHRP. Integrated analysis of copy number variations and microarray data yielded 6 potential markers that were more frequently detected in Gleason pattern 3 of a Gleason score 7 of PCa than in Gleason pattern 3 of a Gleason score 6 PCa. Five of those were cross-validated in an independent sample set with statistically significant areas under the receiver operating characteristic curves (AUCs) (P≤.01). Probes detecting deletions in PTEN and CHD1 had AUCs of 0.87 (95% CI, 0.77-0.97) and 0.73 (95% CI, 0.60-0.86), respectively, and probes detecting gains in ASAP1, MYC, and HDAC9 had AUCs of 0.71 (95% CI, 0.59-0.84), 0.82 (95% CI, 0.71-0.93), and 0.77 (95% CI, 0.66-0.89), respectively (for expansion of gene symbols, use search tool at www.genenames.org). CONCLUSION: Copy number variations in regions encompassing important PCa genes were predictive of cancer significance and have the potential to identify men with LRP PCa by needle biopsy who have IHRP PCa in their prostate gland.


Asunto(s)
Biomarcadores de Tumor/genética , ADN de Neoplasias/genética , Estadificación de Neoplasias , Próstata/patología , Neoplasias de la Próstata/genética , Anciano , Biomarcadores de Tumor/metabolismo , Biopsia con Aguja , Variaciones en el Número de Copia de ADN , Progresión de la Enfermedad , Estudios de Seguimiento , Humanos , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana Edad , Próstata/metabolismo , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/metabolismo , Curva ROC , Estudios Retrospectivos , Factores de Riesgo
7.
DNA Repair (Amst) ; 6(4): 517-29, 2007 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-17129768

RESUMEN

While a role for DNA glycosylase activity in base excision repair (BER) is well understood, there is mounting evidence to implicate cooperation of DNA glycosylases with components of repair pathways other than BER. The mechanisms by which DNA glycosylases interact with non-BER pathways are, in many cases, poorly understood. However, accumulating evidence indicates that crosstalk is common and may be as important in signaling repair as the canonical pathways themselves.


Asunto(s)
Daño del ADN , ADN Glicosilasas/metabolismo , Reparación de la Incompatibilidad de ADN , Enzimas Reparadoras del ADN/metabolismo , Animales , Reparación del ADN , Ratones , Nucleótidos/metabolismo , Oxidación-Reducción , Transcripción Genética
8.
J Cancer Educ ; 23(4): 222-5, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-19058069

RESUMEN

BACKGROUND: Interacting with patients, researchers, and administrators, patient advocates have a unique vantage point. Yet, few prior studies have sought to understand these individuals or to seek their opinions on cancer issues. METHODS: A survey to address the foregoing was developed and mailed to advocates within the National Cancer Institute's Prostate Cancer SPORE Program. RESULTS: A total of 10 of 19 advocates responded. All were men, most were retired, and all had faced a diagnosis of prostate cancer. Two major themes emerged: (1) the importance of patient education in promoting informed clinical decision-making and (2) a perceived need for patient-centered research by providers and educators. CONCLUSIONS: Prostate cancer patient advocates provide a broad range of opinions on the spectrum of cancer care. Similar studies among advocates involved in other malignancies may be worthwhile.


Asunto(s)
Investigación Biomédica/organización & administración , Conocimientos, Actitudes y Práctica en Salud , Defensa del Paciente , Neoplasias de la Próstata , Anciano , Toma de Decisiones , Personal de Salud , Humanos , Masculino , Persona de Mediana Edad , National Cancer Institute (U.S.) , Educación del Paciente como Asunto , Proyectos Piloto , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/terapia , Encuestas y Cuestionarios , Estados Unidos
9.
Oncotarget ; 9(36): 24428-24442, 2018 May 11.
Artículo en Inglés | MEDLINE | ID: mdl-29849951

RESUMEN

The mechanism of prostate cancer (PCa) progression towards the hormone refractory state remains poorly understood. Treatment options for such patients are limited and present a major clinical challenge. Previously, δ-catenin was reported to promote PCa cell growth in vitro and its increased level is associated with PCa progression in vivo. In this study we show that re-arrangements at Catenin Delta 2 (CTNND2) locus, including gene duplications, are very common in clinically significant PCa and may underlie δ-catenin overexpression. We find that δ-catenin in PCa cells exists in a complex with E-cadherin, p120, and α- and ß-catenin. Increased expression of δ-catenin leads to its further stabilization as well as upregulation and stabilization of its binding partners. Resistant to degradation and overexpressed δ-catenin isoform activates Wnt signaling pathway by increasing the level of nuclear ß-catenin and subsequent stimulation of Tcf/Lef transcription targets. Evaluation of responses to treatments, with androgen receptor (AR) antagonist and ß-catenin inhibitors revealed that cells with high levels of δ-catenin are more resistant to killing with single agent treatment than matched control cells. We show that combination treatment targeting both AR and ß-catenin networks is more effective in suppressing tumor growth than targeting a single network. In conclusion, targeting clinically significant PCa with high levels of δ-catenin with anti-androgen and anti ß-catenin combination therapy may prevent progression of the disease to a castration-resistant state and, thus, represents a promising therapeutic strategy.

10.
Oncotarget ; 8(16): 27155-27165, 2017 Apr 18.
Artículo en Inglés | MEDLINE | ID: mdl-28460442

RESUMEN

Achaete-scute homolog 1 (ASCL1) is a neuroendocrine transcription factor specifically expressed in 10-20% of lung adenocarcinomas (AD) with neuroendocrine (NE) differentiation (NED). ASCL1 functions as an upstream regulator of the RET oncogene in AD with high ASCL1 expression (A+AD). RET is a receptor tyrosine kinase with two main human isoforms; RET9 (short) and RET51 (long). We found that elevated expression of RET51 associated mRNA was highly predictive of poor survival in stage-1 A+AD (p=0.0057). Functional studies highlighted the role of RET in promoting invasive properties of A+AD cells. Further, A+AD cells demonstrated close to 10 fold more sensitivity to epidermal growth factor receptor (EGFR) inhibitors, including gefitinib, than AD cells with low ASCL1 expression. Treatment with EGF robustly induced phosphorylation of RET at Tyr-905 in A+AD cells with wild type EGFR. This phosphorylation was blocked by gefitinib and by siRNA-EGFR. Immunoprecipitation experiments found EGFR in a complex with RET in the presence of EGF and suggested that RET51 was the predominant RET isoform in the complex. In the microarray datasets of stage-1 and all stages of A+AD, high levels of EGFR and RET RNA were significantly associated with poor overall survival (p < 0.01 in both analyses). These results implicate EGFR as a key regulator of RET activation in A+AD and suggest that EGFR inhibitors may be therapeutic in patients with A+AD tumors even in the absence of an EGFR or RET mutation.


Asunto(s)
Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Carcinoma Neuroendocrino/genética , Carcinoma Neuroendocrino/metabolismo , Receptores ErbB/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteínas Proto-Oncogénicas c-ret/genética , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Empalme Alternativo , Carcinoma Neuroendocrino/mortalidad , Carcinoma Neuroendocrino/patología , Ciclo Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Receptores ErbB/antagonistas & inhibidores , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Humanos , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Clasificación del Tumor , Fosforilación , Pronóstico , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/genética , ARN Interferente Pequeño/genética
11.
Sci Rep ; 6: 29831, 2016 07 20.
Artículo en Inglés | MEDLINE | ID: mdl-27436510

RESUMEN

Recently, the use of a liquid biopsy has shown promise in monitoring tumor burden. While point mutations have been extensively studied, chromosomal rearrangements have demonstrated greater tumor specificity. Such rearrangements can be identified in the tumor and subsequently detected in the plasma of patients using quantitative PCR (qPCR). In this study we used a whole-genome mate-pair protocol to characterize a landscape of genomic rearrangements in the primary tumors of ten ovarian cancer patients. Individualized tumor-specific primer panels of aberrant chromosomal junctions were identified for each case and detected by qPCR within the cell-free DNA. Selected chromosomal junctions were detected in pre-surgically drawn blood in eight of the ten patients. Of these eight, three demonstrated the continued presence of circulating tumor DNA (ctDNA) post-surgery, consistent with their documented presence of disease, and in five ctDNA was undetectable in the post-surgical blood collection, consistent with their lack of detectable disease. The ctDNA fraction was calculated using a novel algorithm designed for the unique challenges of quantifying ctDNA using qPCR to allow observations of real-time tumor dynamics. In summary, a panel of individualized junctions derived from tumor DNA could be an effective way to monitor cancer patients for relapse and therapeutic efficacy using cfDNA.


Asunto(s)
Ácidos Nucleicos Libres de Células/análisis , Aberraciones Cromosómicas , ADN de Neoplasias/análisis , Neoplasias Ováricas/genética , Ácidos Nucleicos Libres de Células/sangre , Ácidos Nucleicos Libres de Células/genética , ADN de Neoplasias/sangre , ADN de Neoplasias/genética , Femenino , Humanos , Recurrencia Local de Neoplasia/diagnóstico , Recurrencia Local de Neoplasia/genética , Evaluación de Resultado en la Atención de Salud/métodos , Neoplasias Ováricas/sangre , Neoplasias Ováricas/cirugía , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Carga Tumoral/genética
12.
PLoS One ; 10(11): e0142327, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26560244

RESUMEN

Overexpression of TOP2A is associated with risk of systemic progression in prostate cancer patients, and higher levels of TOP2A were found in hormone-resistant cases. To elucidate the mechanism by which high levels of TOP2A contribute to tumor progression we generated TOP2A overexpressing prostate cancer cell lines. We show that TOP2A promotes tumor aggressiveness by inducing chromosomal rearrangements of genes that contribute to a more invasive phenotype. Anti-androgen treatment alone was ineffective in killing TOP2A overexpressing cells due to activation of an androgen receptor network. TOP2A poisons killed tumor cells more efficiently early in the progression course, while at later stages they provided greater benefit when combined with anti-androgen therapy. Mechanistically, we find that TOP2A enhances androgen signaling by facilitating transcription of androgen responsive genes, thereby promoting tumor cell growth. These studies revealed a relationship between TOP2A and androgen receptor signaling pathway that contributes to prostate cancer progression and confers sensitivity to treatments.


Asunto(s)
Adenocarcinoma/metabolismo , Antígenos de Neoplasias/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Invasividad Neoplásica/patología , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/metabolismo , Adenocarcinoma/patología , Línea Celular Tumoral , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Proteínas de Unión a Poli-ADP-Ribosa , Neoplasias de la Próstata/patología , Transducción de Señal/fisiología
13.
Oncotarget ; 6(30): 29087-96, 2015 Oct 06.
Artículo en Inglés | MEDLINE | ID: mdl-26337081

RESUMEN

Massive genomic rearrangements, a result of single catastrophic event termed chromothrispsis or chromosomal catastrophe, have been identified in a variety of human cancers. In a few cancer types, chromothripsis was found to be associated with poor prognosis. We performed mate-pair sequencing and analysis of structural rearrangements in 132 prostate cancer cases which included clinically insignificant Gleason score 6 tumors, clinically significant tumors of higher grade (7+) and high grade prostatic intraepithelial neoplasia. Chromothripsis was observed at least 30 per cent of the samples across different grades. Surprisingly, it was frequently observed in clinically insignificant Gleason score 6 tumors, indicating that chromothripsis does not define more aggressive phenotype. The degree of chromothripsis did not increase significantly in tumors of advanced grades and did not appear to contribute to tumor progression. Our data showed that distribution of chromothriptic rearrangements differed from that of fragile sites but correlated with the size of chromosomes. We also provided evidence that rearrangements resulting from chromothripsis were present in the cells of neighboring Gleason patterns of the same tumor. Our data suggest that that chromothripsis plays role in prostate cancer initiation.


Asunto(s)
Biomarcadores de Tumor/genética , Aberraciones Cromosómicas , Cromosomas Humanos/genética , Reordenamiento Génico , Neoplasias de la Próstata/genética , Sitios Frágiles del Cromosoma , Variaciones en el Número de Copia de ADN , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Masculino , Clasificación del Tumor , Fenotipo , Neoplasias de la Próstata/patología , Transactivadores/genética , Regulador Transcripcional ERG
14.
Methods Mol Biol ; 277: 309-19, 2004.
Artículo en Inglés | MEDLINE | ID: mdl-15201465

RESUMEN

This chapter describes methods for the isolation of specific cell types that reveal how and where expansion can occur. For the hereditary component of expansion, the male germ cell has proved useful in distinguishing processes that can contribute to expansion, as described in our article (Nature Genetics 27, 407, 2001). Mature spermatazoa (SZs) can be isolated directly from the epididymis. Haploid spermatids (STs), diploid spermatagonia (SGs), and tetraploid spermatocytes (SCs) can be removed from the testis and sorted by fluorescence-activity cell sorting (FACS); differences in DNA content and morphology allow resolution by fluorescence and light scattering. Repeat-length measurement can pinpoint the stage at which expansion occurs. Because the timing of meiosis and mitosis with respect to sperm development is known, the analysis can distinguish repair and replication processes. Furthermore, the possible contribution of Y- or X-specific factors can be evaluated by sorting X- and Y-bearing germ cells. To enable analysis of female germ cells, we describe methods for oocyte preparations and a method for the isolation of the eight-cell-stage embryo. Therefore, the methods described here can help to answer such questions as the timing during development of expansion, whether expansion is limited to a single period, whether both replication and repair contribute to instability, and the role of somatic instability in disease. If further expansion of the inherited allele contributes to the phenotype, then intervention in somatic tissue might be therapeutic.


Asunto(s)
Reparación del ADN/genética , Repeticiones de Trinucleótidos , Animales , Femenino , Masculino , Ratones , Ratones Transgénicos , Espermatozoides/citología
15.
Cancer Res ; 73(11): 3275-84, 2013 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-23695551

RESUMEN

Gleason score 7 (GS7) prostate cancer [tumors with both Gleason patterns 3 (GP3) and 4 (GP4)] portends a significantly more aggressive tumor than Gleason score 6 (GS6). It is, therefore, critical to understand the molecular relationship of adjacent GP3 and GP4 tumor cell populations and relate molecular abnormalities to disease progression. To decipher molecular relatedness, we used laser capture microdissection (LCM) and whole-genome amplification (WGA) to separately collect and amplify DNA from adjacent GP3 and GP4 cell populations from 14 cases of GS7 prostate cancer. We then carried out massively parallel mate-pair next generation sequencing (NGS) to examine the landscape of large chromosomal alterations. We identified four to 115 DNA breakpoints in GP3 and 17 to 480 in GP4. Our findings indicate that while GP3 and GP4 from the same tumor each possess unique breakpoints, they also share identical ones, indicating a common origin. Approximately 300 chromosomal breakpoints were localized to the regions affected in at least two tumors, whereas more than 3,000 were unique within the set of 14 tumors. TMPRSS2-ERG was the most recurrent rearrangement present in eight cases, in both GP3 and GP4. PTEN rearrangements were found in five of eight TMPRSS2-ERG fusion-positive cases in both GP3 and GP4. Hierarchical clustering analysis revealed that GP3 has greater breakpoint similarity to its partner GP4 compared with GP3 from different patients. We show evidence that LCM, WGA, and NGS of adjacent tumor regions provide an important tool in deciphering lineage relationships and discovering chromosomal alterations associated with tumor progression.


Asunto(s)
Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Linaje de la Célula , Puntos de Rotura del Cromosoma , ADN de Neoplasias/genética , Progresión de la Enfermedad , Reordenamiento Génico , Estudio de Asociación del Genoma Completo , Humanos , Hibridación Fluorescente in Situ/métodos , Captura por Microdisección con Láser , Masculino , Clasificación del Tumor
16.
Aging (Albany NY) ; 3(5): 509-14, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21566259

RESUMEN

OGG1 and MSH2/MSH3 promote CAG repeat expansion at Huntington's disease (HD) locusin vivo during removal of oxidized bases from DNA. CSB, a transcription-coupled repair (TCR) protein, facilitates repair of some of the same oxidative lesions. In vitro, a knock down CSB results in a reduction of transcription-induced deletions at CAG repeat tract. To test the role of CSB in vivo, we measured intergenerational and somatic expansion of CAG tracts in HD mice lacking CSB, OGG1, or both. We provide evidence that CSB protects CAG repeats from expansion by either active reduction of the tract length during parent-child transmission, or by antagonizing the action of OGG1, which tends to promote expansion in somatic cells. These results raise a possibility that actions of transcription-coupled and base excision repair pathways lead to different outcomes at CAG tracts in vivo.


Asunto(s)
ADN Helicasas/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Expansión de Repetición de Trinucleótido , Animales , ADN Glicosilasas/genética , ADN Glicosilasas/metabolismo , ADN Helicasas/genética , Reparación del ADN , Enzimas Reparadoras del ADN/genética , Femenino , Humanos , Enfermedad de Huntington/genética , Enfermedad de Huntington/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas de Unión a Poli-ADP-Ribosa
17.
Cell Res ; 18(1): 198-213, 2008 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-18166978

RESUMEN

Unstable repeats are associated with various types of cancer and have been implicated in more than 40 neurodegenerative disorders. Trinucleotide repeats are located in non-coding and coding regions of the genome. Studies of bacteria, yeast, mice and man have helped to unravel some features of the mechanism of trinucleotide expansion. Looped DNA structures comprising trinucleotide repeats are processed during replication and/or repair to generate deletions or expansions. Most in vivo data are consistent with a model in which expansion and deletion occur by different mechanisms. In mammals, microsatellite instability is complex and appears to be influenced by genetic, epigenetic and developmental factors.


Asunto(s)
Inestabilidad de Microsatélites , Repeticiones de Trinucleótidos/genética , Repeticiones de Trinucleótidos/fisiología , Animales , Secuencia de Bases , Cromatina/fisiología , Frecuencia de los Genes , Enfermedades Genéticas Congénitas/epidemiología , Enfermedades Genéticas Congénitas/genética , Humanos , Incidencia , Modelos Biológicos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Elementos Reguladores de la Transcripción/fisiología
18.
J Clin Oncol ; 26(24): 3930-6, 2008 Aug 20.
Artículo en Inglés | MEDLINE | ID: mdl-18711181

RESUMEN

PURPOSE: In men who are at high-risk of prostate cancer, progression and death from cancer after radical retropubic prostatectomy (RRP), limited prognostic information is provided by established prognostic features. The objective of this study was to develop a model predictive of outcome in this group of patients. METHODS: Candidate genes were identified from microarray expression data from 102 laser capture microdissected prostate tissue samples. Candidates were overexpressed in tumor compared with normal prostate and more frequently in Gleason patterns 4 and 5 than in 3. A case control study of 157 high-risk patients, matched on Gleason score and stage with systemic progression or death of prostate cancer as the end point, was used to evaluate the expression of candidate genes and build a multivariate model. Tumor was collected from the highest Gleason score in paraffin-embedded blocks and the gene expression was quantified by real-time reverse transcription polymerase chain reaction. Validation of the final model was performed on a separate case-control study of 57 high-risk patients who underwent RRP. RESULTS: A model incorporating gene expression of topoisomerase-2a, cadherin-10, the fusion status based on ERG, ETV1, and ETV4 expression, and the aneuploidy status resulted in a 0.81 area under the curve (AUC) in receiver operating characteristic statistical analysis for the identification of men with systemic progression and death from high grade prostate cancer. The AUC was 0.79 in the independent validation study. CONCLUSION: The model can identify men with high-risk prostate cancer who may benefit from more intensive postoperative follow-up and adjuvant therapies.


Asunto(s)
Modelos Estadísticos , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/cirugía , Estudios de Casos y Controles , Estudios de Cohortes , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Valor Predictivo de las Pruebas , Prostatectomía , Neoplasias de la Próstata/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Resultado del Tratamiento
19.
J Biol Chem ; 279(10): 9389-91, 2004 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-14688268

RESUMEN

The size of the CAG tract at the Huntington's disease (HD) locus upon transmission depends on the gender of the parent. However, the basis for the parent-of-origin effect is unknown. To test whether expansion and contraction in HD are "imprinted" in the germ cells, we isolated the X- and Y-bearing sperm of HD transgenic mice. Here we show that CAG repeat distributions in the X- and Y-bearing spermatozoa of founding fathers do not differ. These data show that gender-dependent changes in CAG repeat length arise in the embryo.


Asunto(s)
Enfermedad de Huntington/genética , Cromosoma X , Cromosoma Y , Animales , Embrión de Mamíferos/fisiología , Humanos , Enfermedad de Huntington/embriología , Enfermedad de Huntington/etiología , Masculino , Ratones , Ratones Transgénicos , Secuencias Repetitivas de Ácidos Nucleicos , Factores Sexuales , Espermatozoides/fisiología , Repeticiones de Trinucleótidos
20.
Proc Natl Acad Sci U S A ; 100(21): 12171-6, 2003 Oct 14.
Artículo en Inglés | MEDLINE | ID: mdl-14527999

RESUMEN

There has been a longstanding debate regarding the role of proteolysis in Huntington's disease. The toxic peptide theory posits that N-terminal cleavage fragments of mutant Huntington's disease protein [mutant huntingtin (mhtt)] enter the nucleus to cause transcriptional dysfunction. However, recent data suggest a second model in which proteolysis of full-length mhtt is inhibited. Importantly, the two competing theories differ with respect to subcellular distribution of mhtt at initiation of toxicity: nuclear if cleaved and cytoplasmic in the absence of cleavage. Using quantitative single-cell analysis and time-lapse imaging, we show here that transcriptional dysfunction is "downstream" of cytoplasmic dysfunction. Primary and reversible toxic events involve destabilization of microtubules mediated by full-length mhtt before cleavage. Restoration of microtubule structure by taxol inhibits nuclear entry and increases cell survival.


Asunto(s)
Enfermedad de Huntington/etiología , Enfermedad de Huntington/metabolismo , Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Muerte Celular , Núcleo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citoplasma/metabolismo , Humanos , Proteína Huntingtina , Enfermedad de Huntington/genética , Microtúbulos/efectos de los fármacos , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/toxicidad , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Proteínas Nucleares/genética , Proteínas Nucleares/toxicidad , Paclitaxel/farmacología
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