Combined low initial DNA damage and high radiation-induced apoptosis confers clinical resistance to long-term toxicity in breast cancer patients treated with high-dose radiotherapy

BACKGROUND. Either higher levels of initial DNA damage or lower levels of radiation-induced apoptosis in peripheral blood lymphocytes have been associated to increased risk for develop late radiation-induced toxicity. It has been recently published that these two predictive tests are inversely related. The aim of the present study was to investigate the combined role of both tests in relation to clinical radiation-induced toxicity in a set of breast cancer patients treated with high dose hyperfractionated radical radiotherapy. METHODS. Peripheral blood lymphocytes were taken from 26 consecutive patients with locally advanced breast carcinoma treated with high-dose hyperfractioned radical radiotherapy. Acute and late cutaneous and subcutaneous toxicity was evaluated using the Radiation Therapy Oncology Group morbidity scoring schema. The mean follow-up of survivors (n = 13) was 197.23 months. Radiosensitivity of lymphocytes was quantified as the initial number of DNA double-strand breaks induced per Gy and per DNA unit (200 Mbp). Radiation-induced apoptosis (RIA) at 1, 2 and 8 Gy was measured by flow cytometry using annexin V/propidium iodide. RESULTS. Mean DSB/Gy/DNA unit obtained was 1.70 ± 0.83 (range 0.63-4.08; median, 1.46). Radiation-induced apoptosis increased with radiation dose (median 12.36, 17.79 and 24.83 for 1, 2, and 8 Gy respectively). We observed that those "expected resistant patients" (DSB values lower than 1.78 DSB/Gy per 200 Mbp and RIA values over 9.58, 14.40 or 24.83 for 1, 2 and 8 Gy respectively) were at low risk of suffer severe subcutaneous late toxicity (HR 0.223, 95%CI 0.073-0.678, P = 0.008; HR 0.206, 95%CI 0.063-0.677, P = 0.009; HR 0.239, 95%CI 0.062-0.929, P = 0.039, for RIA at 1, 2 and 8 Gy respectively) in multivariate analysis. CONCLUSIONS. A radiation-resistant profile is proposed, where those patients who presented lower levels of initial DNA damage and higher levels of radiation induced apoptosis were at low risk of suffer severe subcutaneous late toxicity after clinical treatment at high radiation doses in our series. However, due to the small sample size, other prospective studies with higher number of patients are needed to validate these results.

Role of gene methylation in antitumor immune response: Implication for tumor progression

Cancer immunosurveillance theory has emphasized the role of escape mechanisms in tumor growth. In this respect, a very important factor is the molecular characterization of the mechanisms by which tumor cells evade immune recognition and destruction. Among the many escape mechanisms identified, alterations in classical and non-classical HLA (Human Leucocyte Antigens) class I and class II expression by tumor cells are of particular interest. In addition to the importance of HLA molecules, tumor-associated antigens and accessory/co-stimulatory molecules are also involved in immune recognition. The loss of HLA class I antigen expression and of co-stimulatory molecules can occur at genetic, transcriptional and post-transcriptional levels. Epigenetic defects are involved in at least some mechanisms that preclude mounting a successful host-antitumor response involving the HLA system, tumor-associated antigens, and accessory/co-stimulatory molecules. This review summarizes our current understanding of the role of methylation in the regulation of molecules involved in the tumor immune response.

Epigenetic regulation of human cancer/testis antigen gene, HAGE, in chronic myeloid leukemia.

BACKGROUND AND OBJECTIVES Cancer testis antigens (CTA) provide attractive targets for cancer-specific immunotherapy. Although CTA genes are expressed in some normal tissues, such as the testis, this immunologically protected site lacks MHC I expression and as such, does not present self antigens to T cells. To date, CTA genes have been shown to be expressed in a range of solid tumors via demethylation of their promoter CpG islands, but rarely in chronic myeloid leukemia (CML) or other hematologic malignancies. DESIGN AND METHODS In this study, the methylation status of the HAGE CTA gene promoter was analyzed by quantitative methylation-specific polymerase chain reaction (MSP) and sequencing in four Philadelphia-positive cell lines (TCC-S, K562, KU812 and KYO-1) and in CML samples taken from patients in chronic phase (CP n=215) or blast crisis (BC n=47). HAGE expression was assessed by quantitative reverse transcriptase-polymerase chain reaction. RESULTS The TCC-S cell line showed demethylation of HAGE that was associated with over-expression of this gene. HAGE hypomethylation was significantly more frequent in BC (46%) than in CP (22%) (p=0.01) and was correlated with high expression levels of HAGE transcripts (p<0.0001). Of note, in CP-CML, extensive HAGE hypomethylation was associated with poorer prognosis in terms of cytogenetic response to interferon (p=0.01) or imatinib (p=0.01), molecular response to imatinib (p=0.003) and progression-free survival (p=0.05). INTERPRETATIONS AND CONCLUSION: The methylation status of the HAGE promoter directly correlates with its expression in both CML cell lines and patients and is associated with advanced disease and poor outcome.

Mutational spectrum of the SPG4 (SPAST) and SPG3A (ATL1) genes in Spanish patients with hereditary spastic paraplegia.

BACKGROUND Hereditary Spastic Paraplegias (HSP) are characterized by progressive spasticity and weakness of the lower limbs. At least 45 loci have been identified in families with autosomal dominant (AD), autosomal recessive (AR), or X-linked hereditary patterns. Mutations in the SPAST (SPG4) and ATL1 (SPG3A) genes would account for about 50% of the ADHSP cases. METHODS We defined the SPAST and ATL1 mutational spectrum in a total of 370 unrelated HSP index cases from Spain (83% with a pure phenotype). RESULTS We found 50 SPAST mutations (including two large deletions) in 54 patients and 7 ATL1 mutations in 11 patients. A total of 33 of the SPAST and 3 of the ATL1 were new mutations. A total of 141 (31%) were familial cases, and we found a higher frequency of mutation carriers among these compared to apparently sporadic cases (38% vs. 5%). Five of the SPAST mutations were predicted to affect the pre-mRNA splicing, and in 4 of them we demonstrated this effect at the cDNA level. In addition to large deletions, splicing, frameshifting, and missense mutations, we also found a nucleotide change in the stop codon that would result in a larger ORF. CONCLUSIONS In a large cohort of Spanish patients with spastic paraplegia, SPAST and ATL1 mutations were found in 15% of the cases. These mutations were more frequent in familial cases (compared to sporadic), and were associated with heterogeneous clinical manifestations.

Polymorphisms in DNA-Repair Genes in a Cohort of Prostate Cancer Patients from Different Areas in Spain: Heterogeneity between Populations as a Confounding Factor in Association Studies.

BACKGROUND Differences in the distribution of genotypes between individuals of the same ethnicity are an important confounder factor commonly undervalued in typical association studies conducted in radiogenomics. OBJECTIVE To evaluate the genotypic distribution of SNPs in a wide set of Spanish prostate cancer patients for determine the homogeneity of the population and to disclose potential bias. DESIGN SETTING AND PARTICIPANTS A total of 601 prostate cancer patients from Andalusia, Basque Country, Canary and Catalonia were genotyped for 10 SNPs located in 6 different genes associated to DNA repair: XRCC1 (rs25487, rs25489, rs1799782), ERCC2 (rs13181), ERCC1 (rs11615), LIG4 (rs1805388, rs1805386), ATM (rs17503908, rs1800057) and P53 (rs1042522). The SNP genotyping was made in a Biotrove OpenArray® NT Cycler. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Comparisons of genotypic and allelic frequencies among populations, as well as haplotype analyses were determined using the web-based environment SNPator. Principal component analysis was made using the SnpMatrix and XSnpMatrix classes and methods implemented as an R package. Non-supervised hierarchical cluster of SNP was made using MultiExperiment Viewer. RESULTS AND LIMITATIONS We observed that genotype distribution of 4 out 10 SNPs was statistically different among the studied populations, showing the greatest differences between Andalusia and Catalonia. These observations were confirmed in cluster analysis, principal component analysis and in the differential distribution of haplotypes among the populations. Because tumor characteristics have not been taken into account, it is possible that some polymorphisms may influence tumor characteristics in the same way that it may pose a risk factor for other disease characteristics. CONCLUSION Differences in distribution of genotypes within different populations of the same ethnicity could be an important confounding factor responsible for the lack of validation of SNPs associated with radiation-induced toxicity, especially when extensive meta-analysis with subjects from different countries are carried out.

Prognostic impact of MGMT promoter methylation and MGMT and CD133 expression in colorectal adenocarcinoma.

BACKGROUND New biomarkers are needed for the prognosis of advanced colorectal cancer, which remains incurable by conventional treatments. O6-methylguanine DNA methyltransferase (MGMT) methylation and protein expression have been related to colorectal cancer treatment failure and tumor progression. Moreover, the presence in these tumors of cancer stem cells, which are characterized by CD133 expression, has been associated with chemoresistance, radioresistance, metastasis, and local recurrence. The objective of this study was to determine the prognostic value of CD133 and MGMT and their possible interaction in colorectal cancer patients. METHODS MGMT and CD133 expression was analyzed by immunohistochemistry in 123 paraffin-embedded colorectal adenocarcinoma samples, obtaining the percentage staining and intensity. MGMT promoter methylation status was obtained by using bisulfite modification and methylation-specific PCR (MSP). These values were correlated with clinical data, including overall survival (OS), disease-free survival (DFS), tumor stage, and differentiation grade. RESULTS Low MGMT expression intensity was significantly correlated with shorter OS and was a prognostic factor independently of treatment and histopathological variables. High percentage of CD133 expression was significantly correlated with shorter DFS but was not an independent factor. Patients with low-intensity MGMT expression and ≥50% CD133 expression had the poorest DFS and OS outcomes. CONCLUSIONS Our results support the hypothesis that MGMT expression may be an OS biomarker as useful as tumor stage or differentiation grade and that CD133 expression may be a predictive biomarker of DFS. Thus, MGMT and CD133 may both be useful for determining the prognosis of colorectal cancer patients and to identify those requiring more aggressive adjuvant therapies. Future studies will be necessary to determine its clinical utility.

Mutation prevalence of cerebral cavernous malformation genes in Spanish patients.

OBJECTIVE To study the molecular genetic and clinical features of cerebral cavernous malformations (CCM) in a cohort of Spanish patients. METHODS We analyzed the CCM1, CCM2, and CCM3 genes by MLPA and direct sequencing of exons and intronic boundaries in 94 familial forms and 41 sporadic cases of CCM patients of Spanish extraction. When available, RNA studies were performed seeking for alternative or cryptic splicing. RESULTS A total of 26 pathogenic mutations, 22 of which predict truncated proteins, were identified in 29 familial forms and in three sporadic cases. The repertoire includes six novel non-sense and frameshift mutations in CCM1 and CCM3. We also found four missense mutations, one of them located at the third NPXY motif of CCM1 and another one that leads to cryptic splicing of CCM1 exon 6. We found four genomic deletions with the loss of the whole CCM2 gene in one patient and a partial loss of CCM1and CCM2 genes in three other patients. Four families had mutations in CCM3. The results include a high frequency of intronic variants, although most of them localize out of consensus splicing sequences. The main symptoms associated to clinical debut consisted of cerebral haemorrhage, migraines and epileptic seizures. The rare co-occurrence of CCM with Noonan and Chiari syndromes and delayed menarche is reported. CONCLUSIONS Analysis of CCM genes by sequencing and MLPA has detected mutations in almost 35% of a Spanish cohort (36% of familial cases and 10% of sporadic patients). The results include 13 new mutations of CCM genes and the main clinical symptoms that deserves consideration in molecular diagnosis and genetic counselling of cerebral cavernous malformations.

Una herramienta SIG para el análisis socioeconómico de la Región Metropolitana de Barcelona

Este proyecto ha sido desarrollado en el Institut d'Estudis Territorials, y se plantea como una solución visual que permita mapificar variables estadísticas de la Región Metropolitana de Barcelona, mediante una aplicación Web dinámica con la que el usuario pueda consultar y representar datos en el territorio a través de una cartografía de soporte.El Institut d’Estudis Territorials, a petición del Departament de Política Territorial i Obres Públiques, es el encargado de elaborar el Pla Territorial Metropolità de Barcelona. Esta aplicación complementa a los estudios socioeconómicos que se están llevando a cabo, y pretende ser una herramienta de gestión que permita caracterizar el territorio, hacer diagnósticos y evaluar las diferentes propuestas presentadas para dicho plan.En una etapa preliminar se desarrolló una aplicación Excel, pero pronto se manifestaron las numerosas carencias que esta sufría, tales como la dificultad para acceder a ella de manera concurrente, no ofrecer garantías en lo que a medidas de seguridad se refiere y no poder tratar los datos de manera centralizada. El archivo Excel debía ser distribuido entre los diferentes usuarios por lo que cualquier cambio en los datos implicaba una replicación del archivo y en consecuencia una nueva distribución. Resultó obvia la necesidad de una única base de datos centralizada, y un acceso distribuido a ésta. Se planteó entonces una aplicación Web como solución, la cual incluiría herramientas capaces de gestionar información gráfica (cartografía) y alfa-numérica (datos) de manera conjunta. Conociendo las facilidades que los Sistemas de Información Geográfica (SIG) ofrecen para el manejo de este tipo de datos y su interoperabilidad con Internet, se optó por construir un servicio de mapas con el que el usuario, de manera intuitiva, pudiese construir distintos mapas en función de la variable territorial que desease.Así mismo se plantean mejoras futuras, como la predicción a corto plazo de la evolución de diferentes variables, siempre ofreciendo los mapas como componente principal de la interfaz de usuario.

Estudio Farmacogenético en Pacientes con Cáncer de Pulmón No Microcítico Avanzado tratados con Quimioterapia basada en Platino

a partir de ADN genómico obtenido de las células nucleadas de sangre periférica de 103 pacientes con Cáncer de Pulmón No Microcítico (CPNM) avanzado tratados con quimioterapia basada en platino, hemos analizado la asociación entre supervivencia y cinco SNPs (Single Nucleotide Polymorphism) pertenecientes a dos grupos de genes: i) de la via metabólica del ácido fólico (Timidilato Sintetasa (TS), Metil-tetrahidrofolato Reductasa (MTHFR) y, ii) de la vía de reparación del ADN (Excision repair cross-complemeting group 1 (ERCC1) y Xeroderma pigmentosum group D (XPD).

Aplicación de la genética en la identificación de enfermedades infecciosas bucodentales

La genética ha supuesto una gran revolución en la identificación de seres vivos a través de análisis de ADN. Actualmente se investiga la pos1bilidad de aplicarla en el estudio de nuestros antepasados, desde el hombre prehistórico. En el ámbito de la odontologfa se vislumbran nuevas perspectivas en el estudio de la patologfa infecciosa, gracias a estos avances en biologfia molecular. El presente trabajo pretende repasar cuál ha sido esta patología infecciosa en el hombre del pasado y cuáles son Jos métodos de análisis genético que penniten estudiarla. Fundamentalmente, las infecciones bucodentales del hombre prehistórico se resumen en caries y patología periodontal. La RCP (reacción en cadena de la polimerasa) es la técnica que ha revolucionadola ingeniería genética, pues permite obtener copias del ADN para poder ser analizado y, con ello, ofrece un diagnóstico específico de la etiología de las enfermedades infecciosas, lográndose una identificación más precisa que con el cultivo o con la inmunohistoquímica de las bacterias, hongos y virus que conviven en el medio oral.

El tabaco y la reproducción masculina

Actualmente se estima la población infértil entre el 10%-15%, y el pronóstico es que vaya en ascenso; además los últimos datos muestran que en el mundo, hoy en día, existen un 47% de hombres fumadores. Por este motivo este trabajo tiene como objetivo determinar cómo afecta el hábito de fumar a la reproducción masculina, concretamente a los parámetros seminales, a las hormonas reproductoras y a la fragmentación del ADN espermático. Además, también hemos querido estudiar si el hábito de fumar provoca mutaciones y aneuploidías en la línea germinal, y si éstas, en caso de existir, pueden afectar a la descendencia.Para realizar esta revisión, la cual integra dos meta-análisis, se han utilizado diferentes bases de datos para poder recoger y estudiar artículos epidemiológicos, clínicos y experimentales. Los resultado obtenidos, a partir de un total de 28 estudios, muestran que existe una reducción porcentual media en la concentración de espermatozoides del 12,4% en los fumadores respecto a los no fumadores, en la movilidad espermática la reducción es del 10.3% y en la morfología del 13.6%. También se ha determinado que los fumadores presentan todas las hormonas reproductoras estudiadas incrementadas y que tienen un mayor porcentaje de fragmentación del ADN espermático. Además, mostramos que el tabaco causa aneuploidías y mutaciones en las células germinales, pudiendo causar enfermedades genéticas en la descendencia, como el cáncer.Concluimos que el hecho de no fumar o de dejar de fumar puede proporcionar beneficios en la fertilidad masculina y en la descendencia.

La resolución de casos abierto, exoneraciones y análisis familiares por medio de la genética avanzada. Aspectos forenses, sociales y eticos.

La especialidad de la Genética forense tiene algo más de un siglo, pero con la incorporación de la denominada prueba del ADN hace casi tres décadas, se ha logrado una eficacia que ha revolucionado no solo la investigación policial y las sentencias jurídicas, si no que ha impactado positivamente a toda la sociedad moderna. En este trabajo se analiza el avance que han supuesto las técnicas de identificación a través del ADN, en situaciones legales que en su momento no fueron resueltas (casos abiertos), en las exoneraciones y en los estudios familiares. Las aplicaciones y consecuencias de la Genética forense molecular o del ADN en estos casos, no solo ha dotado de más prestigio y seguridad a la Administración de la Justicia, si no que ha tenido un notable impacto social y ético. Es nuestra convicción, que tanto los profesionales de la Administración de la Justicia, como la sociedad en su totalidad debemos congratularnos y contribuir de la mejor manera posible a que las herramientas forenses en general y las vinculadas a la genética avanzada, se implementen al máximo nivel lo antes y mejor posible en nuestras instituciones.

Ciencia Interactiva.

Resumen basado en la publicación

Cèl.lula i genoma. 'Célula y genoma'.

Resumen tomado de la publicación

Functional studies of the deubiquitinating enzymes USP19, USP4 and UCH-L1

El marcaje de proteínas con ubiquitina, conocido como ubiquitinación, cumple diferentes funciones que incluyen la regulación de varios procesos celulares, tales como: la degradación de proteínas por medio del proteosoma, la reparación del ADN, la señalización mediada por receptores de membrana, y la endocitosis, entre otras (1). Las moléculas de ubiquitina pueden ser removidas de sus sustratos gracias a la acción de un gran grupo de proteasas, llamadas enzimas deubiquitinizantes (DUBs) (2). Las DUBs son esenciales para la manutención de la homeostasis de la ubiquitina y para la regulación del estado de ubiquitinación de diferentes sustratos. El gran número y la diversidad de DUBs descritas refleja tanto su especificidad como su utilización para regular un amplio espectro de sustratos y vías celulares. Aunque muchas DUBs han sido estudiadas a profundidad, actualmente se desconocen los sustratos y las funciones biológicas de la mayoría de ellas. En este trabajo se investigaron las funciones de las DUBs: USP19, USP4 y UCH-L1. Utilizando varias técnicas de biología molecular y celular se encontró que: i) USP19 es regulada por las ubiquitin ligasas SIAH1 y SIAH2 ii) USP19 es importante para regular HIF-1α, un factor de transcripción clave en la respuesta celular a hipoxia, iii) USP4 interactúa con el proteosoma, iv) La quimera mCherry-UCH-L1 reproduce parcialmente los fenotipos que nuestro grupo ha descrito previamente al usar otros constructos de la misma enzima, y v) UCH-L1 promueve la internalización de la bacteria Yersinia pseudotuberculosis.