Gene-Targeted DNA Methylation: Towards Long-Lasting Reprogramming of Gene Expression?

DNA methylation is an essential epigenetic mark, strongly associated with gene expression regulation. Aberrant DNA methylation patterns underlie various diseases and efforts to intervene with DNA methylation signatures are of great clinical interest. Technological developments to target writers or erasers of DNA methylation to specific genomic loci by epigenetic editing resulted in successful gene expression modulation, also in in vivo models. Application of epigenetic editing in human health could have a huge impact, but clinical translation is still challenging. Despite successes for a wide variety of genes, not all genes mitotically maintain their (de)methylation signatures after editing, and reprogramming requires further understanding of chromatin context-dependency. In addition, difficulties of current delivery systems and off-target effects are hurdles to be tackled. The present review describes findings towards effective and sustained DNA (de)methylation by epigenetic editing and discusses the need for multi-effector approaches to achieve highly efficient long-lasting reprogramming.

Antiproliferative Effects of Epigenetic Modifier Drugs Through E-cadherin Up-regulation in Liver Cancer Cell Lines.

INTRODUCTION AND AIM: Epigenetic alterations play an essential role in cancer onset and progression, thus studies of drugs targeting the epigenetic machinery are a principal concern for cancer treatment. Here, we evaluated the potential of the combination of the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5aza-dC) and the pan-deacetylase inhibitor Trichostatin A (TSA), at low cytotoxic concentrations, to modulate the canonical Wnt/ß-catenin pathway in liver cancer cells. MATERIAL AND METHODS: Pyrosequencing was used for DNA methylation analyses of LINE-1 sequences and the Wnt/ß-catenin pathway antagonist DKK3, SFRP1, WIF1 and CDH1. qRT-PCR was employed to verify the expression of the antagonist. Pathway regulation were evaluated looking at the expression of ß-catenin and E-cadherin by confocal microscopy and the antitumoral effects of the drugs was studied by wound healing and clonogenic assays. RESULTS: Our result suggest that 5aza-dC and TSA treatments were enough to induce a significant expression of the pathway antagonists, decrease of ß-catenin protein levels, re-localization of the protein to the plasma membrane, and pathway transcriptional activity reduction. These important effects exerted an antitumoral outcome shown by the reduction of the migration and clonogenic capabilities of the cells. CONCLUSION: We were able to demonstrate Wnt/ ß-catenin pathway modulation through E-cadherin up-regulation induced by 5aza-dC and TSA treatments, under an activation-pathway background, like CTNNB1 and TP53 mutations. These findings provide evidences of the potential effect of epigenetic modifier drugs for liver cancer treatment. However, further research needs to be conducted, to determine the in vivo potential of this treatment regimen for the management of liver cancer.

[Interaction between HIV-1 and GB virus type-C during coinfection status]. / Interacción entre el virus de la inmunodeficiencia humana y el virus GB tipo-C durante el estado de co-infección.

The human immunodeficiency virus (HIV) infection is one of the most important problems in public health. It is estimated that 3 3 million people are infected around the world. HIV and GBV-C share the same transmission route, being frequent the co-infection. Since both viruses replicate in CD4+ lymphocytes, recent studies have described an interaction. Decreasing of HIV viral load and higher CD4 counts have been observed in co-infected patients, leading a better clinical outcome. Nevertheless, some epidemiological studies have shown contradictory results. Additionally, in vitro models report inhibition of HIV by E1, E2, NS3 and NS5A GBV-C proteins, resulting in a decreasing of p24 antigen. This review summarizes the principal findings about co-infection and mechanisms that have been proposed for HIV-1 inhibition.

[Human Pegivirus: Pathogenic potential and non-Hodgkin lymphoma development risk]. / Pegivirus humano: Potencial patogénico y riesgo de desarrollo de linfoma no Hodgkin.

The human pegivirus (HPgV), classified in the Flaviviridae family - Pegivirus genus, is an RNA virus identified in 1995. HPgV is a lymphotrophic virus, with replication sites in bone marrow and lymphoid tissue, as well as in peripheral blood mononuclear cells (PBMCs). Transmission is through sexual and parenteral routes, and recent estimations suggest nearly 750 million people are infected with HPgV worldwide. Almost 25% of infected individuals can develop persistent infection. Until now, HPgV has been considered a non-pathogenic virus; however, epidemiological studies suggest a potential role in lymphoproliferative diseases, particularly in the development of non-Hodgkin lymphoma (NHL). The evidence of this is controversial and the role of HPgV in lymphomagenesis has not yet been demonstrated. Several studies report a high prevalence of HPgV infection in patients with NHL compared to controls and patients with other hematological diseases. Therefore, analytic studies show that HPgV could be related to an increased risk of NHL development. Conversely, other studies indicate no association between HPgV and NHL, so the role of HPgV in lymphomagenesis is not clear. This review summarizes the main findings related to HPgV's pathogenic potential and association with NHL.

Bio-EdIP: An automatic approach for in vitro cell confluence images quantification.

BACKGROUND AND OBJECTIVES: Cell imaging is a widely-employed technique to analyze multiple biological processes. Therefore, simple, accurate and quantitative tools are needed to understand cellular events. For this purpose, Bio-EdIP was developed as a user-friendly tool to quantify confluence levels using cell culture images. METHODS: The proposed algorithm combines a pre-processing step with subsequent stages that involve local processing techniques and a morphological reconstruction-based segmentation algorithm. Segmentation performance was assessed in three constructed image sets, comparing F-measure scores and AUC values (ROC analysis) for Bio-EdIP, its previous version and TScratch. Furthermore, segmentation results were compared with published algorithms using eight public benchmarks. RESULTS: Bio-EdIP automatically segmented cell-free regions from images of in vitro cell culture. Based on mean F-measure scores and ROC analysis, Bio-EdIP conserved a high performance regardless of image characteristics of the constructed dataset, when compared with its previous version and TScratch. Although acquisition quality of the public dataset affected Bio-EdIP segmentation, performance was better in two out of eight public sets. CONCLUSIONS: Bio-EdIP is a user-friendly interface, which is useful for the automatic analysis of confluence levels and cell growth processes using in vitro cell culture images. Here, we also presented new manually annotated data for algorithms evaluation.

Pegivirus humano: potencial patogénico y riesgo de desarrollo de linfoma no Hodgkin / Human Pegivirus: pathogenic potential and non-Hodgkin lymphoma development risk

Resumen El pegivirus humano (HPgV) es un virus ARN que fue identificado en el año 1995. Actualmente se encuentra clasificado dentro de la familia Flaviviridae, género Pegivirus, relacionado filogenéticamente con el virus de la hepatitis C (VHC). El HPgV es un virus linfotrópico, con replicación en médula ósea, tejidos linfoides, y en células mononucleares de sangre periférica. Este virus se transmite por vía parenteral y sexual. Según estimaciones realizadas, en el mundo existen alrededor de 750 millones de personas infectadas por este agente. Se ha evidenciado que hasta en 25% de los casos se presenta una infección persistente, y aunque se considera que el HPgV es un virus no patogénico, existen evidencias epidemiológicas que sugieren una relación con el desarrollo de desórdenes linfoproliferativos, particularmente linfoma no Hodgkin (LNH). Algunos estudios han reportado una alta prevalencia de HPgV en pacientes con LNH comparado con donantes de sangre y/o pacientes con enfermedades hematológicas no malignas, lo que se asocia a un incremento en el riesgo relativo para el desarrollo de LNH en personas infectadas. De otra parte, existen estudios epidemiológicos que contradicen esta asociación, por lo que el rol de HPgV en la aparición de desórdenes lifoproliferativos es un tema actual de debate. En el presente manuscrito se discute el potencial patogénico derivado de los mecanismos de infección persistente del HPgV, así como las principales evidencias sobre la relación entre el HPgV y el riesgo de desarrollo de LNH.

The human pegivirus (HPgV), classified in the Flaviviridae family - Pegivirus genus, is an RNA virus identified in 1995. HPgV is a lymphotrophic virus, with replication sites in bone marrow and lymphoid tissue, as well as in peripheral blood mononuclear cells (PBMCs). Transmission is through sexual and parenteral routes, and recent estimations suggest nearly 750 million people are infected with HPgV worldwide. Almost 25% of infected individuals can develop persistent infection. Until now, HPgV has been considered a non-pathogenic virus; however, epidemiological studies suggest a potential role in lymphoproliferative diseases, particularly in the development of non-Hodgkin lymphoma (NHL). The evidence of this is controversial and the role of HPgV in lymphomagenesis has not yet been demonstrated. Several studies report a high prevalence of HPgV infection in patients with NHL compared to controls and patients with other hematological diseases. Therefore, analytic studies show that HPgV could be related to an increased risk of NHL development. Conversely, other studies indicate no association between HPgV and NHL, so the role of HPgV in lymphomagenesis is not clear. This review summarizes the main findings related to HPgV's pathogenic potential and association with NHL.

Metilación del ADN: implicaciones en carcinogénesis / DNA methylation and implications in carcinogenesis

El cáncer se ha constituido en uno de los principales problemas de la salud pública mundial, no solo por su casuística sino por la alta morbilidad y mortalidad asociadas; solo en 2008 se estimaron más de 12 millones de nuevos casos de cáncer diagnosticados, 7 millones de muertes y 25 millones de personas vivas con este padecimiento. A nivel celular y molecular, el cáncer se define como una alteración de los mecanismos que regulan la división celular. Entre estos mecanismos, la epigenética estudia los cambios heredables que afectan el patrón de la expresión génica, que no son consecuencia de alteraciones en la secuencia nucleotídica del gen (mutaciones), o de sus secuencias reguladoras (promotores). De estos cambios, la metilación del ADN es la mejor caracterizada, asociándose con el silenciamiento o sobreexpresión de genes claves en la regulación del inicio y la progresión del cáncer, como es el caso de los genes involucrados en la vía de señalización Wnt/β-catenina. Comprender los pasos implicados en el inicio y en el establecimiento de alteraciones en la expresión génica mediadas por fenómenos epigenéticos, permitirá desarrollar terapias dirigidas a componentes claves involucrados en este proceso. En el presente manuscrito se analizan algunos mecanismos epigenéticos, su efecto sobre la regulación de la expresión génica, y su papel en la carcinogénesis.

Cancer has become one of the main public health problems worldwide, not only for its incidence, but also due to its high morbidity and mortality. Only in 2008, an estimated 12 million new cancer cases were diagnosed, with 7 million deaths and 25 million people living with the disease. On a cellular and molecular level, cancer is defined as an alteration in the mechanisms regulating cell division. Epigenetics is the study of inheritable changes affecting the gene expression pattern in these mechanisms, which are not a consequence of alterations in the nucleotide sequence of the gene (mutations) or its regulatory sequences (promoters). Among these changes, DNA methylation has been characterized most accurately. It has been associated with the silencing or overexpression of genes performing a key role in regulating the start and progress of cancer, such as the genes involved in the Wnt/β-catenin signaling pathway. Understanding the steps involved in the start and establishment of gene expression alterations mediated by epigenetic phenomena, will make it possible to develop therapies aimed at key components of this process. The present study is an analysis of some epigenetic mechanisms, their effect on gene expression regulation, and their role in carcinogenesis.

Interacción entre el virus de la inmunodeficiencia humana y el virus GB tipo-C durante el estado de co-infección / Interaction between HIV-1 and GB virus type-C during coinfection status

The human immunodeficiency virus (HIV) infection is one of the most important problems in public health. It is estimated that 3 3 million people are infected around the world. HIV and GBV-C share the same transmission route, being frequent the co-infection. Since both viruses replicate in CD4+ lymphocytes, recent studies have described an interaction. Decreasing of HIV viral load and higher CD4 counts have been observed in co-infected patients, leading a better clinical outcome. Nevertheless, some epidemiological studies have shown contradictory results. Additionally, in vitro models report inhibition of HIV by E1, E2, NS3 and NS5A GBV-C proteins, resulting in a decreasing of p24 antigen. This review summarizes the principal findings about co-infection and mechanisms that have been proposed for HIV-1 inhibition.

La infección por el virus de la inmunodeficiencia humana (VIH) continúa siendo uno de los principales problemas en salud pública; se estima que existen actualmente más de 33 millones de personas infectadas en el mundo. El VIH y el virus GB tipo C (GBV-C) comparten la misma vía de transmisión, por lo que es frecuente encontrar individuos co-infectados. Estudios recientes han descrito un efecto inhibitorio asociado a disminución en la carga viral de VIH, altos recuentos de CD4 y mayor tiempo de sobrevida en pacientes co-infectados, resultando en un mejor pronóstico y menor progreso a SIDA; adicionalmente, estudios in vitro indican que las proteínas virales E1, E2, NS3 y NS5A del GBV-C estarían implicadas en la inhibición del VIH-1. En el presente artículo se revisan los principales aspectos de la co-infección, y se describen los mecanismos propuestos para la inhibición de la replicación del VIH-1 mediada por las proteínas virales del GBV-C.

Infección oculta por el virus de la hepatitis C en un paciente retrasplantado por falla hepática de origen desconocido / Occult hepatitis C virus infection in a re-transplanted patient with liver failure of unknown etiology

Recientemente, se ha descrito un nuevo escenario clínico denominado hepatitis C oculta (HCV-Oc); este solo puede ser caracterizado mediante análisis del genoma viral, ya que marcadores serológicos por prueba de ELISA no son detectables. En este trabajo se pretendió identificar el virus de la hepatitis C (HCV) en un paciente retrasplantado por falla hepática de origen desconocido. Para esto, ARN obtenido de los explantes, muestras de suero, plasma y buffy coat, fue analizado mediante una RT-PCR que amplifica la región 5’UTR del HCV, demostrando la presencia del genoma viral solo en el tejido hepático de ambos explantes; al analizar las secuencias, los aislados fueron caracterizados como genotipo 1a.Este estudio obedece al primer reporte en el mundo de HCV-Oc causada por el genotipo 1a del HCV, en un paciente que debió ser trasplantado. Se recomienda tener en cuenta este nuevo escenario para el diagnóstico diferencial en cirrosis hepática, carcinoma hepatocelular y/o falla hepática de origen desconocido.

A new clinical scenario called Occult Hepatitis C Virus infection has been recently described (HCV-Oc); it can only be characterized by molecular analysis, due that serological marker by ELISA are not detected.The aim of the present study was to identify the Hepatitis C Virus infection in a patient re-transplanted by hepatic failure of unknown etiology. For this, RNA obtained from different sources (Liver tissue, serum, plasma and buffy coat) was assessed by a RT-PCR protocol that specifically targeted the HCV 5’UTR, showing the presence of HCV genome just in liver tissue explants. When viral sequences were analyzed, the strains belonged to HCV genotype 1a. Our study corresponds to the first report in the world of HCV-Oc due to HCV genotype 1a, in liver re-transplantation context. We recommend having in mind this new clinical scenario into the differential diagnosis of patients with cirrhosis, hepatocellular carcinoma and/or hepatic failure of unknown etiology.