RESUMEN

Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that causes a devastating neoplastic disease in chickens. MDV has been shown to integrate its genome into the telomeres of latently infected and tumor cells, which is crucial for efficient tumor formation. Telomeric repeat arrays present at the ends of the MDV genome facilitate this integration into host telomeres; however, the integration mechanism remains poorly understood. Until now, MDV integration could only be investigated qualitatively upon infection of chickens. To shed further light on the integration mechanism, we established a quantitative integration assay using chicken T cell lines, the target cells for MDV latency and transformation. We optimized the infection conditions and assessed the establishment of latency in these T cells. The MDV genome was efficiently maintained over time, and integration was confirmed in these cells by fluorescence in situ hybridization (FISH). To assess the role of the two distinct viral telomeric repeat arrays in the integration process, we tested various knockout mutants in our in vitro integration assay. Efficient genome maintenance and integration was thereby dependent on the presence of the telomeric repeat arrays in the virus genome. Taken together, we developed and validated a novel in vitro integration assay that will shed light on the integration mechanism of this highly oncogenic virus into host telomeres.

RESUMEN

Lymphoblastoid cell lines (LCLs) represent a convenient research tool for expanding the amount of biologic material available from an individual. LCLs are commonly used as reference materials, most notably from the Genome in a Bottle Consortium. However, the question remains how faithfully LCL-derived genome assemblies represent the germline genome of the donor individual as compared to the genome assemblies derived from peripheral blood mononuclear cells. We present an in-depth comparison of a large collection of LCL- and peripheral blood mononuclear cell-derived genomes in terms of distributions of coverage and copy number alterations. We found significant differences in the depth of coverage and copy number calls, which may be driven by differential replication timing. Importantly, these copy number changes preferentially affect regions closer to genes and with higher GC content. This suggests that genomic studies based on LCLs may display locus-specific biases, and that conclusions based on analysis of depth of coverage and copy number variation may require further scrutiny.

RESUMEN

Human papillomavirus(HPV)-related head and neck cancer is recognized as a distinct tumor entity with rising incidence reported for several countries. These tumors arise from squamous cells, typically in the oropharynx. In contrast to cancer associated with other risk factors, HPV-related cancer is driven by viral oncoprotein activity and has individual profiles regarding protein expression, and genetic and epigenetic alterations. Molecular characteristics are p16IN4A overexpression, absence of p53 inactivating mutations, and PI3K/AKT and Wnt pathway modulation. Patients with HPV-related head and neck cancer have improved survival compared to those with HPV-negative tumors, and p16INK4A staining has been introduced into tumor staging recently. However, no specific or toxicity-reduced treatment modalities have been established for this entity so far. Although the still incomplete and partially inconsistent data in this field needs further study, particular features of HPV-related cancers such as specific microRNA expression, immunology, or gene methylation patterns certainly have the potential to be implemented in future diagnostic and therapeutic concepts.

Asunto(s)

Biomarcadores de Tumor/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/mortalidad , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/mortalidad , Proteínas de Neoplasias/genética , Infecciones por Papillomavirus/epidemiología , Infecciones por Papillomavirus/genética , Polimorfismo de Nucleótido Simple/genética , Causalidad , Comorbilidad , Medicina Basada en la Evidencia , Femenino , Marcadores Genéticos/genética , Predisposición Genética a la Enfermedad/epidemiología , Predisposición Genética a la Enfermedad/genética , Humanos , Masculino , Prevalencia , Factores de Riesgo , Carcinoma de Células Escamosas de Cabeza y Cuello , Tasa de Supervivencia

RESUMEN

Previous observations that human amniotic fluid cells (AFC) can be transformed by human adenovirus type 5 (HAdV-5) E1A/E1B oncogenes prompted us to identify the target cells in the AFC population that are susceptible to transformation. Our results demonstrate that one cell type corresponding to mesenchymal stem/stroma cells (hMSCs) can be reproducibly transformed by HAdV-5 E1A/E1B oncogenes as efficiently as primary rodent cultures. HAdV-5 E1-transformed hMSCs exhibit all properties commonly associated with a high grade of oncogenic transformation, including enhanced cell proliferation, anchorage-independent growth, increased growth rate, and high telomerase activity as well as numerical and structural chromosomal aberrations. These data confirm previous work showing that HAdV preferentially transforms cells of mesenchymal origin in rodents. More importantly, they demonstrate for the first time that human cells with stem cell characteristics can be completely transformed by HAdV oncogenes in tissue culture with high efficiency. Our findings strongly support the hypothesis that undifferentiated progenitor cells or cells with stem cell-like properties are highly susceptible targets for HAdV-mediated cell transformation and suggest that virus-associated tumors in humans may originate, at least in part, from infections of these cell types. We expect that primary hMSCs will replace the primary rodent cultures in HAdV viral transformation studies and are confident that these investigations will continue to uncover general principles of viral oncogenesis that can be extended to human DNA tumor viruses as well. IMPORTANCE: It is generally believed that transformation of primary human cells with HAdV-5 E1 oncogenes is very inefficient. However, a few cell lines have been successfully transformed with HAdV-5 E1A and E1B, indicating that there is a certain cell type which is susceptible to HAdV-mediated transformation. Interestingly, all those cell lines have been derived from human embryonic tissue, albeit the exact cell type is not known yet. We show for the first time the successful transformation of primary human mesenchymal stromal cells (hMSCs) by HAdV-5 E1A and E1B. Further, we show upon HAdV-5 E1A and E1B expression that these primary progenitor cells exhibit features of tumor cells and can no longer be differentiated into the adipogenic, chondrogenic, or osteogenic lineage. Hence, primary hMSCs represent a robust and novel model system to elucidate the underlying molecular mechanisms of adenovirus-mediated transformation of multipotent human progenitor cells.

Asunto(s)

Proteínas E1A de Adenovirus/genética , Proteínas E1B de Adenovirus/genética , Adenovirus Humanos/genética , Transformación Celular Viral , Regulación Neoplásica de la Expresión Génica , Regulación Viral de la Expresión Génica , Células Madre Mesenquimatosas/virología , Proteínas E1A de Adenovirus/metabolismo , Proteínas E1B de Adenovirus/metabolismo , Adenovirus Humanos/crecimiento & desarrollo , Adenovirus Humanos/metabolismo , Animales , Línea Celular Transformada , Proliferación Celular , Aberraciones Cromosómicas , Células Epiteliales/patología , Células Epiteliales/virología , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Cariotipo , Lentivirus/genética , Lentivirus/metabolismo , Células Madre Mesenquimatosas/patología , Oncogenes , Cultivo Primario de Células , Ratas , Transfección

RESUMEN

BACKGROUND: Prostate cancer cell lines have been used in the search for biomarkers that are suitable for prostate cancer diagnosis. Unfortunately, many cell line studies have only involved single cell lines, partially characterized cell lines or were performed without controls, and this may have been detrimental to effective biomarker discovery. We have analyzed a panel of prostate cancer and nonmalignant control cell lines using current biomarkers and then investigated a set of prospective endosomal and lysosomal proteins to search for new biomarkers. METHODS: Western blotting was used to define the amount of protein and specific molecular forms in cell extracts and culture media from a panel of nonmalignant (RWPE-1, PNT1a, PNT2) and prostate cancer (22RV1, CaHPV10, DU-145, LNCaP) cell lines. Gene expression was determined by qRT-PCR. RESULTS: HPV-18 transfected cell lines displayed a different pattern of protein and gene expression when compared to the other cell lines examined, suggesting that these cell lines may not be the most optimal for prostate cancer biomarker discovery. There was an increased amount of prostatic acid phosphatase and kallikrein proteins in LNCaP cell extracts and culture media, but variable amounts of these proteins in other prostate cancer cell lines. There were minimal differences in the amounts of lysosomal proteins detected in prostate cancer cells and culture media, but two endosomal proteins, cathepsin B and acid ceramidase, had increased gene and protein expression, and certain molecular forms showed increased secretion from prostate cancer cells (P ≤ 0.05). LIMP-2 gene and protein expression was significantly increased in prostate cancer compared to nonmalignant cell lines (P ≤ 0.05). CONCLUSIONS: While the existing prostate cancer biomarkers and lysosomal proteins investigated here were not able to specifically differentiate between a panel of nonmalignant and prostate cancer cell lines, endosomal proteins showed some discriminatory capacity. LIMP-2 is a critical regulator of endosome biogenesis and the increased expression observed in prostate cancer cells indicated that other endosome related proteins may also be upregulated and could be investigated as novel biomarkers.

Asunto(s)

Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral/metabolismo , Próstata/metabolismo , Neoplasias de la Próstata/diagnóstico , Fosfatasa Ácida , Biomarcadores de Tumor/genética , Humanos , Calicreínas/genética , Calicreínas/metabolismo , Masculino , Próstata/patología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo

RESUMEN

Epstein-Barr virus (EBV) is an oncogenic human herpesvirus in the γ-herpesvirinae subfamily that contains a 170-180kb double-stranded DNA genome. In vivo, EBV commonly infects B and epithelial cells and persists for the life of the host in a latent state in the memory B-cell compartment of the peripheral blood. EBV can be reactivated from its latent state, leading to increased expression of lytic genes that primarily encode for enzymes necessary to replicate the viral genome and structural components of the virion. Lytic cycle proteins also aid in immune evasion, inhibition of apoptosis, and the modulation of other host responses to infection. In vitro, EBV has the potential to infect primary human B cells and induce cellular proliferation to yield effectively immortalized lymphoblastoid cell lines, or LCLs. EBV immortalization of B cells in vitro serves as a model system for studying EBV-mediated lymphomagenesis. While much is known about the steady-state viral gene expression within EBV-immortalized LCLs and other EBV-positive cell lines, relatively little is known about the early events after primary B-cell infection. It was previously thought that upon latent infection, EBV only expressed the well-characterized latency-associated transcripts found in LCLs. However, recent work has characterized the early, but transient, expression of lytic genes necessary for efficient transformation and delayed responses in the known latency genes. This chapter summarizes these recent findings that show how dynamic and controlled expression of multiple EBV genes can control the activation of B cells, entry into the cell cycle, the inhibition of apoptosis, and innate and adaptive immune responses.

Asunto(s)

Linfocitos B/virología , Transformación Celular Viral , Regulación Viral de la Expresión Génica , Herpesvirus Humano 4/fisiología , Interacciones Huésped-Patógeno , Herpesvirus Humano 4/patogenicidad , Humanos

RESUMEN

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus associated with a large number of lymphoid and epithelial malignancies. As a successful pathogen it has co-evolved with its human host for millions of years. EBV has the unique ability to establish life-long latent infection in primary human B lymphocytes. During latent infection, a small subset of viral proteins is expressed. These proteins are essential for maintenance of the EBV genome as well as the deregulation of various signaling pathways that facilitate the proliferation and survival of the infected cells. Epstein-Barr nuclear antigen (EBNA)3C is one of the latent proteins shown to be essential for transformation of primary human B lymphocytes in vitro. EBNA3C primarily functions as a transcriptional regulator by interacting with a number of well known cellular and viral transcriptional factors. We have recently identified several binding partners for EBNA3C including proteins that regulate cell cycle and chromatin remodeling. We are actively engaged in discerning the biological significance of these interactions. This review summarizes our current understanding of how EBNA3C usurps cellular pathways that promote B-cell transformation.