Epstein-Barr Virus-Induced Genes and Endogenous Retroviruses in Immortalized B Cells from Patients with Multiple Sclerosis.

The immune pathogenesis of multiple sclerosis (MS) is thought to be triggered by environmental factors in individuals with an unfavorable genetic predisposition. Epstein-Barr virus (EBV) infection is a major risk factor for subsequent development of MS. Human endogenous retroviruses (HERVs) can be activated by EBV, and might be a missing link between an initial EBV infection and the later onset of MS. In this study, we investigated differential gene expression patterns in EBV-immortalized lymphoblastoid B cell lines (LCL) from MS-affected individuals (MSLCL) and controls by using RNAseq and qRT-PCR. RNAseq data from LCL mapped to the human genome and a virtual virus metagenome were used to identify possible biomarkers for MS or disease-relevant risk factors, e.g., the relapse rate. We observed that lytic EBNA-1 transcripts seemed to be negatively correlated with age leading to an increased expression in LCL from younger PBMC donors. Further, HERV-K (HML-2) GAG was increased upon EBV-triggered immortalization. Besides the well-known transactivation of HERV-K18, our results suggest that another six HERV loci are up-regulated upon stimulation with EBV. We identified differentially expressed genes in MSLCL, e.g., several HERV-K loci, ERVMER61-1 and ERV3-1, as well as genes associated with relapses. In summary, EBV induces genes and HERV in LCL that might be suitable as biomarkers for MS or the relapse risk.

Overexpression of Endogenous Retroviruses and Malignancy Markers in Neuroblastoma Cell Lines by Medium-Induced Microenvironmental Changes.

Neuroblastoma (NB) is the commonest solid tumor outside the central nervous system in infancy and childhood with a unique biological heterogeneity. In patients with advanced, metastasizing neuroblastoma, treatment failure and poor prognosis is often marked by resistance to chemo- or immunotherapy. Thus, identification of robust biomarkers seems essential for understanding tumor progression and developing effective therapy. Here, we have studied the expression of human endogenous retroviruses (HERV) as potential targets in NB cell lines during stem-cell medium-induced microenvironmental change. Quantitative PCR revealed that relative expression of the HERV-K family and HERV-W1 ENV were increased in all three NB cell lines after incubation in stem-cell medium. Virus transcriptome analyses revealed the transcriptional activation of three endogenous retrovirus elements: HERV-R ENV (ERV3-1), HERV-E1 and HERV-Fc2 ENV (ERVFC1-1). Known malignancy markers in NB, e.g. proto-oncogenic MYC or MYCN were expressed highly heterogeneously in the three investigated NB cell lines with up-regulation of MYC and MYCN upon medium-induced microenvironmental change. In addition, SiMa cells exclusively showed a phenotype switching from loosely-adherent monolayers to low proliferating grape-like cellular aggregates, which was accompanied by an enhanced CD133 expression. Interestingly, the overexpression of HERV was associated with a significant elevation of immune checkpoint molecule CD200 in both quantitative PCR and RNA-seq analysis suggesting tumor escape mechanism in NB cell lines after incubation in serum-free stem cell medium.

Identification of Differentially Expressed Human Endogenous Retrovirus Families in Human Leukemia and Lymphoma Cell Lines and Stem Cells.

Endogenous retroviruses (ERVs) are becoming more and more relevant in cancer research and might be potential targets. The oncogenic potential of human ERVs (HERVs) has been recognized and includes immunosuppression, cell fusion, antigenicity of viral proteins, and regulation of neighboring genes. To decipher the role of HERVs in human cancers, we used a bioinformatics approach and analyzed RNA sequencing data from the LL-100 panel, covering 22 entities of hematopoietic neoplasias including T cell, B cell and myeloid malignancies. We compared HERV expression in this panel with hematopoietic stem cells (HSCs), embryonic stem cells (ESCs) and normal blood cells. RNA sequencing data were mapped against a comprehensive synthetic viral metagenome with 116 HERV sequences from 14 different HERV families. Of these, 13 HERV families and elements were differently expressed in malignant hematopoietic cells and stem cells. We found transcriptional upregulation of HERVE family in acute megakaryocytic and erythroid leukemia and of HERVFc family in multiple myeloma/plasma cell leukemia (PCL). The HERVFc member HERVFc-1 was found transcriptionally active in the multiple myeloma cell line OPM-2 and also in the Hodgkin lymphoma cell line L-428. The expression of HERVFc-1 in L-428 cells was validated by qRT-PCR. We also confirm transcriptional downregulation of ERV3 in acute megakaryocytic and erythroid leukemia, and HERVK in acute monocytic and myelocytic leukemia and a depression of HERVF in all malignant entities. Most of the higher expressed HERV families could be detected in stem cells including HERVK (HML-2), HERV-like, HERVV, HERVT, ERV9, HERVW, HERVF, HERVMER, ERV3, HERVH and HERVPABLB.

Co-Culture of Peripheral Blood Mononuclear Cells and Endothelial Colony Forming Cells from Cord Blood of Preterm Born Babies.

Umbilical cord blood of neonates is a precious source for many fields of research because of distinct unique features combined with easy accessibility at the time of birth. The number of applications are vast with an emphasis in the field of stem cell therapy and regenerative medicine since cord blood contains relatively large numbers of pluripotent cells. This chapter provides a protocol for developing an autologous co-culture of endothelial-like cells and peripheral blood mononuclear cells from umbilical cord blood of premature born babies and describes an experimental setting to investigate inflammatory processes that are a cornerstone of pathophysiology in the developing organs of preterm born babies.

Melittin Increases Cisplatin Sensitivity and Kills KM-H2 and L-428 Hodgkin Lymphoma Cells.

Hodgkin lymphoma (HL) is neoplasia with high cure rates. However, not all patients can be cured with the current treatment. Chemo-resistance of tumor cells is one factor involved in treatment failure. In addition to its pore-forming activity on lipid bilayer membranes, the toxin melittin from bee venom is an inhibitor of several cancer-related signaling pathways. Moreover, melittin analogs have been shown to inhibit the activity of ATP binding cassette (ABC) transporters which are known to play important roles in the chemo-resistance of tumor cells. Therefore, we tested the toxicity of melittin for HL cell lines KM-H2 and L-428 and whether melittin can increase the chemo-sensitivity of cisplatin-resistant HL cells. We found high toxicity of melittin for KM-H2 and L-428 cells. In co-cultures with normal blood cells, melittin preferentially killed KM-H2 and L-428 cells. In addition, we observed increased cisplatin sensitivity of chemo-resistant L-428 cells after treatment with melittin. ABC transporter activity was not reduced after treatment with melittin. Our data suggest that melittin or melittin analogs might be promising agents for the future development of treatment strategies for HL patients with resistant disease.

Expression of A New Endogenous Retrovirus-Associated Transcript in Hodgkin Lymphoma Cells.

During characterization of a cDNA library from the Hodgkin lymphoma (HL) cell line L-1236, we discovered a new transcript derived from chromosome 1 at the long intergenic non-protein coding RNA 1768 (LINC01768)/colony stimulating factor 1 (CSF1) region. The first exon of this transcript from Hodgkin lymphoma cells (THOLE) starts in the predicted exon 4 of LINC01768 and is part of an endogenous retrovirus (ERV) from the HUERS-P1/LTR8 family. High expression of THOLE was only detectable in HL cell line L-1236. The expression of THOLE in L-1236 cell is another example for ERV/LTR-associated gene expression in HL cells. At the genome level, the HUERS-P1/LTR8 region including THOLE is only present in Hominoidea. The influence of ERV/LTRs on gene expression might explain the characteristic phenotype of human HL.

Impact of rs12917 MGMT Polymorphism on [18F]FDG-PET Response in Pediatric Hodgkin Lymphoma (PHL).

PURPOSE: The enzyme O6-methylguanine-DNA methyltransferase (MGMT) is an important component of the DNA repair machinery. MGMT removes O6-methylguanine from the DNA by transferring the methyl group to a cysteine residue in its active site. Recently, we detected the single nucleotide polymorphism (SNP) rs12917 (C/T) in the MGMT sequence adjacent to the active site in Hodgkin lymphoma (HL) cell line KM-H2. We now investigated whether this SNP is also present in other HL cell lines and patient samples. Furthermore, we asked whether this SNP might have an impact on metabolic response in 2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography ([18F]FDG-PET), and on overall treatment outcome based on follow-up intervals of at least 34 months. PROCEDURES: We determined the frequency of this MGMT polymorphism in 5 HL cell lines and in 29 pediatric HL (PHL) patients. The patient cohort included 17 female and 12 male patients aged between 4 and 18 years. After characterization of the sequence, we tested a possible association between rs12917 and age, gender, Ann Arbor stage, treatment group, metabolic response following two courses of OEPA (vincristine, etoposide, prednisone, and doxorubicin) chemotherapy, radiotherapy indication, and relapse status. RESULTS: We detected the minor T allele in four of five HL cell lines. 11/29 patients carried the minor T allele whereas 18/29 patients showed homozygosity for the major C allele. Interestingly, we observed significantly better metabolic response in PHL patients carrying the rs12917 C allele resulting in a lower frequency of radiotherapy indication. CONCLUSION: MGMT polymorphism rs12917 seems to affect chemotherapy response in PHL. The prognostic value of this polymorphism should be investigated in a larger patient cohort.

Differentiation-Dependent Regulation of Human Endogenous Retrovirus K Sequences and Neighboring Genes in Germ Cell Tumor Cells.

Under physiological conditions, most human endogenous retroviruses (HERVs) are transcriptionally silent. However, re-activation of HERVs is observed under pathological conditions like inflammation or cancer. In addition to expression of HERV sequences, an impact of HERV-loci on expression of adjacent genes has been suggested as probably important patho-physiological mechanism. A candidate for such a gene is PRODH (proline dehydrogenase 1), which is located on chromosome 22 adjacent to HERVK-24. Germ cell tumors (GCTs) are known to express high level of HERVK sequences. In addition, non-seminomatous GCT are useful models to study HERV expression in the context of differentiation since they reflect aspects of cellular development during embryogenesis and usually contain different cell types. This is due to the embryonal carcinoma (EC) cells, which are the stem cell component of GCT. They are pluripotent, show high expression of pluripotency markers like OCT4 and LIN28A and can differentiate into either somatic derivatives (teratoma cells) or choriocarcinoma or yolk-sac tumor cells reflecting extra-embryonal differentiation. OCT4 is lost upon differentiation. We used GCT derived cell lines of varying differentiation stages to analyze expression of HERVK and PRODH. Differentiation status and cellular relationship of GCT cells was determined using microarray analysis and western blotting of the embryonic pluripotency markers OCT4 and LIN28A. The highest expression of HERVK was found in undifferentiated EC cells, which retain a stem cell phenotype and express both OCT4 and LIN28. In contrast, the lowest expression of HERVK was observed in somatic differentiated GCT cells which also lack OCT4 and LIN28A whereas GCT cells with differentiation characteristics of yolk-sac tumor expressed LIN28A but not OCT4 and showed intermediate level of HERVK. A similar pattern was found for PRODH. Differentiation of EC cells by siRNA mediated knock-down of OCT4 or treatment with differentiation inducing medium decreased expression of HERVK and PRODH. Treatment of differentiated GCT cells with 5'-azacytidine and trichostatin A increased expression of HERVK and PRODH, indicating that epigenetic mechanisms are responsible for altered expression of these genes. Our data suggest that HERVK expression is dependent on cellular differentiation stages regulated by epigenetic mechanisms, which can also affect expression of neighboring genes.

Characterization of Alstrom Syndrome 1 (ALMS1) Transcript Variants in Hodgkin Lymphoma Cells.

The Alstrom syndrome gene (ALMS1) is one of the largest disease associated genes identified today in the human genome and is implicated in cell cycle control, ciliogenesis, endosome recycling and intracellular transport mechanisms. ALMS1 mutations cause Alstrom syndrome, a rare genetic disorder. However, its function is not completely understood. DNA microarray analysis suggested that ALMS1 might be differentially expressed between Hodgkin lymphoma (HL) cells and normal tissues. By using reverse transcription-polymerase chain reaction (RT-PCR) we detected low but variable expression of ALMS1 in HL cell lines with highest expression in KM-H2 cells. Immunofluorescence indicated centrosomal accumulation of ALMS1 protein in HL cells. Knock-down of ALMS1 in KM-H2 cells had no impact on viability or cytotoxic drug sensitivity of these cells. Sequencing of RT-PCR products from HL cell lines identified three variable regions in ALMS1 transcripts that affect exons 2, 13, and 23. One of these variants was characterized by splicing out of exon 13. The other variants are characterized by two alternative 5 prime ends or alternative 3 prime ends. Structure prediction of the corresponding RNAs and proteins suggest that the different transcript variants might affect posttranscriptional regulation and ligand binding.

Endogenous Retrovirus 3 - History, Physiology, and Pathology.

Endogenous viral elements (EVE) seem to be present in all eukaryotic genomes. The composition of EVE varies between different species. The endogenous retrovirus 3 (ERV3) is one of these elements that is present only in humans and other Catarrhini. Conservation of ERV3 in most of the investigated Catarrhini and the expression pattern in normal tissues suggest a putative physiological role of ERV3. On the other hand, ERV3 has been implicated in the pathogenesis of auto-immunity and cancer. In the present review we summarize knowledge about this interesting EVE. We propose the model that expression of ERV3 (and probably other EVE loci) under pathological conditions might be part of a metazoan SOS response.

Glutathione-S-transferases and Chemotherapy Resistance of Hodgkin's Lymphoma Cell Lines.

BACKGROUND: Glutathione-S-transferases (GSTs) are associated with multidrug resistance of tumor cells and are involved in drug detoxification and control of apoptosis. We analyzed the impact of GSTs on apoptosis of Hodgkin's lymphoma (HL) cells. MATERIALS AND METHODS: Expression of GST isoforms in HL cell lines was assessed by analysis of DNA microarray data and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The impact of the GST inhibitor ethacrynic acid (EA) on HL cell survival was analyzed in vitro. RESULTS: DNA microarray analysis and qRT-PCR analysis demonstrated higher expression of GST isoforms in chemoresistant HL cells. Therefore, GSTs may contribute to chemoresistance of HL cells. Incubation of GST-expressing chemoresistant L-1236 HL cells with EA significantly enhanced the activity of cisplatin against these cells. CONCLUSION: Our data suggest that the combined treatment with chemotherapy and GST inhibitors such as EA might be an interesting option for patients with chemoresistant HL.

Stimulation of the hypoxia pathway modulates chemotherapy resistance in Hodgkin's lymphoma cells.

Hodgkin's lymphoma (HL) is a malignant disease of the lymphatic system. The therapy has been improved during the last decades but there are still patients who cannot be cured, and the therapy is associated with several adverse late effects. Therefore, we asked which genes might be involved in the chemotherapy resistance of HL cells. We observed that HL cells became more resistant against cisplatin after treatment with cobalt chloride. Therefore, we analyzed which genes were differentially expressed between cells incubated in medium with or without cobalt chloride. We found several genes which were up- or downregulated in the presence of cobalt chloride and might be involved in the modulation of chemotherapy resistance. Cobalt chloride is a hypoxia-mimetic agent. Therefore, we tested chemo-resistance and gene expression of HL cells under hypoxic conditions and confirmed the results from the cobalt chloride experiments. Taken together, activation of the hypoxia pathway led to altered gene expression and drug resistance of HL cells. Differentially expressed genes might be interesting targets for the development of future treatment strategies against drug-resistant HL.

Functional analysis and molecular characterization of spontaneously outgrown human lymphoblastoid cell lines.

In vitro, the infection of human B-cells with the lymphotropic gammaherpesvirus Epstein-Barr virus (EBV) induces formation of permanently growing lymphoblastoid cell lines (LCL). In a spontaneously outgrown LCL (cell line CSIII), we detected nucleotide sequence variations of the EBV nuclear antigen 1 (EBNA1) RNA that was different from the reference sequence of EBNA1 in the prototypic EBV strain B95-8. In the present study, we molecularly and functionally characterized this virus isolate in comparison to LCL with the prototypic nucleotide sequence. Although we detected high functional similarity between CSIII and the other LCL, our data suggest that the lytic cycle might be ineffective in the CSIII LCL. DNA microarray analysis indicated that RNA binding motif, single stranded interacting protein 1 (RBMS1), which is typically expressed in latency III of EBV to prevent the lytic cycle, was the most overexpressed gene in CSIII LCL.

Expression of dual-specificity phosphatase 5 pseudogene 1 (DUSP5P1) in tumor cells.

Sequencing of individual clones from a newly established cDNA library from the chemoresistant Hodgkin's lymphoma cell line L-1236 led to the isolation of a cDNA clone corresponding to a short sequence from chromosome 1. Reverse transcriptase-polymerase chain reaction indicated high expression of this sequence in Hodgkin's lymphoma derived cell lines but not in normal blood cells. Further characterization of this sequence and the surrounding genomic DNA revealed that this sequence is part of a human endogenous retrovirus locus. The sequence of this endogenous retrovirus is interrupted by a pseudogene of the dual specificity phosphatase 5 (DUSP5). Reverse transcriptase-polymerase chain reaction revealed high expression of this pseudogene (DUSP5P1) in HL cell lines but not in normal blood cells or Epstein-Barr virus-immortalized B cells. Cells from other tumor types (Burkitt's lymphoma, leukemia, neuroblastoma, Ewing sarcoma) also showed a higher DUSP5P1/DUSP5 ratio than normal cells. Furthermore, we observed that higher expression of DUSP5 in relation to DUSP5P1 correlated with the expression of the pro-apoptotic factor B cell leukemia/lymphoma 2-like 11 (BCL2L11) in peripheral blood cells and HL cells. Knock-down of DUSP5 in HL cells resulted in down-regulation of BCL2L11. Thus, the DUSP5/DUSP5P1 system could be responsible for regulation of BCL2L11 leading to inhibition of apoptosis in these tumor cells.

Curcuma Contra Cancer? Curcumin and Hodgkin's Lymphoma.

Curcumin, a phytochemical isolated from curcuma plants which are used as coloring ingredient for the preparation of curry powder, has several activities which suggest that it might be an interesting drug for the treatment or prevention of cancer. Curcumin targets different pathways which are involved in the malignant phenotype of tumor cells, including the nuclear factor kappa B (NFKB) pathway. This pathway is deregulated in multiple tumor entities, including Hodgkin's lymphoma (HL). Indeed, curcumin can inhibit growth of HL cell lines and increases the sensitivity of these cells for cisplatin. In this review we summarize curcumin activities with special focus on possible activities against HL cells.

MondoA is highly overexpressed in acute lymphoblastic leukemia cells and modulates their metabolism, differentiation and survival.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. To identify novel candidates for targeted therapy, we performed a comprehensive transcriptome analysis identifying MondoA (MLXIP) - a transcription factor regulating glycolysis - to be overexpressed in ALL compared to normal tissues. Using microarray-profiling, gene-set enrichment analysis, RNA interference and functional assays we show that MondoA overexpression increases glucose catabolism and maintains a more immature phenotype, which is associated with enhanced survival and clonogenicity of leukemia cells. These data point to an important contribution of MondoA to leukemia aggressiveness and make MondoA a potential candidate for targeted treatment of ALL.

Down-regulation of achaete-scute complex homolog 1 (ASCL1) in neuroblastoma cells induces up-regulation of insulin-like growth factor 2 (IGF2).

Neuroblastoma (NB) is the most common extra-cranial solid pediatric tumor. The prognosis of patients with NB has been improved during the last decades. However, treatment results for patients with advanced tumor stages are still unsatisfying. NB cells are characterized by a high tendency for spontaneous or induced differentiation. During differentiation, down-regulation of the basic helix-loop-helix transcription factor achaete-scute complex homolog 1 (ASCL1) has been observed but the consequences of ASCL1 down-regulation have not been elucidated. We used RNA interference to knock-down ASCL1 in NB cells. DNA microarray analysis was used for the identification of ASCL1-regulated genes. Furthermore, conventional and quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used for validation of ASCL1-regulated genes. Down-regulation of ASCL1 influenced the expression of several genes. After down-regulation of ASCL1, we observed very high expression of insulin-like growth factor 2 (IGF2), a factor that is known to be induced during differentiation of NB cells. RT-PCR indicated up-regulation of multiple IGF2 transcript variants after ASCL1 knock-down. Our data suggest that the ASCL1-pathway is responsible for the up-regulation of IGF2 during NB differentiation.

Retinoic acid enhances sensitivity of neuroblastoma cells for imatinib mesylate.

BACKGROUND: Retinoids can induce differentiation of neuroblastoma (NB) cells and are in clinical use for the treatment of patients with NB. Despite improvements of standard treatment during the last years, many patients with NB still relapse and new treatment options for these patients are required. PROCEDURE: We analyzed NB cells after incubation with retinoids by using Affymetrix HG_U133A microarrays, reverse transcription-polymerase chain reaction (RT-PCR), and flow cytometry. Sequencing of RT-PCR products was applied for determination of CD117 mRNA sequences from NB cell lines. In addition, we tested sensitivity of NB cells for the kinase inhibitor imatinib mesylate after treatment with retinoids. RESULTS: Treatment of NB cells with retinoids induced expression of several genes including the retinoid metabolizing enzymes CYP26A1 and CYP26B1. In addition, we observed up-regulation of CD117 (KIT), particularly after long-term treatment with retinoids. Sequencing of CD117 mRNA from NB cell lines revealed heterozygosity for a non-synonymous single nucleotide polymorphism in SH-SY5Y NB cells. Up-regulation of CD117 in NB cells correlated with increased sensitivity for the kinase inhibitor imatinib mesylate. CONCLUSIONS: The combination of retinoids with kinase inhibitors might be worth exploring further for the treatment of NB patients.

High expression of the evolutionarily conserved alpha/beta hydrolase domain containing 6 (ABHD6) in Ewing tumors.

Despite improvements in the treatment of patients with Ewing family tumors (EFT), the prognosis for patients with advanced disease is still unsatisfactory. Recently, we identified lipase I as an EFT-associated gene that might be interesting for the development of new immunological or pharmacological treatment strategies. Lipase I is a member of the large protein superfamilies of alpha/beta hydrolases and serine hydrolases. In the present paper we describe high expression of another member of these superfamilies in EFT. By DNA microarray data base mining we found exceptional high expression of alpha/beta hydrolase domain containing 6 (ABHD6) in EFT but not in other sarcomas. Expression of ABHD6 in EFT correlated with expression of another EFT-associated gene, aristaless. Analysis of ABHD6-associated GGAA microsatellites revealed shorter microsatellites in EFT with lack of ABHD6 expression. ABHD6 homologues were found in varying chordata but not in other animal species. Based on homology modeling we predicted the 3D-structure of ABHD6, which shows high similarity with bacterial homoserine transacetylases. High expression of ABHD6 in EFT in comparison to normal tissues and other tumors suggests that ABHD6 might be an interesting new diagnostic or therapeutic target for EFT. However, knock down of ABHD6 in EFT cells did not inhibit tumor cell growth.

Loss of detectability of Charcot-Leyden crystal protein transcripts in blood cells after treatment with dimethyl sulfoxide.

Charcot-Leyden crystal protein (CLC) is a major secretory effector protein of eosinophils. In addition, CLC has been identified as marker for regulatory T-cells and differential expression of CLC has been described under diverse pathological conditions. By analysis of DNA microarray data from peripheral blood mononuclear cells (PBMC) we found differences for the expression of CLC between PBMC that had been cryopreserved or had been used for RNA isolation immediately after cell separation. Reverse transcriptase-polymerase chain reaction (RT-PCR) of separated cell populations indicated that contaminating granulocytes were the main source of CLC transcripts in PBMC. CLC was only detectable in fresh PBMC and not in cryopreserved material. Transcripts corresponding to CLC were also detectable by RT-PCR only in fresh PBMC and eosinophils. Loss of CLC transcripts in PBMC was induced by a short pulse with dimethyl sulfoxide (DMSO), indicating that the freezing medium was responsible for this phenomenon. We conclude that CLC transcripts are lost during cryopreservation in the presence of DMSO and can never be identified as differentially expressed in cryopreserved samples.