Carl Virtanen

DNA methylation profiles in monozygotic and dizygotic twins

Twin studies have provided the basis for genetic and epidemiological studies in human complex traits. As epigenetic factors can contribute to phenotypic outcomes, we conducted a DNA methylation analysis in white blood cells (WBC), buccal epithelial cells and gut biopsies of 114 monozygotic (MZ) twins as well as WBC and buccal epithelial cells of 80 dizygotic (DZ) twins using 12K CpG island microarrays. Here we provide the first annotation of epigenetic metastability of ∼6,000 unique genomic regions in MZ twins. An intraclass correlation (ICC)-based comparison of matched MZ and DZ twins showed significantly higher epigenetic difference in buccal cells of DZ co-twins (P = 1.2 × 10−294). Although such higher epigenetic discordance in DZ twins can result from DNA sequence differences, our in silico SNP analyses and animal studies favor the hypothesis that it is due to epigenomic differences in the zygotes, suggesting that molecular mechanisms of heritability may not be limited to DNA sequence differences.

IDH1(R132H) mutation increases murine haematopoietic progenitors and alters epigenetics

Mutations in the IDH1 and IDH2 genes encoding isocitrate dehydrogenases are frequently found in human glioblastomas and cytogenetically normal acute myeloid leukaemias (AML). These alterations are gain-of-function mutations in that they drive the synthesis of the ‘oncometabolite’ R-2-hydroxyglutarate (2HG). It remains unclear how IDH1 and IDH2 mutations modify myeloid cell development and promote leukaemogenesis. Here we report the characterization of conditional knock-in (KI) mice in which the most common IDH1 mutation, IDH1(R132H), is inserted into the endogenous murine Idh1 locus and is expressed in all haematopoietic cells (Vav-KI mice) or specifically in cells of the myeloid lineage (LysM-KI mice). These mutants show increased numbers of early haematopoietic progenitors and develop splenomegaly and anaemia with extramedullary haematopoiesis, suggesting a dysfunctional bone marrow niche. Furthermore, LysM-KI cells have hypermethylated histones and changes to DNA methylation similar to those observed in human IDH1- or IDH2-mutant AML. To our knowledge, our study is the first to describe the generation and characterization of conditional IDH1(R132H)-KI mice, and also the first report to demonstrate the induction of a leukaemic DNA methylation signature in a mouse model. Our report thus sheds light on the mechanistic links between IDH1 mutation and human AML.

Intra- and Interindividual Epigenetic Variation in Human Germ Cells

Epigenetics represents a secondary inheritance system that has been poorly investigated in human biology. The objective of this study was to perform a comprehensive analysis of DNA methylation variation between and within the germlines of normal males. First, methylated cytosines were mapped using bisulphite modification-based sequencing in the promoter regions of the following disease genes: presenilins (PSEN1 and PSEN2), breast cancer (BRCA1 and BRCA2), myotonic dystrophy (DM1), and Huntington disease (HD). Major epigenetic variation was detected within samples, since the majority of sperm cells of the same individual exhibited unique DNA methylation profiles. In the interindividual analysis, 41 of 61 pairwise comparisons revealed distinct DNA methylation profiles (P=.036 to 6.8 x 10(-14)). Second, a microarray-based epigenetic profiling of the same sperm samples was performed using a 12,198-feature CpG island microarray. The microarray analysis has identified numerous DNA methylation-variable positions in the germ cell genome. The largest degree of variation was detected within the promoter CpG islands and pericentromeric satellites among the single-copy DNA fragments and repetitive elements, respectively. A number of genes, such as EED, CTNNA2, CALM1, CDH13, and STMN2, exhibited age-related DNA methylation changes. Finally, allele-specific methylation patterns in CDH13 were detected. This study provides evidence for significant epigenetic variability in human germ cells, which warrants further research to determine whether such epigenetic patterns can be efficiently transmitted across generations and what impact inherited epigenetic individuality may have on phenotypic outcomes in health and disease.

Integrated classification of lung tumors and cell lines by expression profiling

The utility of cancer cell lines depends largely on their accurate classification, commonly based on histopathological diagnosis of the cancers from which they were derived. However, because cancer is often heterogeneous, the cell line, which also has the opportunity to alter in vitro, may not be representative. Yet without the overall architecture used in histopathological diagnosis of fresh samples, reclassification of cell lines has been difficult. Gene-expression profiling accurately reproduces histopathological classification and is readily applicable to cell lines. Here, we compare the gene-expression profiles of 41 cell lines with 44 tumors from lung cancer. These profiles were generated after hybridization of samples to four replicate 7,685-element cDNA microarrays. After removal of genes that were uniformly up- or down-regulated in fresh compared with cell-line samples, cluster analysis produced four major branch groups. Within these major branches, fresh tumor samples essentially clustered according to pathological type, and further subclusters were seen for both adenocarcinoma (AC) and small cell lung carcinoma (SCLC). Four of eight squamous cell carcinoma (SCC) cell lines clustered with fresh SCC, and 11 of 13 SCLC cell lines grouped with fresh SCLC. In contrast, although none of the 11 AC cell lines clustered with AC tumors, three clustered with SCC tumors and six with SCLC tumors. Although it is possible that preexisting SCC or SCLC cells are being selected from AC tumors after establishment of cell lines, we propose that, even in situ, AC will ultimately progress toward one of two poorly differentiated phenotypes with expression profiles resembling SCC or SCLC.

Global proteomic assessment of the classical protein-tyrosine phosphatome and "Redoxome".

Protein-tyrosine phosphatases (PTPs), along with protein-tyrosine kinases, play key roles in cellular signaling. All Class I PTPs contain an essential active site cysteinyl residue, which executes a nucleophilic attack on substrate phosphotyrosyl residues. The high reactivity of the catalytic cysteine also predisposes PTPs to oxidation by reactive oxygen species, such as H(2)O(2). Reversible PTP oxidation is emerging as an important cellular regulatory mechanism and might contribute to diseases such as cancer. We exploited these unique features of PTP enzymology to develop proteomic methods, broadly applicable to cell and tissue samples, that enable the comprehensive identification and quantification of expressed classical PTPs (PTPome) and the oxidized subset of the PTPome (oxPTPome). We find that mouse and human cells and tissues, including cancer cells, display distinctive PTPomes and oxPTPomes, revealing additional levels of complexity in the regulation of protein-tyrosine phosphorylation in normal and malignant cells.

Three-dimensional culture and cAMP signaling promote the maturation of human pluripotent stem cell-derived hepatocytes

Human pluripotent stem cells (hPSCs) represent a novel source of hepatocytes for drug metabolism studies and cell-based therapy for the treatment of liver diseases. These applications are, however, dependent on the ability to generate mature metabolically functional cells from the hPSCs. Reproducible and efficient generation of such cells has been challenging to date, owing to the fact that the regulatory pathways that control hepatocyte maturation are poorly understood. Here, we show that the combination of three-dimensional cell aggregation and cAMP signaling enhance the maturation of hPSC-derived hepatoblasts to a hepatocyte-like population that displays expression profiles and metabolic enzyme levels comparable to those of primary human hepatocytes. Importantly, we also demonstrate that generation of the hepatoblast population capable of responding to cAMP is dependent on appropriate activin/nodal signaling in the definitive endoderm at early stages of differentiation. Together, these findings provide new insights into the pathways that regulate maturation of hPSC-derived hepatocytes and in doing so provide a simple and reproducible approach for generating metabolically functional cell populations.

The NF-κB regulator MALT1 determines the encephalitogenic potential of Th17 cells

Effector functions of inflammatory IL-17-producing Th (Th17) cells have been linked to autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS). However, what determines Th17 cell encephalitogenicity is still unresolved. Here, we show that after EAE induction, mice deficient for the NF-κB regulator MALT1 (Malt1-/- mice) exhibit strong lymphocytic infiltration in the CNS, but do not develop any clinical signs of EAE. Loss of Malt1 interfered with expression of the Th17 effector cytokines IL-17 and GM-CSF both in vitro and in vivo. In line with their impaired GM-CSF secretion, Malt1-/- Th cells failed to recruit myeloid cells to the CNS to sustain neuroinflammation, whereas autoreactive WT Th cells successfully induced EAE in Malt1-/- hosts. In contrast, Malt1 deficiency did not affect Th1 cells. Despite their significantly decreased secretion of Th17 effector cytokines, Malt1-/- Th17 cells showed normal expression of lineage-specific transcription factors. Malt1-/- Th cells failed to cleave RelB, a suppressor of canonical NF-κB, and exhibited altered cellular localization of this protein. Our results indicate that MALT1 is a central, cell-intrinsic factor that determines the encephalitogenic potential of inflammatory Th17 cells in vivo.

Low malignant potential tumors with micropapillary features are molecularly similar to low-grade serous carcinoma of the ovary

OBJECTIVE Low-grade serous carcinoma (LGSC) is a chemoresistant ovarian neoplasm thought to potentially arise in a background of low malignant potential tumors (LMP), which are typically non-aggressive. However, LMP with micropapillary features (LMP-MP) have more aggressive clinical behavior and may represent an intermediate in progression to LGSC. The objective of this study was to obtain and compare gene expression profiles of LMP, LMP-MP and LGSC to determine if LMP-MP more closely resembles LGSC, and to identify genes involved in LGSC carcinogenesis. METHODS Epithelial cells from LMP (n=17), LMP-MP (n=9) and LGSC (n=11) were isolated by laser capture microdissection. RNA was extracted, reverse transcribed to cDNA, amplified and hybridized to Affymetrix U133 Plus2 genechip arrays. Gene expression data were checked for quality, filtered and significantly altered genes between subgroups were identified. Differential expression of selected genes was verified by RT-qPCR and immunohistochemistry. RESULTS Gene expression analysis identified differential expression between LMP and LMP-MP, LMP and LGSC but not LMP-MP and LGSC. Integration of differentially expressed genes into the protein interaction database CytoScape highlighted gene products in the MAPK pathway as differentially regulated between LMP and LGSC. Four genes were selected and validated by RT-qPCR performed on microarray samples (n=15) and immunohistochemistry on a representative microarray (n=57). CONCLUSION The gene expression profile of LMP-MP is similar to LGSC and distinct from LMP, reflecting their more aggressive clinical behavior. Candidate genes in the MAPK pathway were highlighted which may play a role in LGSC carcinogenesis and indicate potential therapeutic targets.

Plasminogen Activator uPA Is a Direct Transcriptional Target of the JAG1-Notch Receptor Signaling Pathway in Breast Cancer

Aberrant activation of the Notch receptor signaling pathway and overexpression of the Notch ligand JAG1 are associated with poor outcome in breast cancer. The plasminogen activator system, which includes urokinase-type plasminogen activator (uPA), has been validated as a marker of recurrence, high metastasis risk and death in breast malignancy. By using microarray profiling of breast cancer cell lines that had undergone siRNA-mediated abrogation of Notch signaling we uncovered a link between activated Notch signaling and uPA expression. An association between elevated expression of the Notch ligand JAG1, uPA, and the basal-like breast cancer subtype was confirmed in breast cancer cell lines. The association between JAG1 and uPA expression persisted in a survey of primary carcinomas of the breast. We found that Notch knockdown reduced transcription of uPA and phenocopied uPA knockdown in breast cancer cells. Through mutational analysis we identified a CBF-1 binding site in the uPA promoter that is required for direct transcriptional regulation by Notch. These data suggest that JAG1-induced Notch activation results in breast cancer progression through upregulation of the plasminogen activator system, directly linking these 2 important pathways of poor prognosis.

MicroRNA Expression Profiling of Esophageal Cancer Before and After Induction Chemoradiotherapy

BACKGROUND The prognosis for esophageal cancer is poor but may be improved by neoadjuvant therapy. A complete pathologic response (pCR) is associated with improved survival. We conducted a study to profile the expression of microRNAs (miRNAs) in esophageal cancer before and after induction therapy. Our aims were to identify those miRNAs that are differentially regulated after induction therapy and attempt to describe a miRNA pattern that could predict pCR. METHODS Total RNA was extracted from pretreatment and posttreatment specimens from 25 patients who underwent trimodal therapy using concurrent irinotecan/cisplatin and radiotherapy followed by surgical treatment. miRNAs were labeled and hybridized to the Illumina miRNA BeadChip microarray (Illumina, Inc, San Diego, CA). Expression data was quantified using BeadStudio software (Illumina), using a cutoff for significant gene differences of p less than 0.05 with a 2-fold difference in expression. Survival analysis was performed using SPSS, version 18 (SPSS, Inc, Chicago, IL). RESULTS Using pretreatment biopsy specimens, 71 miRNAs were significantly different between pCR and non-pCR groups. Of these, 5 miRNAs were greater than 2-fold differentially regulated, including miR-296, recently shown to be of prognostic significance in esophageal carcinoma. After induction therapy, 568 miRNAs were found to be significantly upregulated or downregulated, 111 of which had a 2-fold difference. Patients with high levels of miR-135b or miR-145 in the posttreatment biopsy specimens had significantly shorter median disease-free survival (DFS) than did those with low levels (11.5 versus 5.1 months; p=0.04; 11.5 versus 2.8 months; p=0.03). CONCLUSIONS miRNA expression profiling of pretreatment biopsy specimens revealed 5 miRNAs differentially expressed in patients with pCR compared with patients without pCR. We have also identified 111 miRNAs significantly upregulated or downregulated after induction therapy, some of which may be predictive of outcome. Further study of these miRNAs may elucidate a novel understanding of mechanisms of resistance to chemotherapy or radiotherapy.