Genome-wide gene expression profiling of testicular carcinoma in situ progression into overt tumours

The carcinoma in situ (CIS) cell is the common precursor of nearly all testicular germ cell tumours (TGCT). In a previous study, we examined the gene expression profile of CIS cells and found many features common to embryonic stem cells indicating that initiation of neoplastic transformation into CIS occurs early during foetal life. Progression into an overt tumour, however, typically first happens after puberty, where CIS cells transform into either a seminoma (SEM) or a nonseminoma (N-SEM). Here, we have compared the genome-wide gene expression of CIS cells to that of testicular SEM and a sample containing a mixture of N-SEM components, and analyse the data together with the previously published data on CIS. Genes showing expression in the SEM or N-SEM were selected, in order to identify gene expression markers associated with the progression of CIS cells. The identified markers were verified by reverse transcriptase–polymerase chain reaction and in situ hybridisation in a range of different TGCT samples. Verification showed some interpatient variation, but combined analysis of a range of the identified markers may discriminate TGCT samples as SEMs or N-SEMs. Of particular interest, we found that both DNMT3B (DNA (cytosine-5-)-methyltransferase 3 beta) and DNMT3L (DNA (cytosine-5-)-methyltransferase 3 like) were overexpressed in the N-SEMs, indicating the epigenetic differences between N-SEMs and classical SEM

Genomic copy number and expression patterns in testicular germ cell tumours

Testicular germ cell tumours of adults and adolescents (TGCT) include seminomas (SE) and nonseminomas (NS), with spermatocytic seminomas (SSE) representing a distinct entity in older men. SE and NS have gain of 12p material in all cases, whereas SSE are associated with overrepresentation of chromosome 9. Here, we compare at the chromosomal level, copy number imbalances with global expression changes, identified by comparative expressed sequence hybridisation analyses, in seven SE, one combined tumour, seven NS and seven cell lines. Positive correlations were found consistent with copy number as a main driver of expression change, despite reported differences in methylation status in SE and NS. Analysis of chromosomal copy number and expression data could not distinguish between SE and NS, in-keeping with a similar genetic pathogenesis. However, increased expression from 4q22, 5q23.2 and 9p21 distinguished SSE from SE and NS and decreased copy number and expression from 2q36–q37 and 6q24 was a specific feature of NS-derived cell lines. Our analysis also highlights 19 regions with both copy number and expression imbalances in greater than 40% of cases. Mining available expression array data identified genes from these regions as candidates for involvement in TGCT development. Supplementary data is available at http://www.crukdmf.icr.ac.uk/array/array.html

Prioritizing single-nucleotide variations that potentially regulate alternative splicing

Recent evidence suggests that many complex diseases are caused by genetic variations that play regulatory roles in controlling gene expression. Most genetic studies focus on nonsynonymous variations that can alter the amino acid composition of a protein and are therefore believed to have the highest impact on phenotype. Synonymous variations, however, can also play important roles in disease pathogenesis by regulating pre-mRNA processing and translational control. In this study, we systematically survey the effects of single-nucleotide variations (SNVs) on binding affinity of RNA-binding proteins (RBPs). Among the 10,113 synonymous SNVs identified in 697 individuals in the 1,000 Genomes Project and distributed by Genetic Analysis Workshop 17 (GAW17), we identified 182 variations located in alternatively spliced exons that can significantly change the binding affinity of nine RBPs whose binding preferences on 7-mer RNA sequences were previously reported. We found that the minor allele frequencies of these variations are similar to those of nonsynonymous SNVs, suggesting that they are in fact functional. We propose a workflow to identify phenotype-associated regulatory SNVs that might affect alternative splicing from exome-sequencing-derived genetic variations. Based on the affecting SNVs on the quantitative traits simulated in GAW17, we further identified two and four functional SNVs that are predicted to be involved in alternative splicing regulation in traits Q1 and Q2, respectively

Splice variants as novel targets in pancreatic ductal adenocarcinoma

The study was funded by the MolDiagPaCa European Union Framework Programme and CR-UK Programme grant A12008 from CR-UK (C. Chelala, T. Crnogorac-Jurcevic, and N.R. Lemoine). Italian Cancer Genome Project – Ministry of University [FIRB RBAP10AHJB]; Associazione Italiana Ricerca Cancro [grant number: 12182]; FP7 European Community Grant Cam-Pac [no: 602783]; Italian Ministry of Health [FIMPCUP_J33G13000210001]. The funders were not involved in the design of the study, collection, analysis, and interpretation of data and in writing of the manuscript. We thank Tracy Chaplin-Perkins for help with running the Affymetrix experiments

Aza-deoxycytidine induces apoptosis or differentiation via DNMT3B and targets embryonal carcinoma cells but not their differentiated derivatives

Background: Teratocarcinoma is a malignant male germ cell tumour, which contains stem cells and differentiated cancer tissues. DNMT3B has been shown to be highly expressed in human teratocarcinoma stem cells, and to mediate cytotoxicity of Aza-deoxycytidine (Aza-dC) in a pluripotent stem cell line NTERA2. Methods: We have established DNMT3B or POU5F1 (hereafter referred to as OCT4) knockdown in teratocarcinoma stem cells N2102Ep and TERA1 and in the pluripotent NTERA2 by a doxycycline-inducible system, and tested the cytotoxicity induced by Aza-dC. Results: Silencing of DNMT3B led to apoptosis of human teratocarcinoma stem cells N2102Ep and TERA1. Further, we found that induction of apoptosis or differentiation in NTERA2 and human embryonic stem cells by Aza-dC requires DNMT3B. To test whether Aza-dC inhibits proliferation of differentiated teratocarcinoma cells, we depleted OCT4 expression in N2102Ep and TERA1 cells treated with Aza-dC. Treatment with Aza-dC reduced cell number of differentiated cells to a lesser extent than their undifferentiated parental stem cells. Moreover, in contrast to the stem cells, Aza-dC failed to induce apoptosis of differentiated cells. Conclusions: Our finding suggests that DNMT3B acts as an antiapoptotic gene in teratocarcinoma stem cells, and mediates apoptosis and differentiation of human pluripotent stem cells induced by Aza-dC, and that Aza-dC specifically induces apoptosis of teratocarcinoma stem cells

Increased expression of urokinase plasminogen activator and its cognate receptor in human seminomas

<p>Abstract</p> <p>Background</p> <p>The urokinase plasminogen activating system (uPAS) is implicated in neoplastic progression and high tissue levels of uPAS components correlate with a poor prognosis in different human cancers. Despite that, relative few studies are available on the expression and function of the uPAS components in human seminomas. In the present study we characterized the expression of the urokinase plasminogen activator (uPA), its cognate receptor (uPAR) and the uPA inhibitors PAI-1 and PAI-2 in normal human testis and seminomas.</p> <p>Methods</p> <p>The expression of the above genes was evaluated by means of quantitative RT-PCR, western blot, zymographic analysis and immunohistochemistry.</p> <p>Results</p> <p>Quantitative RT-PCR analysis of 14 seminomas demonstrated that uPA and uPAR mRNAs were, with respect to control tissues, increased in tumor tissues by 3.80 ± 0.74 (p < 0.01) and 6.25 ± 1.18 (p < 0.01) fold, respectively. On the other hand, PAI-1 mRNA level was unchanged (1.02 ± 0.24 fold), while that of PAI-2 was significantly reduced to 0.34 ± 0.18 (p < 0.01) fold. Western blot experiments performed with protein extracts of three seminomas and normal tissues from the same patients showed that uPA protein levels were low or undetectable in normal tissues and induced in tumor tissues. On the same samples, zymographic analysis demonstrated increased uPA activity in tumor tissue extracts. Western blot experiments showed that also the uPAR protein was increased in tumor tissues by 1.83 ± 0.15 fold (p < 0.01). The increased expression of uPA and uPAR was further confirmed by immunohistochemical staining performed in 10 seminomas and autologous uninvolved peritumoral tissues. Finally, variation in the mRNA level of PAI-1 significantly correlated with tumor size.</p> <p>Conclusions</p> <p>We demonstrated the increased expression of uPA and uPAR in human seminomas with respect to normal testis tissues, which may be relevant in testicular cancer progression.</p

Human pluripotent embryonal carcinoma NTERA2 cl.D1 cells maintain their typical morphology in an angiomyogenic medium

BACKGROUND: Pluripotent embryonal carcinomas are good potential models, to study, "in vitro," the mechanisms that control differentiation during embryogenesis. The NTERA2cl.D1 (NT2/D1) cell line is a well known system of ectodermal differentiation. Retinoic acid (RA) induces a dorsal pattern of differentiation (essentially neurons) and bone morphogenetic protein (BMP) or hexamethylenebisacetamide (HMBA) induces a more ventral (epidermal) pattern of differentiation. However, whether these human cells could give rise to mesoderm derivatives as their counterpart in mouse remained elusive. We analyzed the morphological characteristics and transcriptional activation of genes pertinent in cardiac muscle and endothelium differentiation, during the growth of NT2/D1 cells in an inductive angiomyogenic medium with or without Bone Morphogenetic Protein 2 (BMP2). RESULTS: Our experiments showed that NT2/D1 maintains their typical actin organization in angiomyogenic medium. Although the beta myosin heavy chain gene was never detected, all the other 15 genes analyzed maintained their expression throughout the time course of the experiment. Among them were early and late cardiac, endothelial, neuronal and teratocarcinoma genes. CONCLUSION: Our results suggest that despite the NT2/D1 cells natural tendency to differentiate into neuroectodermal lineages, they can activate genes of mesodermal lineages. Therefore, we believe that these pluripotent cells might still be a good model to study biological development of mesodermal derivatives, provided the right culture conditions are met

Width of Gene Expression Profile Drives Alternative Splicing

Alternative splicing generates an enormous amount of functional and proteomic diversity in metazoan organisms. This process is probably central to the macromolecular and cellular complexity of higher eukaryotes. While most studies have focused on the molecular mechanism triggering and controlling alternative splicing, as well as on its incidence in different species, its maintenance and evolution within populations has been little investigated. Here, we propose to address these questions by comparing the structural characteristics as well as the functional and transcriptional profiles of genes with monomorphic or polymorphic splicing, referred to as MS and PS genes, respectively. We find that MS and PS genes differ particularly in the number of tissues and cell types where they are expressed.We find a striking deficit of PS genes on the sex chromosomes, particularly on the Y chromosome where it is shown not to be due to the observed lower breadth of expression of genes on that chromosome. The development of a simple model of evolution of cis-regulated alternative splicing leads to predictions in agreement with these observations. It further predicts the conditions for the emergence and the maintenance of cis-regulated alternative splicing, which are both favored by the tissue specific expression of splicing variants. We finally propose that the width of the gene expression profile is an essential factor for the acquisition of new transcript isoforms that could later be maintained by a new form of balancing selection