Linfomas Double-Hit em casuística de Linfomas Não-Hodgkin B de alto grau: estudo clínico, morfológico, imunoistoquímico e citogenético

Pós-graduação em Patologia - FMB

Assessing the gain of biological data integration in gene networks inference

Background: A current challenge in gene annotation is to define the gene function in the context of the network of relationships instead of using single genes. The inference of gene networks (GNs) has emerged as an approach to better understand the biology of the system and to study how several components of this network interact with each other and keep their functions stable. However, in general there is no sufficient data to accurately recover the GNs from their expression levels leading to the curse of dimensionality, in which the number of variables is higher than samples. One way to mitigate this problem is to integrate biological data instead of using only the expression profiles in the inference process. Nowadays, the use of several biological information in inference methods had a significant increase in order to better recover the connections between genes and reduce the false positives. What makes this strategy so interesting is the possibility of confirming the known connections through the included biological data, and the possibility of discovering new relationships between genes when observed the expression data. Although several works in data integration have increased the performance of the network inference methods, the real contribution of adding each type of biological information in the obtained improvement is not clear. Methods: We propose a methodology to include biological information into an inference algorithm in order to assess its prediction gain by using biological information and expression profile together. We also evaluated and compared the gain of adding four types of biological information: (a) protein-protein interaction, (b) Rosetta stone fusion proteins, (c) KEGG and (d) KEGG+GO. Results and conclusions: This work presents a first comparison of the gain in the use of prior biological information in the inference of GNs by considering the eukaryote (P. falciparum) organism. Our results indicates that information based on direct interaction can produce a higher improvement in the gain than data about a less specific relationship as GO or KEGG. Also, as expected, the results show that the use of biological information is a very important approach for the improvement of the inference. We also compared the gain in the inference of the global network and only the hubs. The results indicates that the use of biological information can improve the identification of the most connected proteins.

Mechanical loading induces the expression of a Pol I regulon at the onset of skeletal muscle hypertrophy

von Walden F, Casagrande V, Ostlund Farrants AK, Nader GA. Mechanical loading induces the expression of a Pol I regulon at the onset of skeletal muscle hypertrophy. Am J Physiol Cell Physiol 302: C1523-C1530, 2012. First published March 7, 2012; doi:10.1152/ajpcell.00460.2011.-The main goal of the present study was to investigate the regulation of ribosomal DNA (rDNA) gene transcription at the onset of skeletal muscle hypertrophy. Mice were subjected to functional overload of the plantaris by bilateral removal of the synergist muscles. Mechanical loading resulted in muscle hypertrophy with an increase in rRNA content. rDNA transcription, as determined by 45S pre-rRNA abundance, paralleled the increase in rRNA content and was consistent with the onset of the hypertrophic response. Increased transcription and protein expression of c-Myc and its downstream polymerase I (Pol I) regulon (POL1RB, TIF-1A, PAF53, TTF1, TAF1C) was also consistent with the increase in rRNA. Similarly, factors involved in rDNA transcription, such as the upstream binding factor and the Williams syndrome transcription factor, were induced by mechanical loading in a corresponding temporal fashion. Chromatin immunoprecipitation revealed that these factors, together with Pol I, were enriched at the rDNA promoter. This, in addition to an increase in histone H3 lysine 9 acetylation, demonstrates that mechanical loading regulates rRNA synthesis by inducing a gene expression program consisting of a Pol I regulon, together with accessory factors involved in transcription and chromatin remodeling at the rDNA promoter. Altogether, these data indicate that transcriptional and epigenetic mechanisms take place in the regulation of ribosome production at the onset of muscle hypertrophy.

Combined snake venomics and venom gland transcriptomic analysis of Bothropoides pauloensis

Unraveling the repertoire of venom toxins of Bothropoides pauloensis was assessed by snake venomics and venom gland transcriptomic surveys. Both approaches yielded converging overall figures, pointing to metalloproteinases (similar to 37%), PLA(2)s (26-32%), and vasoactive (bradykinin-potentiating) peptides (12-17%) as the major toxin classes. The high occurrence of SVMPs, PLA(2) molecules, vasoactive peptides, along with serine proteinases, explains the local and systemic effects observed in envenomations by B. pauloensis. Minor (<3%) C-type lectin, serine proteinase, L-amino acid oxidase, nerve growth factor, and CRISP molecules were also identified in the transcriptome and the proteome. Low abundance (0.3%) EST singletons coding for vascular endothelial growth factor (svVEGF), ohanin, hyaluronidase, and 5' nucleotidase were found only in the venom gland cDNA library. At the molecular level, the transcriptomic and proteomic datasets display low compositional concordance. In particular, although there is good agreement between transcriptome and proteome in the identity of BPPs, PLA(2) molecules and L-amino acid oxidase, both datasets strongly depart in their C-type lectin and SVMP complements. These data support the view that venom composition is influenced by transcriptional and translational mechanisms and emphasize the value of combining proteomic and transcriptomic approaches to acquire a more complete understanding of the toxinological profile and natural history of the snake venom. (C) 2012 Elsevier B.V. All rights reserved.

Translocation capture sequencing: A method for high throughput mapping of chromosomal rearrangements

Chromosomal translocations require formation and joining of DNA double strand breaks (DSBs). These events disrupt the integrity of the genome and are involved in producing leukemias, lymphomas and sarcomas. Translocations are frequent, clonal and recurrent in mature B cell lymphomas, which bear a particularly high DNA damage burden by virtue of activation-induced cytidine deaminase (AID) expression. Despite the ubiquity of genomic rearrangements, the forces that underlie their genesis are not well understood. Here, we provide a detailed description of a new method for studying these events, translocation capture sequencing (TC-Seq). TC-Seq provides the means to document chromosomal rearrangements genome-wide in primary cells, and to discover recombination hotspots. Demonstrating its effectiveness, we successfully estimate the frequency of c-myc/IgH translocations in primary B cells, and identify hotspots of AID-mediated recombination. Furthermore. TC-Seq can be adapted to generate genome-wide rearrangement maps in any cell type and under any condition. (C) 2011 Elsevier B.V. All rights reserved.

Clinical implication of 14-3-3 epsilon expression in gastric cancer

AIM: To evaluate for the first time the protein and mRNA expression of 14-3-3 epsilon in gastric carcinogenesis. METHODS: 14-3-3 epsilon protein expression was determined by western blotting, and mRNA expression was examined by real-time quantitative RT-PCR in gastric tumors and their matched non-neoplastic gastric tissue samples. RESULTS: Authors observed a significant reduction of 14-3-3 epsilon protein expression in gastric cancer (GC) samples compared to their matched non-neoplastic tissue, Reduced levels of 14-3-3 epsilon were also associated with diffuse-type GC and early-onset of this pathology. Our data suggest that reduced 14-3-3 epsilon may have a role in gastric carcinogenesis process. CONCLUSION: Our results reveal that the reduced 14-3-3 epsilon expression in GC and investigation of 14-3-3 epsilon interaction partners may help to elucidate the carcinogenesis process. (C) 2012 Baishideng. All rights reserved.

Forced Expression of Nanog in Human Bone Marrow-Derived Endothelial Cells Activates Other Six Pluripotent Genes

Human endothelial cells (ECs) have the ability to make up the lining of blood vessels. These cells are also capable of neovascularization and revascularization and have been applied in various clinical situations. With the aim of understanding the effect of NANOG superexpression on ECs, we transduced the Nanog gene into the ECs. Nanog is highly expressed in embryonic stem cells (ESCs) and is essential for pluripotency and self-renewal. However, Nanog can also be expressed in somatic stem cells, and this gene is related to great expansion capacity in vitro. We found that ECs expressing Nanog showed expression of other stemness genes, such as Sox2, FoxD3, Oct4, Klf4, c-myc, and beta-catenin, that are not normally expressed or are expressed at very low levels in ECs. Nanog is one of the stemness genes that can activate other stemness genes, and the upregulation of the Nanog gene seems to be critical for reprogramming cells. In this study, the introduction of Nanog was sufficient to alter the expression of key genes of the pluripotent pathway. The functional importance of Nanog for altering the cell expression profile and morphology was clearly demonstrated by our results.

Biochemical, physicochemical and molecular characterization of a genuine 2-Cys-peroxiredoxin purified from cowpea [Vigna unguiculata (L.) Walpers] leaves

Background: Peroxiredoxins have diverse functions in cellular defense-signaling pathways. 2-Cys-peroxiredoxins (2-Cys-Prx) reduce H2O2 and alkyl-hydroperoxide. This study describes the purification and characterization of a genuine 2-Cys-Prx from Vigna unguiculata (Vu-2-Cys-Prx). Methods: Vu-2-Cys-Prx was purified from leaves by ammonium sulfate fractionation, chitin affinity and ion exchange chromatography. Results: Vu-2-Cys-Prx reduces H2O2 using NADPH and DTT. Vu-2-Cys-Prx is a 44 kDa (SDS-PAGE)/46 kDa (exclusion chromatography) protein that appears as a 22 kDa molecule under reducing conditions, indicating that it is a homodimer linked intermolecularly by disulfide bonds and has a pI range of 4.56-4.72; its NH2-terminal sequence was similar to 2-Cys-Prx from Phaseolus vulgaris (96%) and Populus tricocarpa (96%). Analysis by ESI-Q-TOF MS/MS showed a molecular mass/pI of 28.622 kDa/5.18. Vu-2-Cys-Prx has 8% alpha-helix, 39% beta-sheet, 22% of turns and 31% of unordered forms. Vu-2-Cys-Prx was heat stable, has optimal activity at pH 7.0, and prevented plasmid DNA degradation. Atomic force microscopy shows that Vu-2-Cys-Prx oligomerized in decamers which might be associated with its molecular chaperone activity that prevented denaturation of insulin and citrate synthase. Its cDNA analysis showed that the redox-active Cys(52) residue and the amino acids Pro(45), Thr(49) and Arg(128) are conserved as in other 2-Cys-Prx. General significance: The biochemical and molecular features of Vu-2-Cys-Prx are similar to other members of 2-Cys-Prx family. To date, only one publication reported on the purification of native 2-Cys-Prx from leaves and the subsequent analysis by N-terminal Edman sequencing, which is crucial for construction of stromal recombinant 2-Cys-Prx proteins. (C) 2012 Elsevier B.V. All rights reserved.

Funktionelle Zusammenhänge zwischen Expressionsveränderungen des von Hippel-Lindau Tumorsuppressorgens und dessen potentiellen Zielgenen in sporadischen Nierenzellkarzinomen

ZusammenfassungNierenzellkarzinome (NZK) sind häufig gekennzeichnet durch den Funktionsverlust des von Hippel-Lindau Tumorsuppressorgens (VHL TSG). Man kennt heute eine Reihe potentieller Kandidatengene, die eventuell in Wechselwirkung mit dem VHL-Genprodukt stehen (VEGF, c-fos, c-myc). Zur Aufklärung der molekularen Vorgänge, die zur Pathogenese der gut vaskularisierten Nierenzellkarzinome beitragen, wurden NZK-Zellinien untersucht. Vektorkonstrukte wurden generiert, um das in vitro und in vivo Wachstumsverhalten vor und nach Transfektion zu beobachten. Mit Hilfe eines GFP-Fusionsproteins wurden zusätzlich Hinweise auf die intrazelluläre Lokalisation des VHL-Genproduktes (pVHL) geliefert. Nach Abschluß der vorliegenden Studien zeigte sich eine direkte Abhängigkeit der beiden Gene VHL und VEGF. Sowohl die in vitro als auch die in vivo Ansätze bewiesen eine Interaktion der beiden Gene. Die Frage nach einer Interaktion von pVHL mit den Genprodukten Fos und Myc läßt sich jedoch nicht eindeutig beantworten. Die Expressionsveränderungen dieser Gene erfolgten scheinbar willkürlich, eine Interaktion konnte nicht bestätigt werden.Die Lokalisation des VHL-Genproduktes ist von Fall zu Fall unterschiedlich, ein Zusammenhang mit den verschiedenen Funktionen des VHL-Gens ist demnach wahrscheinlich. Zusammenfassend läßt sich sagen, daß das VHL TSG eine Rolle bei der Kontrolle der Angiogenese spielt, eine Interaktion mit VEGF ist mehr als wahrscheinlich. Zukünftig sind bei der Aufklärung des Pathomechanismus von Nierenzellkarzinomen deshalb vor allem Studien nötig, die dem VHL TSG als Angiogeneseinhibitor eine zentrale Bedeutung bei der Heilung sporadischer Tumoren mit starker Vaskularisierung zukommen lassen.

Untersuchungen zur Pathogenese der "Bypass graft disease"

Untersuchung zur Pathogenese der 'Bypass graft disease' Histomorphologische Untersuchungen und in vitro Zellkulturanalysen bilden die Grundlage für Fortschritte im Verständnis der pathologischen Mechanismen der aortokoronaren 'Bypass graft disease'. In der vorliegenden Arbeit wurde die pathomorphologische Veränderung der Gefäßanatomie im Verlauf der 'Bypass graft disease' an Hand histologischer Präparate explantierter humaner venöser Bypass-Läsionen analysiert. Erstmalig wurde ein histomorphologisches Klassifizierungsschema (Typ I - Typ III) beschrieben. Morphometrische Analysen zeigten, dass die Fläche der Neointima und Media im Verlauf der pathologischen Umgestaltung der Bypass-Architektur (Typ I zu Typ III) deutlich zunimmt. Bestimmungen der Zelldichte dokumentierten eine deutlich größere Zellzahl in allen Gefäßwandschichten der Bypass-Läsionen bei der Gegenüberstellung mit einer Kontrollgruppe nativer Venen, wobei im Verlauf der 'Bypass graft disease' (Typ I zu Typ III) eine Abnahme der Zelldichte zu beobachten war. Erstmalig durchgeführte Untersuchungen zur Proliferationsaktivität in aortokoronaren Bypass-Läsionen im Vergleich zu nativen Gefäßen präsentierten eine deutlich höhere zelluläre Proliferation in den Bypass-Präparaten. Diese war am stärksten in Typ III Läsionen ausgeprägt. Expressionsstudien im in vitro Zellkulturmodellsystem identifiziereten die homodimeren Isotypen (AA / BB) des Wachstumsfaktors PDGF als Stimulatoren der Transkriptionsfaktoren c-fos und c-myc in primärkultivierten humanen Muskelzellen der Aorta.

Transcriptional functions of DNA Topoisomerases at a genome-wide scale and a single gene levels

The DNA topology is an important modifier of DNA functions. Torsional stress is generated when right handed DNA is either over- or underwound, producing structural deformations which drive or are driven by processes such as replication, transcription, recombination and repair. DNA topoisomerases are molecular machines that regulate the topological state of the DNA in the cell. These enzymes accomplish this task by either passing one strand of the DNA through a break in the opposing strand or by passing a region of the duplex from the same or a different molecule through a double-stranded cut generated in the DNA. Because of their ability to cut one or two strands of DNA they are also target for some of the most successful anticancer drugs used in standard combination therapies of human cancers. An effective anticancer drug is Camptothecin (CPT) that specifically targets DNA topoisomerase 1 (TOP 1). The research project of the present thesis has been focused on the role of human TOP 1 during transcription and on the transcriptional consequences associated with TOP 1 inhibition by CPT in human cell lines. Previous findings demonstrate that TOP 1 inhibition by CPT perturbs RNA polymerase (RNAP II) density at promoters and along transcribed genes suggesting an involvement of TOP 1 in RNAP II promoter proximal pausing site. Within the transcription cycle, promoter pausing is a fundamental step the importance of which has been well established as a means of coupling elongation to RNA maturation. By measuring nascent RNA transcripts bound to chromatin, we demonstrated that TOP 1 inhibition by CPT can enhance RNAP II escape from promoter proximal pausing site of the human Hypoxia Inducible Factor 1 (HIF-1) and c-MYC genes in a dose dependent manner. This effect is dependent from Cdk7/Cdk9 activities since it can be reversed by the kinases inhibitor DRB. Since CPT affects RNAP II by promoting the hyperphosphorylation of its Rpb1 subunit the findings suggest that TOP 1inhibition by CPT may increase the activity of Cdks which in turn phosphorylate the Rpb1 subunit of RNAP II enhancing its escape from pausing. Interestingly, the transcriptional consequences of CPT induced topological stress are wider than expected. CPT increased co-transcriptional splicing of exon1 and 2 and markedly affected alternative splicing at exon 11. Surprisingly despite its well-established transcription inhibitory activity, CPT can trigger the production of a novel long RNA (5’aHIF-1) antisense to the human HIF-1 mRNA and a known antisense RNA at the 3’ end of the gene, while decreasing mRNA levels. The effects require TOP 1 and are independent from CPT induced DNA damage. Thus, when the supercoiling imbalance promoted by CPT occurs at promoter, it may trigger deregulation of the RNAP II pausing, increased chromatin accessibility and activation/derepression of antisense transcripts in a Cdks dependent manner. A changed balance of antisense transcripts and mRNAs may regulate the activity of HIF-1 and contribute to the control of tumor progression After focusing our TOP 1 investigations at a single gene level, we have extended the study to the whole genome by developing the “Topo-Seq” approach which generates a map of genome-wide distribution of sites of TOP 1 activity sites in human cells. The preliminary data revealed that TOP 1 preferentially localizes at intragenic regions and in particular at 5’ and 3’ ends of genes. Surprisingly upon TOP 1 downregulation, which impairs protein expression by 80%, TOP 1 molecules are mostly localized around 3’ ends of genes, thus suggesting that its activity is essential at these regions and can be compensate at 5’ ends. The developed procedure is a pioneer tool for the detection of TOP 1 cleavage sites across the genome and can open the way to further investigations of the enzyme roles in different nuclear processes.

Analisi molecolare dei linfomi cutanei aggressivi

I linfomi primitivi cutanei riconosciuti nella classificazione della WHO/EORTC si presentano come “entità cliniche distinte” su base clinica, morfologica, immunofenotipica e molecolare. Il fenotipo linfocitario T helper CD4+ caratterizza i CTCL, ma alcune entità a prognosi aggressiva presentano un immunofenotipo citotossico CD8+. Numerosi studi di citogenetica (CGH) e gene-expression profiling (GEP) sono stati condotti negli ultimi anni sui CTCL e sono state riscontrate numerose aberrazioni cromosomiche correlate ai meccanismi di controllo del ciclo cellulare. Scopo del nostro studio è la valutazione delle alterazioni genomiche coinvolte nella tumorigenesi di alcuni CTCL aggressivi: il linfoma extranodale NK/T nasal-type, il linfoma primitivo cutaneo aggressivo epidermotropo (AECTCL) e il gruppo dei PTCL/NOS pleomorfo CD8+. Il materiale bioptico dei pazienti è stato sottoposto alla metodica dell’array-CGH per identificare le anomalie cromosomiche; in alcuni casi di AECTCL è stata applicata la GEP, che evidenzia il profilo di espressione genica delle cellule neoplastiche. I dati ottenuti sono stati valutati in modo statistico, evidenziando le alterazioni cromosomiche comuni significative di ogni entità. In CGH, sono state evidenziate alcune aberrazioni comuni fra le entità studiate, la delezione di 9p21.3, l’amplificazione di 17q, 19p13, 19q13.11-q13.32 , 12q13 e 16p13.3, che determinano la delezione dei geni CDKN2A e CDKN2B e l’attivazione del JAK/STAT signaling pathway. Altre alterazioni definiscono l’amplificazione di c-MYC (8q24) e CCND1/CDK4-6 (11q13). In particolare, sono state evidenziate numerose anomalie genomiche comuni in casi di AECTCL e PTCL/NOS pleomorfo. L’applicazione della GEP in 5 casi di AECTCL ha confermato l’alterata espressione dei geni CDKN2A, JAK3 e STAT6, che potrebbero avere un ruolo diretto nella linfomagenesi. Lo studio di un numero maggiore di casi in GEP e l’introduzione delle nuove indagini molecolari come l’analisi dei miRNA, della whole-exome e whole genome sequences consentiranno di evidenziare alterazioni molecolari correlate con la prognosi, definendo anche nuovi target terapeutici.

Spezifische Inhibition des Wnt-Signalwegs und die Analyse der Konsequenzen auf zellbiologischer Ebene

Der kanonische Wnt Signalweg ist durch Regulation einer Vielzahl von Zielgenen in unterschiedliche Prozesse wie Entwicklung, Wachstum und Differenzierung involviert. Fehlregulation des Signalwegs kann zur Tumorentstehung führen. Die exakte Rolle des Wnt Signalwegs und seiner Zielgene in der karzinogenen Kaskade ist noch nicht genau bekannt. In dieser Arbeit sollte die Beteiligung der Wnt Zielgene c-MYC, CCND1 (kodiert Cyclin D1) und VEGF an der Karzinogenese untersucht werden. Um die Funktionen der Wnt Zielgene und ihre zellulären Effekte unabhängig voneinander untersuchen zu können, wurden die Mengen der entsprechenden Transkriptionsprodukte durch siRNA (short interfering RNA) gezielt verringert. Die Konsequenzen der Inaktivierung wurden in Kolon- und Zervixkarzinomzelllinien untersucht, wobei die zellulären Parameter Proliferation, Apoptose, Metabolismus sowie Migration und Adhäsion untersucht wurden. Dabei konnte beobachtet werden, dass der Wnt Signalweg mit seinen Zielgenen Cyclin D1 und c-MYC die Proliferation mit dem Energiemetabolismus von Tumorzellen verknüpft. Darüber hinaus konnte gezeigt werden, dass Cyclin D1 an der Regulation der zelluläre Migration und Adhäsion beteiligt ist, während VEGF die Apoptose abhängig vom zellulären Kontext inhibiert. Diese Ergebnisse liefern erste Hinweise auf die funktionelle Rolle der verschiedenen Zielgene im Prozess der Karzinogenese in Tumoren mit aktiviertem Wnt Signalweg. Damit ist diese Arbeit ein möglicher Ausgangspunkt für Studien mit dem Ziel der gezielten therapeutischen Beeinflussung des Wnt Signalwegs auf Ebene der Zielgene.

The KIT gene in familial mastocytosis

Familial cutaneous mastocytosis is an exceptional condition of unknown etiology. In this study we report the largest series of patients with familial cutaneous mastocytosis without other manifestations (18 affected subjects from seven unrelated families), and we investigate the role of germ-line KIT mutations in the pathogenesis of the disease. The mean age at onset was 5.4 years (range from birth to 22 years), and the clinical behavior was variable over a mean follow up period of 15.1 years (range 2-36): improvement in seven, stability in eight and worsening in the remaining three patients. The pattern of inheritance was compatible with an autosomal dominant trait with incomplete penetrance; a female preponderance (14 females vs 4 males, ratio 3.5:1) was noted; among the six women who have been pregnant at least once, three experienced important clinical changes during pregnancy. No germ-line mutation was found in the exons 10, 11, and 17 of the KIT proto-oncogene, which are the most commonly mutated exons in sporadic mastocytosis. However, in the majority of affected subjects we found the Met541Leu polymorphic variant of the KIT gene, which seems to confer a growth advantage to mast cells in vitro. This observation further suggests that the Met541Leu may be a predisposing factor of cutaneous mastocytosis, although it seems to be neither necessary nor sufficient for the development of the disease.

Ruolo della Interleuchina-6 nella colite ulcerosa long-standing: regolazione della p53 ed induzione delle cellule mesenchimali epiteliali

L’infiammazione cronica è un fattore di rischio di insorgenza del cancro, e la citochina infiammatoria IL-6 gioca un ruolo importante nella tumorigenesi. In questo studio abbiamo dimostrato che L’IL-6 down-regola l'espressione e l'attività di p53. In linee cellulari umane, IL-6 stimola la trascrizione dell’rRNA mediante espressione della proteina c-myc a livello post-trascrizionale in un meccanismo p38MAPK-dipendente. L'up-regolazione della biogenesi ribosomiale riduce l'espressione di p53 attraverso l'attivazione della via della proteina ribosomale-MDM2. La down-regolazione di p53 produce l’acquisizione di modifiche fenotipiche e funzionali caratteristiche della epitelio mesenchimale di transizione, un processo associato a trasformazione maligna e progressione tumorale. I nostri dati mostrano che questi cambiamenti avvengono anche nelle cellule epiteliali del colon di pazienti affetti da colite ulcerosa, un esempio rappresentativo di una infiammazione cronica soggetta a trasformazione neoplastica, che scompaiono dopo trattamento con farmaci antinfiammatori. Questi risultati svelano un nuovo effetto oncogenico indotto dall’IL-6 che può contribuire notevolmente ad aumentare il rischio di sviluppare il cancro non solo in pazienti con infiammazioni croniche, ma anche in quei pazienti con condizioni patologiche caratterizzate da elevato livello di IL-6 nel plasma, quali l'obesità e e il diabete mellito di tipo 2.