AFV1, a novel virus infecting hyperthermophilic archaea of the genus acidianus

AbstractWe describe a novel virus, AFV1, of the hyperthermophilic archaeal genus Acidianus. Filamentous virions are covered with a lipid envelope and contain at least five different proteins with molecular masses in the range of 23–130 kDa and a 20.8-kb-long linear double-stranded DNA. The virus has been assigned to the family Lipothrixviridae on the basis of morphotypic characteristics. Host range is confined to several strains of Acidianus and the virus persists in its hosts in a stable carrier state. The latent period of virus infection is about 4 h. Viral DNA was sequenced and sequence similarities were found to the lipothrixvirus SIFV, the rudiviruses SIRV1 and SIRV2, as well as to conjugative plasmids and chromosomes of the genus Sulfolobus. Exceptionally for the linear genomes of archaeal viruses, many short direct repeats, with the sequence TTGTT or close variants thereof, are closely clustered over 300 bp at each end of the genome. They are reminiscent of the telomeric ends of linear eukaryal chromosomes

Molekularbiologische Untersuchungen von dysreguliert-exprimierten Genen im kolorektalen Karzinom

Diese Dissertation befasst sich mit fehlregulierten Genen im kolorektalen Karzinom (CRC). Das Ziel der Arbeit war die molekularbiologische Charakterisierung dysreguliert-exprimierter Gene im CRC, um neue Einblicke in die Biologie von kolorektalen Tumoren zu gewinnen und um neue diagnostische und prädiktive Marker zu identifizieren. Die Analysen von Expression, Funktion und Bedeutung von Maspin, das in CRC unerforscht war, stellten den Schwerpunkt dieser Dissertation dar. Maspin wird in CRC tumorspezifisch überexprimiert. Besonders hohe Expression zeigten undifferenzierte Tumore, Mikrosatelliten-instabile (MSI) Tumore, MSI CRC Zelllinien, Tumorzellen der Invasionsfront und disseminierte Tumorzellen. Die Aktivierung der Maspinexpression erfolgt durch die Demethylierung des Maspinpromotors. Die Überexpression bewirkte die Ausbildung von Pseudopodien, die Änderung von der Wuchsform als Monolayer zu einer sphäroiden Wuchsform mit starker interzellulärer Adhäsion und hatte eine Verringerung der Proliferationsrate sowie eine langsamere Adhäsion zur Folge. Die Maspin-supprimierten Klone bildeten die Pseudopodien zurück und zeigten beschleunigte Proliferation. Eine hohe Maspinexpression korrelierte mit gesteigerter Motilität und Invasivität, wobei Maspin seine invasionsverstärkende Wirkung an der Zelloberfläche entfaltet. In vitro korreliert eine hohe Maspinexpression mit einer höheren Resistenz gegen das Chemotherapeutikum 5-Fluoruracil (5-FU). Dabei beeinflusste die Maspinexpression die Expression der Gene des 5-FU-Stoffwechsels. Die Expression der 5-FU-katabolisierenden Dihydropyrimidindehydrogenase war in den überexprimierenden Klonen stark erhöht, in den supprimierten Klonen hingegen vollständig blockiert. Bei dem Hauptzielmolekül von 5-FU, der Thymidylatsynthase, wurde als Folge der Maspin-Überexpression eine erhöhte Expression und bei den supprimierten Klonen entsprechend eine erniedrigte Expression festgestellt. Die Maspin-Überexpression bewirkte zudem die Expressionsblockierung der Thymidinphosphorylase, welche die 5-FU aktiviert. Ferner war die Maspin-Überexpression mit einer transkriptionellen Blockierung von der MMP2 und MMP3 assoziiert und die Maspin-Suppression ging mit dem Expressionsausfall von TIMP1 und TIMP3 einher. Die Daten der Array-basierten Transkriptomanalysen an CRC zeigten einen signifikanten Expressionsanstieg von TS, DPD, TP, MMP1, TIMP1 und MMP10 bei Tumoren mit starker Maspinexpression. Die Sequenzanalysen von Tumoren, Normalgewebe und CRC-Zelllinien deckten einen S/P Polymorphismus der Aminosäure 144 auf, konnten aber keinen Hinweis auf die Entstehung eines Kernlokalisationssignals, auf Mutationen innerhalb der reaktiven Seitenkette von Maspin oder auf nonsense-Mutationen liefern. Die nukleäre Lokalisation von Maspin tritt spezifisch bei dedifferenzierten Tumoren und invasiven Tumorzellen auf. In einer retrospektiven Studie an Stadium III Kolonkarzinom-Patienten erwies sich die nukleäre Maspinlokalisation als ein unabhängiger Marker für eine schlechte Prognose ohne eine 5-FU-basierte Chemotherapie. Nur Patienten mit positiver Maspin-Kernfärbung profitierten von einer Chemotherapie. In dieser Dissertation wurden zwei neue quantitative DNA-Methylierungsanalysen etabliert: QAMMOD (Quantitative Methylierungsanalyse von modifizierter DNA) und QESD (Quantifizierung Endonuklease-resistenter DNA). Die DNA-Promotormethylierungen von P16- und MGMT zeigten zwischen den MSI Tumoren und den MSS CRC keine signifikanten Unterschiede, korrelierten aber innerhalb der MSI CRC signifikant mit der Methylierung von MLH1. Mit Hilfe der QAMMOD und der QESD wurde erstmals die MLH1-Promotormethylierung in hereditären nicht-polypösen kolorektalen Karzinomen (HNPCC) und sporadischen MSI Tumoren analysiert. Dabei wurde bei den HNPCC-Tumoren negative oder schwache MLH1-Methylierung festgestellt. Der Grad der MLH1-Methylierung war bei den HNPCC-Tumoren signifikant niedriger als bei den am MLH1-Promotor durchwegs stark methylierten sporadischen MSI CRC. Mit Hilfe der neuen quantitativen Methylierungsanalysen konnten erstmals Grenzwerte der MLH1-Methylierung definiert werden, welche methylierungsnegative HNPCC-Kandidaten von den methylierungspositiven sporadischen MSI CRC unterscheiden und für weitere diagnostische Untersuchungen selektionieren. Die QESD ist eine universelle Methode der quantitativen Methylierungsanalyse und wurde innerhalb dieser Dissertation zusätzlich für die quantitative Methylierungsanalyse der Gene GSTP1, CA4, RASSF1, SFRP1, PITX2, CDH3, APC, DNMT1, DNMT3A, DNMT3B und TS etabliert

Archaeal tetrathionate hydrolase goes viral: secretion of a sulfur metabolism enzyme in the form of virus-like particles

International audienceIn the course of screening for virus-host systems in extreme thermal environments, we have isolated a strain of the hyperthermophilic archaeaon Acidianus hospitalis producing unusual filamentous particles with a zipper-like appearance. The particles were shown to represent a secreted form of a genuine cellular enzyme, tetrathionate hydrolase, involved in sulfur metabolism

Evidence of prognostic relevant expression profiles of heat-shock proteins and glucose-regulated proteins in oesophageal adenocarcinomas

A high percentage of oesophageal adenocarcinomas show an aggressive clinical behaviour with a significant resistance to chemotherapy. Heat-shock proteins (HSPs) and glucose-regulated proteins (GRPs) are molecular chaperones that play an important role in tumour biology. Recently, novel therapeutic approaches targeting HSP90/GRP94 have been introduced for treating cancer. We performed a comprehensive investigation of HSP and GRP expression including HSP27, phosphorylated (p)-HSP27((Ser15)), p-HSP27((Ser78)), p-HSP27((Ser82)), HSP60, HSP70, HSP90, GRP78 and GRP94 in 92 primary resected oesophageal adenocarcinomas by using reverse phase protein arrays (RPPA), immunohistochemistry (IHC) and real-time quantitative RT-PCR (qPCR). Results were correlated with pathologic features and survival. HSP/GRP protein and mRNA expression was detected in all tumours at various levels. Unsupervised hierarchical clustering showed two distinct groups of tumours with specific protein expression patterns: The hallmark of the first group was a high expression of p-HSP27((Ser15, Ser78, Ser82)) and low expression of GRP78, GRP94 and HSP60. The second group showed the inverse pattern with low p-HSP27 and high GRP78, GRP94 and HSP60 expression. The clinical outcome for patients from the first group was significantly improved compared to patients from the second group, both in univariate analysis (p = 0.015) and multivariate analysis (p = 0.029). Interestingly, these two groups could not be distinguished by immunohistochemistry or qPCR analysis. In summary, two distinct and prognostic relevant HSP/GRP protein expression patterns in adenocarcinomas of the oesophagus were detected by RPPA. Our approach may be helpful for identifying candidates for specific HSP/GRP-targeted therapies

Correction to: MicroRNA expression profiling for the prediction of resistance to neoadjuvant radiochemotherapy in squamous cell carcinoma of the esophagus

Following publication of the original article [1], the authors reported that for one of the authors, Stephanie E. Combs, the middle name was accidentally omitted. They also reported that for two of the authors, Daniel Habermehl and Stephanie E. Combs, two affiliations were accidentally omitted. In this Correction the incorrect and correct author name are shown and the two omitted affiliations are listed

MicroRNA expression profiling for the prediction of resistance to neoadjuvant radiochemotherapy in squamous cell carcinoma of the esophagus

BACKGROUND: MicroRNAs (miRNAs) play an important role in cancer biology. Neoadjuvant radiochemotherapy followed by surgery is a standard treatment for locally advanced esophageal squamous cell carcinoma (ESCC). However, a subset of patients do not respond. We evaluated whether miRNA profiles can predict resistance to radiochemotherapy. METHODS: Formalin-fixed, paraffin-embedded pretherapeutic biopsies of patients treated by radiochemotherapy followed by esophagectomy were analyzed. The response was determined by histopathological tumor regression grading. miRNA profiling was performed by microarray analysis (Agilent platform) in 16 non-responders and 15 responders. Differentially expressed miRNAs were confirmed by real-time quantitative PCR (qRT-PCR) in an expanded cohort of 53 cases. RESULTS: The miRNA profiles within and between non-responders and responders were highly similar (r = 0.96, 0.94 and 0.95). However, 12 miRNAs were differentially expressed (> twofold; p ≤ 0.025): non-responders showed upregulation of hsa-miR-1323, hsa-miR-3678-3p, hsv2-miR-H7-3p, hsa-miR-194*, hsa-miR-3152, kshv-miR-K12-4-3p, hsa-miR-665 and hsa-miR-3659 and downregulation of hsa-miR-126*, hsa-miR-484, hsa-miR-330-3p and hsa-miR-3653. qRT-PCR analysis confirmed the microarray findings for hsa-miR-194* and hsa-miR-665 (p < 0.001 each) with AUC values of 0.811 (95% CI 0.694-0.927) and 0.817 (95% CI 0.704-0.930), respectively, in ROC analysis. CONCLUSIONS: Our results indicate that miRNAs are involved in the therapeutic response in ESCC and suggest that miRNA profiles could facilitate pretherapeutic patient selection

Epidermal growth factor receptor, phosphatidylinositol-3-kinase catalytic subunit/PTEN, and KRAS/NRAS/BRAF in primary resected esophageal adenocarcinomas: loss of PTEN is associated with worse clinical outcome

Alterations of the epidermal growth factor receptor (EGFR) can be observed in a significant subset of esophageal adenocarcinomas (EACs), and targeted therapy against EGFR may become an interesting approach for the treatment of these tumors. Mutations of KRAS, NRAS, BRAF, and phosphatidylinositol-3-kinase catalytic subunit (PIK3CA) and deregulation of PTEN expression influence the responsiveness against anti-EGFR therapy in colorectal carcinomas. We investigated the prevalence of these events in a collection of 117 primary resected EACs, correlated the findings with EGFR expression and amplification, and determined their clinicopathologic impact. KRAS mutations were detected in 4 (3%) of 117 tumors (3× G12D and 1 G12V mutation). One tumor had a PIK3CA E545K mutation. Neither NRAS nor BRAF mutations were detected. Sixteen (14%) of 117 cases were negative for PTEN expression, determined by immunohistochemistry. Loss of PTEN was observed predominantly in advanced tumor stages (P = .004). There was no association between PTEN and EGFR status. Loss of PTEN was associated with shorter overall and disease-free survival (P < .001 each) and also an independent prognostic factor in multivariate analysis (P = .015). EGFR status had no prognostic impact in this case collection. In summary, loss of PTEN can be detected in a significant subset of EAC and is associated with an aggressive phenotype. Therefore, PTEN may be useful as a prognostic biomarker. In contrast, mutations of RAS/RAF/PIK3CA appear only very rarely, if at all, in EAC. A possible predictive role of PTEN in anti-EGFR treatment warrants further investigations, whereas determination of RAS/RAF/PIK3CA mutations may only have a minor impact in this context

MethyQESD, a robust and fast method for quantitative methylation analyses in HNPCC diagnostics using formalin-fixed and paraffin-embedded tissue samples

Promoter hypermethylation occurs in various tumors and leads to silencing of tumor-relevant genes. Thus, promoter methylation analysis (MA) has been established as an important tool in cancer research and diagnostics. Here we present MethyQESD (methylation-quantification of endonuclease-resistant DNA) as a fast, easy, precise and reliable method for quantitative MA without the need of bisulfite-treatment or fluorescent probes. Though MethyQESD principally works with any gene promoter we established MethyQESD for the mismatch repair gene MLH1 and tested its utility to differentiate between sporadic microsatellite unstable (MSI-H) colorectal cancer and hereditary nonpolyposis colorectal cancer (HNPCC) by quantitative MLH1 MA. We investigated formalin-fixed and paraffin-embedded tissue samples from a previously published, well-characterized tumor collective comprising 25 HNPCC, 14 sporadic MSI-H CRC and 16 sporadic microsatellite stable (MSS) CRC. We found a high accuracy of MethyQESD by spiking experiments with dilution series of methylated (SW48 cancer cell line) and unmethylated (blood) DNA (Pearson's r=0.9997 (proximal MLH1 promoter region), r=0.9976 (distal MLH1 promoter region)). MethyQESD and conventional quantitative MA using of 96 formalin-fixed and paraffin-embedded CRC showed a high degree of concordance of both methods (Pearson's r=0.885). HNPCC tumors showed either null MLH1 methylation or a significantly lower degree of MLH1 methylation than sporadic MSI-H CRC (P<0.001). MLH1 methylation was negative in all MSS tumors. Receiver operating characteristic (ROC) curve analyses defined a cutoff value of 16.5% MLH1 methylation for specific and sensitive identification of sporadic MSI-H CRC (area under ROC curve: 1.000; asymptotic significance: P<0.001). Thus, quantitative MLH1 MA by MethyQESD provides a simple, fast and valuable tool to identify HNPCC candidates. Furthermore, MethyQESD works reliably with formalin-fixed paraffin-embedded tissue and simplifies DNA MA both for research and diagnostic purposes