Adaptation and validation of a quantitative vanA/vanB DNA screening assay on a high-throughput PCR system.

Vancomycin resistant enterococci (VRE) are a leading cause of ICU-acquired bloodstream infections in Europe. The bacterial load in enteral colonization may be associated with a higher probability of transmission. Here, we aimed to establish a quantitative vanA/vanB DNA real-time PCR assay on a high-throughput system. Limits of detection (LOD), linear range and precision were determined using serial bacterial dilutions. LOD was 46.9 digital copies (dcp)/ml for vanA and 60.8 dcp/ml for vanB. The assay showed excellent linearity between 4.7 × 101 and 3.5 × 105 dcp/ml (vanA) and 6.7 × 102 and 6.7 × 105 dcp/ml (vanB). Sensitivity was 100% for vanA and vanB, with high positive predictive value (PPV) for vanA (100%), but lower PPV for vanB (34.6%) likely due to the presence of vanB DNA positive anerobic bacteria in rectal swabs. Using the assay on enriched VRE broth vanB PPV increased to 87.2%. Quantification revealed median 2.0 × 104 dcp/ml in PCR positive but VRE culture negative samples and median 9.1 × 104 dcp/ml in VRE culture positive patients (maximum: 107 dcp/ml). The automated vanA/B_UTC assay can be used for vanA/vanB detection and quantification in different diagnostic settings and may support future clinical studies assessing the impact of bacterial load on risk of infection and transmission.

High expression of micro RNA-135A in hepatocellular carcinoma is associated with recurrence within 12 months after resection.

BACKGROUND: Hepatocellular carcinoma has a dismal prognosis due to recurrence rates of up to 70% after curative resection. Early recurrence is driven by synchronous microscopic intrahepatic metastases. The predictive value of histological parameters is discussed controversially and adjuvant therapy is not established. The aim of this study was to identify patients at high risk for early intrahepatic recurrence by expression profiling of selected micro RNAs. METHODS: In 52 patients undergoing HCC resection between 2011 and 2014, liver and tumor tissue was collected during surgery. Twelve patients with incomplete data regarding HCC recurrence, secondary liver transplantation, or perioperative death were excluded, leaving 40 patients with early recurrence <12 months (R+) or without recurrence for >24 months (R-) to compare grading, T, L, V, and R status. If tissue quality permitted, micro RNAs were measured in HCC and liver tissue. RESULTS: Ten women and 30 men (64.0 ± 10.2 years) were analyzed. R+ occurred in 29 patients 6.2 ± 4.5 months after resection. Surveillance of R- was 26.2 ± 5.2 months. High intratumoral expression of miR-135a was associated with high risk of recurrence (HR = 4.2, p = 0.024, time to recurrence 8.8 ± 2.0 vs. 24.8 ± 4.4 months in patients with low miR-135a expression). As expected, T3 status was correlated with early recurrence, while other histological parameters and expression of miR-21, miR-122, and miR-125a did not. CONCLUSIONS: We show a significant association between high expression of miR-135a and early HCC recurrence. Therefore, high intratumoral miR-135a expression might serve as a novel biomarker to identify patients urgently requiring adjuvant therapy post resection.

Multi-color RGB marking enables clonality assessment of liver tumors in a murine xenograft model.

We recently introduced red-green-blue (RGB) marking for clonal cell tracking based on individual color-coding. Here, we applied RGB marking to study clonal development of liver tumors. Immortalized, non-tumorigenic human fetal hepatocytes expressing the human telomerase reverse transcriptase (FH-hTERT) were RGB-marked by simultaneous transduction with lentiviral vectors encoding mCherry, Venus, and Cerulean. Multi-color fluorescence microscopy was used to analyze growth characteristics of RGB-marked FH-hTERT in vitro and in vivo after transplantation into livers of immunodeficient mice with endogenous liver damage (uPA/SCID). After initially polyclonal engraftment we observed oligoclonal regenerative nodules derived from transplanted RGB-marked FH-hTERT. Some mice developed monochromatic invasive liver tumors; their clonal origin was confirmed both on the molecular level, based on specific lentiviral-vector insertion sites, and by serial transplantation of one tumor. Vector insertions in proximity to the proto-oncogene MCF2 and the transcription factor MITF resulted in strong upregulation of mRNA expression in the respective tumors. Notably, upregulated MCF2 and MITF expression was also observed in 21% and 33% of 24 human hepatocellular carcinomas analyzed. In conclusion, liver repopulation with RGB-marked FH-hTERT is a useful tool to study clonal progression of liver tumors caused by insertional mutagenesis in vivo and will help identifying genes involved in liver cancer.

Constitutive gp130 activation rapidly accelerates the transformation of human hepatocytes via an impaired oxidative stress response.

Pro-inflammatory signaling pathways, especially interleukin 6 (IL-6), and reactive oxygen species (ROS) promote carcinogenesis in the liver. In order to elucidate the underlying oncogenic mechanism, we activated the IL-6 signal transducer glycoprotein 130 (gp130) via stable expression of a constitutively active gp130 construct (L-gp130) in untransformed telomerase-immortalized human fetal hepatocytes (FH-hTERT). As known from hepatocellular adenomas, forced gp130 activation alone was not sufficient to induce malignant transformation. However, additional challenge of FH-hTERT L-gp130 clones with oxidative stress resulted in 2- to 3-fold higher ROS levels and up to 6-fold more DNA-double strand breaks (DSB). Despite increased DNA damage, ROS-challenged FH-hTERT L-gp130 clones displayed an enhanced proliferation and rapidly developed colony growth capabilities in soft agar. As driving gp130-mediated oncogenic mechanism, we detected a decreased expression of antioxidant genes, in particular glutathione peroxidase 3 and apolipoprotein E, and an absence of P21 upregulation following ROS-conferred induction of DSB. In summary, an impaired oxidative stress response in hepatocytes with gp130 gain-of-function mutations, as detected in dysplastic intrahepatic nodules and hepatocellular adenomas, is one of the central oncogenic mechanisms in chronic liver inflammation.

Specific genomic and transcriptomic aberrations in tumors induced by partial hepatectomy of a chronically inflamed murine liver.

Resection of hepatocellular carcinoma (HCC) tumors by partial hepatectomy (PHx) is associated with promoting hepatocarcinogenesis. We have previously reported that PHx promotes hepatocarcinogenesis in the Mdr2-knockout (Mdr2-KO) mouse, a model for inflammation-mediated HCC. Now, to explore the molecular mechanisms underlying the tumor-promoting effect of PHx, we compared genomic and transcriptomic profiles of HCC tumors developing in the Mdr2-KO mice either spontaneously or following PHx. PHx accelerated HCC development in these mice by four months. PHx-induced tumors had major chromosomal aberrations: all were amplifications affecting multiple chromosomes. Most of these amplifications were located near the acrocentric centromeres of murine chromosomes. Four different chromosomal regions were amplified each in at least three tumors. The human orthologs of these common amplified regions are known to be amplified in HCC. All tumors of untreated mice had chromosomal aberrations, including both deletions and amplifications. Amplifications in spontaneous tumors affected fewer chromosomes and were not located preferentially at the chromosomal edges. Comparison of gene expression profiles revealed a significantly enriched expression of oncogenes, chromosomal instability markers and E2F1 targets in the post-PHx compared to spontaneous tumors. Both tumor groups shared the same frequent amplification at chromosome 18. Here, we revealed that one of the regulatory genes encoded by this amplified region, Crem, was over-expressed in the nuclei of murine and human HCC cells in vivo, and that it stimulated proliferation of human HCC cells in vitro. Our results demonstrate that PHx of a chronically inflamed liver directed tumor development to a discrete pathway characterized by amplification of specific chromosomal regions and expression of specific tumor-promoting genes. Crem is a new candidate HCC oncogene frequently amplified in this model and frequently over-expressed in human HCC.

Transduction of fetal mice with a feline lentiviral vector induces liver tumors which exhibit an E2F activation signature.

Lentiviral vectors are widely used in basic research and clinical applications for gene transfer and long-term expression; however, safety issues have not yet been completely resolved. In this study, we characterized hepatocarcinomas that developed in mice 1 year after in utero administration of a feline-derived lentiviral vector. Mapped viral integration sites differed among tumors and did not coincide with the regions of chromosomal aberrations. Furthermore, gene expression profiling revealed that no known cancer-associated genes were deregulated in the vicinity of viral integrations. Nevertheless, five of the six tumors exhibited highly significant upregulation of E2F target genes, of which a majority are associated with oncogenesis, DNA damage response, and chromosomal instability. We further show in vivo and in vitro that E2F activation occurs early on following transduction of both fetal mice and cultured human hepatocytes. On the basis of the similarities in E2F target gene expression patterns among tumors and the lack of evidence implicating insertional mutagenesis, we propose that transduction of fetal mice with a feline lentiviral vector induces E2F-mediated major cellular processes that drive hepatocytes toward uncontrolled proliferation culminating in tumorigenesis.

The NTPase/helicase domain of hepatitis C virus nonstructural protein 3 inhibits protein kinase C independently of its NTPase activity.

Helicase motif VI is a short arginine-rich motif within the NTPase/helicase domain of the non-structural protein 3 (NS3) of the hepatitis C virus (HCV). We previously demonstrated that it reduces the catalytic activity and intracellular shuttling of protein kinase C (PKC). Thus, NS3-mediated PKC inhibition may be involved in HCV-associated hepatocellular carcinoma (HCC). In this study, we expand on our earlier results, which were obtained in experiments with short fragments of NS3, to show for the first time that the catalytically active, longer C-terminal NTPase/helicase of NS3 acts as a potent PKC inhibitor in vitro. PKC inhibition assays with the NTPase-inactive mutant NS3h-D1316A revealed a mixed type kinetic inhibition pattern. A broad range of 11 PKC isotypes was tested and all of the PKC isotypes were inhibited with IC50-values in the low micromolar range. These findings were confirmed for the wild-type NTPase/helicase domain in a non-radiometric PKC inhibition assay with ATP regeneration to rule out any effect of ATP hydrolysis caused by its NTPase activity. PKCα was inhibited with a micromolar IC50 in this assay, which compares well with our result for NS3h-D1316A (IC50 = 0.7 µM). In summary, these results confirm that catalytically active NS3 NTPase/helicase can act in an analogous manner to shorter NS3 fragments as a pseudosubstrate inhibitor of PKC.

Forced activation of ß-catenin signaling supports the transformation of hTERT-immortalized human fetal hepatocytes.

Hepatocarcinogenesis is a multistep process driving the progressive transformation of normal liver cells into highly malignant derivatives. Unlimited proliferation and telomere maintenance have been recognized as prerequisites for the development of liver cancer. Moreover, recent studies identified illegitimate ß-catenin signaling as relevant hit in a considerable subset of patients. To further investigate the currently not well-understood malignant evolution driven by telomerase and ß-catenin, we monitored cytogenetic and phenotypic alterations in untransformed telomerase-immortalized human fetal hepatocytes following forced activation of ß-catenin signaling. As expected, constitutive activation of ß-catenin signaling significantly enhanced proliferation with decreasing serum dependence. Previously intact contact inhibition was almost completely eliminated. Interestingly, after several passages in cell culture, immortalized clones with dominant-positive ß-catenin signaling acquired additional chromosomal aberrations, in particular translocations, anchorage-independent growth capabilities, and formed tumors in athymic nude mice. In further support for the driving role of ß-catenin during hepatocarcinogenesis, improved colony growth in soft agar and accelerated tumor formation was also confirmed in Huh7 cells following stable expression of the constitutively active S33Y ß-catenin mutant. Telomerase inhibition showed that short-term expansion of transformed clones was not telomerase dependent. Finally, cancer pathway profiling in derived tumors revealed upregulation of characteristic genes associated with invasion and angiogenesis. In conclusion, illegitimate activation of ß-catenin signaling enhances the transformation from immortalization to malignant growth in human fetal hepatocytes. Our data functionally confirm a permissive role for ß-catenin signaling in the initial phase of hepatocarcinogenesis.

Telomeres and telomerase in chronic myeloid leukaemia: impact for pathogenesis, disease progression and targeted therapy.

Telomeres are specialized structures localized at the end of human chromosomes. Due to the end replication problem, each cell division results in a loss of telomeric repeats in normal somatic cells. In germ line and stem cells, the multicomponent enzyme telomerase maintains the length of telomere repeats. However, elevated telomerase activity has also been reported in the majority of solid tumours as well as in acute and chronic leukaemia. Chronic myeloid leukaemia (CML) serves as a model disease to study telomere biology in clonal myeloproliferative disorders. In CML, telomere shortening correlates with disease stage, duration of chronic phase (CP), prognosis measured by the Hasford risk score and the response to disease-modifying therapeutics such as the tyrosine kinase inhibitor Imatinib. In addition, telomerase activity (TA) is already increased in CP CML and further upregulated with disease progression to accelerated phase and blast crisis (BC). Furthermore, a correlation of TA with increased genetic instability as well as a shorter survival of the patients has been reported. Here, we review the current state of knowledge of the role of telomere and telomerase biology in CML and discuss the possible impact of novel treatment approaches.