Denise Heim

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. Copyright

Forced Activation of β-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 β-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 β-catenin, we monitored cytogenetic and phenotypic alterations in untransformed telomerase-immortalized human fetal hepatocytes following forced activation of β-catenin signaling. As expected, constitutive activation of β-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 β-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 β-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 β-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 β-catenin signaling enhances the transformation from immortalization to malignant growth in human fetal hepatocytes. Our data functionally confirm a permissive role for β-catenin signaling in the initial phase of hepatocarcinogenesis. Mol Cancer Res; 9(9); 1222–31. ©2011 AACR.

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.

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.

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.

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.

P0302 : Constitutive gp130 activation accelerates transformation of proliferating human hepatocytes via increased levels of oxidative stress

Background and Aims: Transforming growth factor (TGF)-b is a ubiquitously expressed cytokine with fundamental roles in various aspects of cell physiology. In carcinogenesis, TGF-b signaling plays a dual role. While it suppresses the proliferation of epithelial cells and adenoma cells at early stages by inducing growth arrest and apoptosis, it triggers epithelial to mesenchymal transition (EMT) and gain of metastatic abilities at later stages of carcinoma development. The molecular mechanisms underlying this ’TGF-bswitch’ are only beginning to be unravelled. To mimic the pathophysiological situation as closely as possible, we exposed the cells to TGFb long-term. Through this approach we aimed to identify those cooperating factors and signaling pathways that cause HCC cells to interpret the TGF-b signal in a tumor progressive way. Methods: In vitro comparison of migratory behaviour of various HCC cell lines treated long-term (>10 days) with TGF-b. Analysis of regulatory networks and target genes underlying the TGF-b treatment. Results: HCC cell lines that have undergone EMT secrete TGF-b and show elevated levels of Smad2/3 phosphorylation indicating an autocrine regulatory feedback loop. Inhibition of TGF-b by LY2109 abrogates autocrine stimulation and diminishes the migratory potential of mesenchymal HCC cells. Silencing of either TGF-bR1 or Smad4 indicated the importance of canonical TGF-b/Smad signaling HCC cell migration. Short-term treatment of cells with TGF-b could not improve migratory abilities. Interestingly, long-term TGF-b treatment revealed crucial differences between mesenchymal HCC cell lines. While HLF cells showed an increase in migration when treated with TGF-b for more than 10 days, SNU449 displayed a dramatic reduction in migration. However, both cell lines displayed no modulation in Smad phosphorylation, indicating a change in the utilization of TGF-b signaling in long-term treated SNU449 cells. Conclusions: EMT-transformed HCC cells establish an autocrine TGF-b loop which stimulates migration. However, TGF-b cannot add up to the autocrine loop but causes a different, even opposing reaction, over time. Interpretation of long-term TGF-b signaling, which mimics the patient’s situation more closely, depends on duration and intensity and is controlled by co-acting factors and signaling pathways. P0301 Withdrawn

1091 MULTI-COLOUR RGB MARKING TO ASSESS THE CLONALITY OF LIVER TUMOURS

Methods: CD133 cells were isolated by magnetic bead sorting after Huh-7 cells were genetically labeled with green fluorescent protein (GFP) or red fluorescent protein (RFP). In this scheme, CD133 cells were labeled with GFP and CD133 cells were labeled with RFP. The same number of GFP CSCs and the RFP non-CSCs were mixed and injected subcutaneously or in the spleen of nude mice. Results: CSCs had higher proliferative potential compared to nonCSCs in vitro. CSCs performed a higher in vitro proliferative potential and lower mRNA expressions of mature hepatocyte markers, glutamine synthetase and cytochrome P450 3A4, than non-CSCs. When either CD133 or CD133 cells were subcutaneously injected into SCID mice, CD133 cells formed tumors, whereas CD133 cells induced either a very small number of tumors or none at all. GFP-CSCs were highly tumorigenic and metastatic as well as highly resistant to chemotherapy in vivo compared to RFP-non-CSCs. Conclusions: The identification of CD133 cells could thus be a potentially powerful tool to investigate the tumorigenic process in the hepatoma system and to also develop effective therapies targeted against hepatocellular carcinoma. The ability to specifically distinguish CSCs in vivo in real time provides a visual target for prevention of metastasis and drug resistance.