Bettina Grasl-Kraupp

Rapid communicationIn situ detection of fragmented DNA (tunel assay) fails to discriminate among apoptosis, necrosis, and autolytic cell death: A cautionary note☆

Detection of DNA fragments in situ using the terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay is increasingly applied to investigate active cell death (apoptosis). We studied the specificity of the assay in well-defined models of apoptosis and necrosis as well as in postmortem autolysis in rat liver. During involution of liver hyperplasia, which follows stopping treatment with the hepatomitogens cyproterone acetate (CPA) or nafenopin (NAF), numerous apoptotic hepatocytes could be observed with TUNEL-positive chromatin residues. A similar TUNEL-positive reaction appeared in necrotic hepatocytes after a cytotoxic dose of carbon tetrachloride (CC14) or N-nitrosomorpholine (NNM). Also, in insufficiently fixed, autolytic livers TUNEL-positive nuclei were observed. Thus, DNA fragmentation is common to different kinds of cell death; its detection in situ should not be considered a specific marker of apoptosis.

Fibroblast growth factor receptor-mediated signals contribute to the malignant phenotype of non-small cell lung cancer cells: therapeutic implications and synergism with epidermal growth factor receptor inhibition

Fibroblast growth factors (FGF) and their high-affinity receptors (FGFR) represent an extensive cellular growth and survival system. Aim of this study was to evaluate the contribution of FGF/FGFR-mediated signals to the malignant growth of non-small cell lung cancer (NSCLC) and to assess their potential as targets for therapeutic interventions. Multiple FGFR mRNA splice variants were coexpressed in NSCLC cells (n = 16) with predominance of FGFR1. Accordingly, both expression of a dominant-negative FGFR1 (dnFGFR1) IIIc-green fluorescent protein fusion protein and application of FGFR small-molecule inhibitors (SU5402 and PD166866) significantly reduced growth, survival, clonogenicity, and migratory potential of the majority of NSCLC cell lines. Moreover, dnFGFR1 expression completely blocked or at least significantly attenuated s.c. tumor formation of NSCLC cells in severe combined immunodeficient mice. Xenograft tumors expressing dnFGFR1 exhibited significantly reduced size and mitosis rate, enhanced cell death, and decreased tissue invasion. When FGFR inhibitors were combined with chemotherapy, antagonistic to synergistic in vitro anticancer activities were obtained depending on the application schedule. In contrast, simultaneous blockage of FGFR- and epidermal growth factor receptor-mediated signals exerted synergistic effects. In summary, FGFR-mediated signals in cooperation with those transmitted by epidermal growth factor receptor are involved in growth and survival of human NSCLC cells and should be considered as targets for combined therapeutic approaches. [Mol Cancer Ther 2008;7(10):3408–19]

Nuclear β-Catenin Induces an Early Liver Progenitor Phenotype in Hepatocellular Carcinoma and Promotes Tumor Recurrence

Transforming growth factor-beta cooperates with oncogenic Ras to activate nuclear beta-catenin during the epithelial to mesenchymal transition of hepatocytes, a process relevant in the progression of hepatocellular carcinoma (HCC). In this study we investigated the role of beta-catenin in the differentiation of murine, oncogene-targeted hepatocytes and in 133 human HCC patients scheduled for orthotopic liver transplantation. Transforming growth factor-beta caused dissociation of plasma membrane E-cadherin/beta-catenin complexes and accumulation of nuclear beta-catenin in Ras-transformed, but otherwise normal hepatocytes in p19(ARF)-/- mice. Both processes were inhibited by Smad7-mediated disruption of transforming growth factor-beta signaling. Overexpression of constitutively active beta-catenin resulted in high levels of CK19 and M2-PK, whereas ablation of beta-catenin by axin overexpression caused strong expression of CK8 and CK18. Therefore, nuclear beta-catenin resulted in dedifferentiation of neoplastic hepatocytes to immature progenitor cells, whereas loss of nuclear beta-catenin led to a differentiated HCC phenotype. Poorly differentiated human HCC showed cytoplasmic redistribution or even loss of E-cadherin, suggesting epithelial to mesenchymal transition. Analysis of 133 HCC patient samples revealed that 58.6% of human HCC exhibited strong nuclear beta-catenin accumulation, which correlated with clinical features such as vascular invasion and recurrence of disease after orthotopic liver transplantation. These data suggest that activation of beta-catenin signaling causes dedifferentiation to malignant, immature hepatocyte progenitors and facilitates recurrence of human HCC after orthotopic liver transplantation.

Up‐regulation of the fibroblast growth factor 8 subfamily in human hepatocellular carcinoma for cell survival and neoangiogenesis

Fibroblast growth factors (FGFs) and their high‐affinity receptors [fibroblast growth factor receptors (FGFRs)] contribute to autocrine and paracrine growth stimulation in several nonliver cancer entities. Here we report that at least one member of the FGF8 subfamily (FGF8, FGF17, and FGF18) was up‐regulated in 59% of 34 human hepatocellular carcinoma (HCC) samples that we investigated. The levels of the corresponding receptors (FGFR2, FGFR3, and FGFR4) were also elevated in the great majority of the HCC cases. Overall, 82% of the HCC cases showed overexpression of at least one FGF and/or FGFR. The functional implications of the deregulated FGF/FGFR system were investigated by the simulation of an insufficient blood supply. When HCC‐1.2, HepG2, or Hep3B cells were subjected to serum withdrawal or the hypoxia‐mimetic drug deferoxamine mesylate, the expression of FGF8 subfamily members increased dramatically. In the serum‐starved cells, the incidence of apoptosis was elevated, whereas the addition of FGF8, FGF17, or FGF18 impaired apoptosis, which was associated with phosphorylation of extracellular signal‐regulated kinase 1/2 and ribosomal protein S6. In contrast, down‐modulation of FGF18 by small interfering RNA (siRNA) significantly reduced the viability of the hepatocarcinoma cells. siRNA targeting FGF18 also impaired the cells potential to form clones at a low cell density or in soft agar. With respect to the tumor microenvironment, FGF17 and FGF18 stimulated the growth of HCC‐derived myofibroblasts, and FGF8, FGF17, and FGF18 induced the proliferation and tube formation of hepatic endothelial cells. Conclusion: FGF8, FGF17, and FGF18 are involved in autocrine and paracrine signaling in HCC and enhance the survival of tumor cells under stress conditions, malignant behavior, and neoangiogenesis. Thus, the FGF8 subfamily supports the development and progression of hepatocellular malignancy. (HEPATOLOGY 2011)

Fibroblast Growth Factor Receptors as Therapeutic Targets in Human Melanoma: Synergism with BRAF Inhibition

Cutaneous melanoma is a tumor with rising incidence and a very poor prognosis at the disseminated stage. Melanomas are characterized by frequent mutations in BRAF and also by overexpression of fibroblast growth factor 2 (FGF2), offering opportunities for therapeutic intervention. We investigated inhibition of FGF signaling and its combination with dacarbazine or BRAF inhibitors as an antitumor strategy in melanoma. The majority of melanoma cell lines displayed overexpression of FGF2 but also FGF5 and FGF18 together with different isoforms of FGF receptors (FGFRs) 1-4. Blockade of FGF signals with dominant-negative receptor constructs (dnFGFR1, 3, or 4) or small-molecule inhibitors (SU5402 and PD166866) reduced melanoma cell proliferation, colony formation, as well as anchorage-independent growth, and increased apoptosis. DnFGFR constructs also significantly inhibited tumor growth in vivo. Combination of FGF inhibitors with dacarbazine showed additive or antagonistic effects, whereas synergistic drug interaction was observed when combining FGFR inhibition with the multikinase/BRAF inhibitor sorafenib or the V600E mutant-specific BRAF inhibitor RG7204. In conclusion, FGFR inhibition has antitumor effects against melanoma cells in vitro and in vivo. Combination with BRAF inhibition offers a potential for synergistic antimelanoma effects and represents a promising therapeutic strategy against advanced melanoma.

Apoptosis and multistage carcinogenesis in rat liver.

Apoptosis is a type of active cell death. It is involved in the homeostasis of cell number in tissues and is controlled by the growth regulatory network in the organism. It is also involved in the active removal of damaged cells. We have studied the role of apoptosis in cancer pre-stages and overt cancer in vivo, using rat liver as our main model system. Quantitative determination of apoptosis in histological specimens revealed that the rate of apoptosis tends to increase from normal to (pre)neoplastic to malignant cells. Thereby active cell death largely counterbalances the increasing replicative activity in developing malignancy. Tumor promoters shift the balance in favor of cell replication, whereas promoter withdrawal, fasting or TGF-beta 1 favor apoptosis (anti-promotion). Preneoplastic cells are more susceptible than normal liver cells to stimulation of both cell replication or cell death. Consequentially (pre)neoplastic tissue may preferentially grow or die during the appropriate treatment. Regimens that favor apoptosis and lower cell replication are shown to result in the elimination of preneoplastic cell clones from the liver (anti-initiation) and to reduce the cancer risk of the animal.

Targeting fibroblast-growth-factor-receptor-dependent signaling for cancer therapy

Introduction : Fibroblast growth factors (FGF) exert a combination of biological effects that contribute to four of the six essential hallmarks of cancer. It is no surprise that FGF-dependent signaling has increasingly moved to the center of cancer therapy research during the past decade. This is illustrated by the large number of publications focusing on various aspects of this theme that have been published in the past 5 years. Areas covered : Information from these sources as well as ongoing work from the authors groups is used to outline the physiological functions of FGF signaling and to highlight how the high oncogenic effects of deregulated FGFs and FGFRs derive from their physiological functions. The biological effect of deregulated FGFR signaling in malignant diseases is described and the current state of therapeutic targeting of FGFR is summarized. Expert opinion : Strategies for targeting FGFR-signaling for cancer therapy are very promising, but need to be carefully developed based on the physiological roles of FGF signaling. Preventive measures may be necessary for protection from FGF-related side effects. Combined targeting of several receptor tyrosine kinases or combination with other therapies may be a useful way of avoiding or ameliorating side effects. FGF-related markers of prognosis and therapy response still need to be investigated.

Concepts of Cell Death and Application to Carcinogenesis

The occurrence of cell death as a physiologic event in multicellular organisms has been known for more than 150 yr. In 1972, the term apoptosis was introduced on morphological grounds. The hypothesis that all kinds of cell death can be categorized as either apoptotic or necrotic is not generally confirmed. Cells seem to use different pathways for suicide, as reflected by different morphology: condensation-prominent, Type I or apoptosis; autophagy-prominent, Type II; and so forth. Type II cell death was found in mammary tissue and mammary tumor cells and in a variety of other organs. For unequivocal identification of the various types of cell death, morphological, biochemical, and functional criteria may be used in combination. During tumor development in various organs of animals and humans, not only rates of cell proliferation but also rates of cell death may increase with increasing malignancy. Morphological and functional criteria (antipromotion, withdrawal of survival factors) indicate that cell death in tumors frequently is of an active nature.

Xanthohumol, a prenylated flavonoid contained in beer, prevents the induction of preneoplastic lesions and DNA damage in liver and colon induced by the heterocyclic aromatic amine amino-3-methyl-imidazo[4,5-f]quinoline (IQ)

Xanthohumol (XN) is a hop derived prenylated flavonoid contained in beer. Earlier findings indicated that it has promising chemopreventive properties and protects cells against DNA damage by carcinogens via inhibition of their activation. Furthermore, it was found that XN inhibits DNA synthesis and proliferation of cancer cells in vitro, inactivates oxygen radicals and induces apoptosis. Since evidence for its chemoprotective properties is restricted to results from in vitro experiments, we monitored the impact of XN on the formation of amino-3-methyl-imidazo[4,5-f]quinoline (IQ)-induced preneoplastic foci in livers and colons of rats (9/group). Additionally, we studied its effects on IQ-induced DNA damage in colonocytes and hepatocytes in single cell gel electrophoresis assays and on the activities of a panel of drug metabolising enzymes. Consumption of the drinking water supplemented with XN (71 microg/kg b.w.) before and during carcinogen treatment led to a significant reduction of the number of GST-p+ foci in the liver by 50% and also to a decrease of the foci area by 44%. DNA migration was decreased significantly in both, colon mucosa and liver cells, but no alterations of the activities of different phases I and II enzymes were found in hepatic tissue. Our findings indicate that XN protects against DNA damage and cancer induced by the cooked food mutagen. Since the effects were observed with low doses of XN which are reached after consumption of brews with high XN levels, our findings may be relevant for humans.

Potent protection of gallic acid against DNA oxidation: results of human and animal experiments.

Gallic acid (3,4,5-trihydroxybenzoic acid, GA) is a constituent of plant derived foods, beverages and herbal remedies. We investigated its DNA protective properties in a placebo controlled human intervention trial in single cell gel electrophoresis experiments. Supplementation of drinking water with GA (12.8 mg/person/d) for three days led to a significant reduction of DNA migration attributable to oxidised pyrimidines (endonuclease III sensitive sites) and oxidised purines (formamidopyrimidine glycosylase sensitive sites) in lymphocytes of healthy individuals by 75% and 64% respectively. Also DNA damage caused by treatment of the cells with reactive oxygen species (ROS) was reduced after GA consumption (by 41%). These effects were paralleled by an increase of the activities of antioxidant enzymes (superoxide dismutase, glutathione peroxidase and glutathion-S-transferase-π) and a decrease of intracellular ROS concentrations in lymphocytes, while no alterations of the total antioxidant capacity (TAC), of malondialdehyde levels in serum and of the urinary excretion of isoprostanes were found. Experiments with rats showed that GA reduces oxidatively damaged DNA in lymphocytes, liver, colon and lungs and protects these organs against γ-irradiation-induced strand breaks and formation of oxidatively damaged DNA-bases. Furthermore, the number of radiation-induced preneoplastic hepatic foci was decreased by 43% after oral administration of the phenolic. Since we did not find alterations of the TAC in plasma and lipid peroxidation of cell membranes but intracellular effects it is likely that the antioxidant properties of GA seen in vivo are not due to direct scavenging of radicals but rather to indirect mechanisms (e.g. protection against ROS via activation of transcription factors). As the amount of GA used in the intervention trial is similar to the daily intake in Middle Europe (18 mg/person/day), our findings indicate that it may contribute to prevention of formation of oxidatively damaged DNA in humans.