Katja Storch

Three-Dimensional Cell Growth Confers Radioresistance by Chromatin Density Modification

Cell shape and architecture are determined by cell-extracellular matrix interactions and have profound effects on cellular behavior, chromatin condensation, and tumor cell resistance to radiotherapy and chemotherapy. To evaluate the role of chromatin condensation for radiation cell survival, tumor cells grown in three-dimensional (3D) cell cultures as xenografts and monolayer cell cultures were compared. Here, we show that increased levels of heterochromatin in 3D cell cultures characterized by histone H3 deacetylation and induced heterochromatin protein 1alpha expression result in increased radiation survival and reduced numbers of DNA double strand breaks (DSB) and lethal chromosome aberrations. Intriguingly, euchromatin to heterochromatin-associated DSBs were equally distributed in irradiated 3D cell cultures and xenograft tumors, whereas irradiated monolayer cultures showed a 2:1 euchromatin to heterochromatin DSB distribution. Depletion of histone deacetylase (HDAC) 1/2/4 or application of the class I/II pharmacologic HDAC inhibitor LBH589 induced moderate or strong chromatin decondensation, respectively, which was translated into cell line-dependent radiosensitization and, in case of LBH589, into an increased number of DSBs. Neither growth conditions nor HDAC modifications significantly affected the radiation-induced phosphorylation of the important DNA repair protein ataxia telangiectasia mutated. Our data show an interrelation between cell morphology and cellular radiosensitivity essentially based on chromatin organization. Understanding the molecular mechanisms by which chromatin structure influences the processing of radiation-induced DNA lesions is of high relevance for normal tissue protection and optimization of cancer therapy.

Differential expression of anti‐Müllerian hormone (amh) and anti‐Müllerian hormone receptor type II (amhrII) in the teleost medaka

In mammals, the anti‐Müllerian hormone (Amh) is responsible for the regression of the Müllerian ducts; therefore, Amh is an important factor of male sex differentiation. The amh gene has been cloned in various vertebrates, as well as in several teleost species. To date, all described species show a sexually dimorphic expression of amh during sex differentiation or at least in differentiated juvenile gonads. We have identified the medaka amh ortholog and examined its expression pattern. Medaka amh shows no sexually dimorphic expression pattern. It is expressed in both developing XY male and XX female gonads. In adult testes, amh is expressed in the Sertoli cells and in adult ovaries in granulosa cells surrounding the oocytes, like in mammals. To better understand the function of amh, we cloned the anti‐Müllerian hormone receptor type II (amhrII) ortholog and compared its expression pattern with amh, aromatase (cyp19a1), and scp3. During gonad development, amhrII is coexpressed with medaka amh in somatic cells of the gonads and shows no sexually dimorphic expression. Only the expression level of the Amh type II receptor gene was decreased noticeably in adult female gonads. These results suggest that medaka Amh and AmhrII are involved in gonad formation and maintenance in both sexes. Developmental Dynamics 236:271–281, 2007.

Caveolin-1 mediated radioresistance of 3D grown pancreatic cancer cells.

BACKGROUND AND PURPOSE Resistance of pancreatic ductal adenocarcinoma (PDAC) to chemo- and radiotherapy is a major obstacle. The integral membrane protein Caveolin-1 (Cav-1) has been suggested as a potent target in human pancreatic carcinoma cells. MATERIALS AND METHODS Human pancreatic tumor cells were examined in a three-dimensional (3D) cell culture model with regard to clonogenic survival, apoptosis, radiogenic DNA-double strand breaks and protein expression and phosphorylation under siRNA-mediated knockdown of Cav-1 without and in combination with irradiation (X-rays, 0-6Gy). Immunohistochemistry was used to assess Cav-1 expression in biopsies from patients with PDAC. RESULTS Tumor cells in PDAC showed significantly higher Cav-1 expression relative to tumor stroma. Cav-1 knockdown significantly reduced beta1 integrin expression and Akt phosphorylation, induced Caspase 3- and Caspase 8-dependent apoptosis and enhanced the radiosensitivity of 3D cell cultures. While cell cycling and Cav-1 promoter activity remained stable, Cav-1 knockdown-induced radiosensitization correlated with elevated numbers of residual DNA-double strand breaks. CONCLUSIONS Our data strongly support the concept of Cav-1 as a potent target in pancreatic carcinoma cells due to radiosensitization and Cav-1 overexpression in tumor cells of PDAC. 3D cell cultures are powerful and useful tools for the testing of novel targeting strategies to optimize conventional radio- and chemotherapy regimes for PDAC.

The Small Molecule Inhibitor QLT0267 Radiosensitizes Squamous Cell Carcinoma Cells of the Head and Neck

Background The constant increase of cancer cell resistance to radio- and chemotherapy hampers improvement of patient survival and requires novel targeting approaches. Integrin-Linked Kinase (ILK) has been postulated as potent druggable cancer target. On the basis of our previous findings clearly showing that ILK transduces antisurvival signals in cells exposed to ionizing radiation, this study evaluated the impact of the small molecule inhibitor QLT0267, reported as putative ILK inhibitor, on the cellular radiation survival response of human head and neck squamous cell carcinoma cells (hHNSCC). Methodology/Principal Findings Parental FaDu cells and FaDu cells stably transfected with a constitutively active ILK mutant (FaDu-IH) or empty vectors, UTSCC45 cells, ILK floxed/floxed(fl/fl) and ILK −/− mouse fibroblasts were used. Cells grew either two-dimensionally (2D) on or three-dimensionally (3D) in laminin-rich extracellular matrix. Cells were treated with QLT0267 alone or in combination with irradiation (X-rays, 0–6 Gy single dose). ILK knockdown was achieved by small interfering RNA transfection. ILK kinase activity, clonogenic survival, number of residual DNA double strand breaks (rDSB; γH2AX/53BP1 foci assay), cell cycle distribution, protein expression and phosphorylation (e.g. Akt, p44/42 mitogen-activated protein kinase (MAPK)) were measured. Data on ILK kinase activity and phosphorylation of Akt and p44/42 MAPK revealed a broad inhibitory spectrum of QLT0267 without specificity for ILK. QLT0267 significantly reduced basal cell survival and enhanced the radiosensitivity of FaDu and UTSCC45 cells in a time- and concentration-dependent manner. QLT0267 exerted differential, cell culture model-dependent effects with regard to radiogenic rDSB and accumulation of cells in the G2 cell cycle phase. Relative to corresponding controls, FaDu-IH and ILK fl/fl fibroblasts showed enhanced radiosensitivity, which failed to be antagonized by QLT0267. A knockdown of ILK revealed no change in clonogenic survival of the tested cell lines as compared to controls. Conclusions/Significance Our data clearly show that the small molecule inhibitor QLT0267 has potent cytotoxic and radiosensitizing capability in hHNSCC cells. However, QLT0267 is not specific for ILK. Further in vitro and in vivo studies are necessary to clarify the potential of QLT0267 as a targeted therapeutic in the clinic.

Fully automated interpretation of ionizing radiation-induced γH2AX foci by the novel pattern recognition system AKLIDES®

Purpose: Assessment of phosphorylated histone H2AX (γH2AX) foci as a measure for double-strand breaks (DSB) is a common technique. Since visual interpretation is time-consuming and influenced by subjective factors, we adapted the pattern recognition algorithms of autoantibodies to automated reading of γH2AX foci. Materials and methods: DSB formation was assessed by detection of γH2AX foci after exposition of thyreocyte rat cell line to 188Re. We used pattern recognition algorithms of the automated fluorescence interpretation system AKLIDES® for evaluation of γH2AX foci. Manual investigation was performed by three laboratories involving five observers. The results were compared by determining correlation and inter-laboratory variability. Results: The study confirmed the adaptation of automated interpretation system AKLIDES® to automated assessment of γH2AX foci in irradiated cells. Both manual and automated quantification resulted in increasing focus numbers depending on dose. Comparison of automated reading with visual assessment for five manual observers resulted in a determination coefficient of R2 = 0.889. The inter-laboratory variability for five manual investigators of three laboratories was 38.4 %. Conclusion: The interpretation system AKLIDES® demonstrated a high correlation with visually observed results. High inter-laboratory variability found for manual investigations revealed the usefulness for a standardized technique for evaluation of γH2AX foci.

Pharmacological inhibition of EGFR tyrosine kinase affects ILK-mediated cellular radiosensitization in vitro

Purpose: Integrin-linked kinase (ILK) mediates signals from β integrins and links integrins to epidermal growth factor receptor (EGFR). Previous studies have identified an antisurvival effect of ILK in irradiated cells. The aim of this study was to evaluate the role of EGFR tyrosine kinase (tk) activity for ILK-mediated radiosensitization. Materials and methods: Human FaDu squamous cell carcinoma (SCC) cells stably transfected with hyperactive ILK (ILK-hk) and ILKfl/fl and ILK−/− mouse fibroblasts were treated with the pharmacological EGFR-tk inhibitor BIBX1382BS without or in combination with single doses of X-rays. Clonogenic radiation survival, protein expression and phosphorylation (EGFR, v-akt murine thymoma viral oncogene homolog 1 (Akt), p42/44 mitogen-activated protein kinase), DNA-double strand break (DSB) repair measured by γH2AX foci, cell morphology and cell cycle distribution were examined. Results: Expression of ILK-hk or ILKfl/fl status resulted in significant radiosensitization relative to vector controls or ILK−/−. Following BIBX1382BS, clonogenic survival of normal fibroblasts and vector controls remained unaffected while ILK-hk-related radiosensitization was significantly diminished. In contrast to BIBX1382BS, which did not affect DNA-DSB repair, ILK-hk-mediated radiosensitization was associated with reduced DNA-DSB repair. At 10 days after BIBX1382BS treatment, FaDu transfectants, in contrast to fibroblasts, showed reduced cell size, accumulation of G1 phase cells and reduced Akt-serine(S)473 phosphorylation. Conclusions: Our findings confirm ILK as a cell type-independent antisurvival factor in irradiated cells, which actions in terms of radiosensitization critically depend on proper EGFR-tk activity.

Cetuximab Attenuates Its Cytotoxic and Radiosensitizing Potential by Inducing Fibronectin Biosynthesis

Inherent and acquired resistance to targeted therapeutics continues to emerge as a major clinical obstacle. For example, resistance to EGF receptor targeting occurs commonly, more so than was expected, on the basis of preclinical work. Given emerging evidence that cancer cell-substrate interactions are important determinants of therapeutic sensitivity, we examined the impact of cell-fibronectin interactions on the efficacy of the EGF receptor antibody cetuximab, which is used widely for lung cancer treatment. Our results revealed the potential for cell-fibronectin interactions to induce radioresistance of human non-small cell lung cancer cells. Cell adhesion to fibronectin enhanced tumor cell radioresistance and attenuated the cytotoxic and radiosensitizing effects of cetuximab. Both in vitro and in vivo, we found that cetuximab treatment led to a remarkable induction of fibronectin biosynthesis. Mechanistic analyses revealed the induction was mediated by a p38-MAPK-ATF2 signaling pathway and that RNAi-mediated inhibition of fibronectin could elevate the cytotoxic and radiosensitizing potential of cetuximab. Taken together, our findings show how cell adhesion blunts cetuximab, which, by inducing fibronectin, generates a self-attenuating mechanism of drug resistance.

Three-dimensional Invasion of Human Glioblastoma Cells Remains Unchanged by X-ray and Carbon Ion Irradiation In Vitro

PURPOSE Cell invasion represents one of the major determinants that treatment has failed for patients suffering from glioblastoma. Contrary findings have been reported for cell migration upon exposure to ionizing radiation. Here, the migration and invasion capability of glioblastoma cells on and in collagen type I were evaluated upon irradiation with X-rays or carbon ions. METHODS AND MATERIALS Migration on and invasion in collagen type I were evaluated in four established human glioblastoma cell lines exposed to either X-rays or carbon ions. Furthermore, clonogenic radiation survival, proliferation (5-bromo-2-deoxyuridine positivity), DNA double-strand breaks (γH2AX/53BP1-positive foci), and expression of invasion-relevant proteins (eg, β1 integrin, FAK, MMP2, and MMP9) were explored. Migration and invasion assays for primary glioblastoma cells also were carried out with X-ray irradiation. RESULTS Neither X-ray nor carbon ion irradiation affected glioblastoma cell migration and invasion, a finding similarly observed in primary glioblastoma cells. Intriguingly, irradiated cells migrated unhampered, despite DNA double-strand breaks and reduced proliferation. Clonogenic radiation survival was increased when cells had contact with extracellular matrix. Specific inhibition of the β1 integrin or proliferation-associated signaling molecules revealed a critical function of JNK, PI3K, and p38 MAPK in glioblastoma cell invasion. CONCLUSIONS These findings indicate that X-rays and carbon ion irradiation effectively reduce proliferation and clonogenic survival without modifying the migration and invasion ability of glioblastoma cells in a collagen type I environment. Addition of targeted agents against members of the MAPK and PI3K signaling axis to conventional chemoradiation therapy seems potentially useful to optimize glioblastoma therapy.

The novel HDAC inhibitor NDACI054 sensitizes human cancer cells to radiotherapy.

BACKGROUND AND PURPOSE Inhibition of histone deacetylases (HDACs) has preclinically and clinically shown promise to overcome radio- and chemoresistance of tumor cells. NDACI054 is a novel HDAC inhibitor, which has been evaluated here for its effects on cell survival and radiosensitization of human tumor cell lines from different origins cultured under more physiological three-dimensional (3D), extracellular matrix (ECM)-based conditions. MATERIAL AND METHODS A549 lung, DLD-1 colorectal, MiaPaCa2 pancreatic and UT-SCC15 head and neck squamous cell carcinoma cells were treated with increasing NDACI054 concentrations (0-50 nM, 24 h) either alone or in combination with X-rays (single dose, 0-6 Gy). Subsequently, 3D clonogenic cell survival, HDAC activity, histone H3 acetylation, apoptosis, residual DNA damage (γH2AX/p53BP1 foci assay 24h post irradiation) and phosphorylation kinetics of Ataxia telangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK), Caspase-3 and Poly(ADP-ribose)-Polymerase 1 (PARP 1) cleavage were analyzed. RESULTS NDACI054 potently decreased HDAC activity with concomitant increase in acetyl-histone H3 levels, mediated significant cytotoxicity and radiosensitization. These effects were accompanied by a significant increase of residual γH2AX/p53BP1-positive foci, slightly elevated levels of Caspase-3 and PARP 1 cleavage but no induction of apoptosis. CONCLUSIONS Our data show potent antisurvival and radiosensitizing effects of the novel HDAC inhibitor NDACI054 encouraging further preclinical examinations on this compound for future clinical use.

Focal Adhesion-Chromatin Linkage Controls Tumor Cell Resistance to Radio- and Chemotherapy

Cancer resistance to therapy presents an ongoing and unsolved obstacle, which has clear impact on patients survival. In order to address this problem, novel in vitro models have been established and are currently developed that enable data generation in a more physiological context. For example, extracellular-matrix- (ECM-) based scaffolds lead to the identification of integrins and integrin-associated signaling molecules as key promoters of cancer cell resistance to radio- and chemotherapy as well as modern molecular agents. In this paper, we discuss the dynamic nature of the interplay between ECM, integrins, cytoskeleton, nuclear matrix, and chromatin organization and how this affects the response of tumor cells to various kinds of cytotoxic anticancer agents.