Regulation

Prof. Dr Gerhard Thiel (TU Darmstadt, biology)

Background: Exposure of cells to ionizing irradiation often affects the regulation of the cell cycle; depending on the type of irradiation and on the type cells radiation can cause an elevated proliferation or an arrest in cell division. Several reports have in the past shows that the regulation of the cell cycle also involves several types of ion channels. Well investigated in this respect is the voltage dependent K+ channel Eag. The activity of this channel is crucial for the transition from the arrest phase to the G2 phase and this can be modified by several stress factors. An elevated cell proliferation of many tumors is causally linked to an elevated activity and density of this type of ion channel (1). In pilot experiments we have tested the effect of ionizing radtion on the activity of ion channels. Electrophysiological recordings on A-549 cells (tumor cells from human lung cancer) show that already very low doses of x-ray radiation result already within a minute in an activation of two types of K+ channels (2). This generates a hyperpolarization of the cells by ca. 20 mV. Such a hyperpolarization is in other cells known as a requirement for the progression in the cell cycle (3). In preliminary experiments we found that also the cell cycle of A-549 cells requires K+ channel activity; an inhibition of the K+ channels by conventional channel blockers resulted in A-549 cells in a halt of cell division.

Goal of the project: We want to examine the effect of ionizing irradiation on Eag channels and the consequence of this on cell cycle regulation.

Working strategy: Cultured human cells, which express Eag channels, will be exposed to x-ray and/or heavy ion irradiation. A mobile patch clamp apparatus will allow a recording of ion channel activity with high temporal resolution before and after radiation treatment. This will provide detailed information on the dependency of Eag channel activity on doses and type of radiation. Complementary experiments will address the question whether this treatment is changing the membrane voltage of cells and if this is causally related to the progression in cell cycle. Cell biological measurements will uncover the signal transduction cascades which link the primary radiation stress to the regulation of ion channels. In further experiments we will examine also the long-term effect of radiation on Eag activity. For this reason we will monitor the expression of Eag channels in radiation-stressed cells. This question will be tackled by electrophysiological recording of Eag channel activity and by complementary cell biological methods, which allow a quantification of the channel protein in the radiated cells. The required methods are available in the laboratory (4).

References:

(1) Pardo et al. (2005) J Membr Biol; 205:115-24.

(2) Roth et al. (2010) Nanion-User-Meeting.

(3) Strobl et al. (1995) Gen Pharmacol; 26:1643-9.

(4) Meckel et al. (2004) Plant J; 39:182-93.

(5) Hurstet et al. (2004) Plant J; 37:391-7.

Dr. Claudia Fournier (GSI, Biophysics)

Background: One of the most frequent side effects during the cancer radiotherapy is fibrosis, which is an alteration of the connective tissue often preceded by acute inflammation. Although the molecular basis of fibrosis is not completely understood, it has been shown that the interaction of epithelial and endothelial cells as well as different cell types of the stroma (macrophages, fibroblasts) via cytokines, growth factors and othe signalling molecules of the extracellular matrix are involved. Many components play also a role in inflammation and wound healing. Our previous investigations of the fibrosis inducing potential of heavy ions revealed that the efficiency of heavy ions compared to photons increases with ionizing density of the radiation quality (1). At the other hand, an anti-inflammatory effect is observed during low dose treatment of chronic inflammation diseases using the α-particle emitter radon. This indicates a complex dependence of the regulation of inflammation related processes on ionizing density of the radiation quality, dose, and fraction of irradiated cells in the exposed tissue and interaction of the different cell types.

Goal of the project: After the establishment of a 3D model of the epithelial layer and stroma the potential anti-inflammatory effects of heavy ions should be investigated by irradiation of single cells, which corresponds to a low dose.

Working plan: Ongoing experiments in cocultures of endothelial cells, monocytes and lymphocytes are planned to be extended on keratinocytes, fibroblasts and other cell types of the connective tissue. Based on the obtained results, a 3D model of the epithelial layer and the connective tissue (lung or skin, EpiAirway and EpiDermFT, MatTek) should be adapted for heavy ion irradiation. Conditions for the future use of the GSI microbeam for targeted exposure of single cells (2) should be taken into account in the concept of the 3D model.

In a first step, the impact of irradiation on proliferation, cell cycle regulation, differentiation and apoptosis will be assessed in situ (morphological features after differential staining of nucleus and cytoplasm, immuno-detection in slices in combination with cell type specific markers like integrins and cadherins).

In a second step, production and activation of cytokines and anchoring of the cells in the extracellular matrix will be investigated by immuno-detection. This will be performed in a close collaboration with project 1F (T. Meckel, integrins, extracellular matrix). The obtained data can be used by project 3A (B. Drossel) for the modelling of the impact of radiation on cellular effects (cell cycle regulation, differentiation, apoptosis). Furthermore, ongoing collaborations with projects 2D (P. Layer, differentiation), 2A (G. Thiel, signal transduction) and 2C (N. Dencher, oxidative stress) will be extended.

References:

(1) Fournier et al. (2001) Int J Radiat Biol; 77:713-22.

(2) Heiss et al. (2006) Radiat Res; 165:231-9.

Prof. Dr. Norbert A. Dencher (TU Darmstadt, Chemie)

Hintergrund: Um die Wirkung von Strahlung auf die Physiologie der Zelle bis auf das molekulare Niveau besser zu verstehen und um auftretenden Schädigungen ggf. in Zukunft entgegenwirken zu können, müssen an Zellkulturen und daraus isolierten Zellkomponenten Untersuchungen über Veränderungen des Proteoms und Lipidoms sowie zellphysiologischer Aktivitätsparameter durchgeführt werden. Die Untersuchungen von biologischen Membranen und von Mitochondrien sind dabei von besonderem Interesse, da die Energiebereitstellung und Signaltransduktion durch sie erfolgen, aber auch zahlreiche Krankheiten (z.B. Morbus Alzheimer und Parkinson) sowie das Altern durch deren Fehlfunktionen ausgelöst werden.

Ziel des Projektes: In diesem Projekt soll untersucht werden, ob und durch welche Mechanismen strahleninduzierte Veränderungen im Proteom, im Lipidom, im oxidativen Status und in der Aktivität von Zellen und Mitochondrien auftreten und wie diese durch O2, CO2 und Sauerstoffradikale beeinflusst werden. Weiterhin soll einerseits zwischen der direkten Einwirkung der Strahlung auf Proteine und Lipide und andererseits deren sekundären Veränderungen nach Schädigung der Nukleinsäuren im Zellkern bzw. in den Mitochondrien differenziert werden. Auf diese Weise sollen direkte Aussagen auf Protein- und Membranebene zum Mechanismus der Strahlenwirkung auf Tumorzellen erhalten werden.

Arbeitsplan: Als Vorarbeiten zu diesem Teilprojekt dienen die Charakterisierung verschiedener menschlicher Zellen nach Einwirkung ionisierender Strahlung im Rahmen unseres BMBF-Projektes in Zusammenarbeit mit der GSI (“Induktion und Transmission von genetischen Schäden nach Hoch-LET-Bestrahlung: In vivo und in vitro Untersuchungen“). Humane Fibroblasten und spontan immortalisierte Oligodendrozyten aus Rattenhirn (OLN-93) werden im Rahmen dieses Projektes nach Exposition mit ionisierender Strahlung (Röntgenstrahlung und Kohlenstoffionen) mit analytischen und zellbiologischen Methoden charakterisiert bzw. dienen der Isolation von Membranen und Mitochondrien. Weiterhin sollen strahleninduzierte Veränderungen des Proteoms, des Lipidoms und zellphysiologischer Aktivitätsparameter an Zellen und Zellkomponenten unter dem Einfluss der vorgegebenen Konzentration an O2 und CO2 untersucht werden. Eine Quantifizierung der durch Strahlung induzierten Veränderungen der Protein-Quantität, der posttranslationalen Modifikationsmuster und der Protein-Protein-Interaktionen soll mittels nativer 2D-Gelelektrophorese und Massenspektrometrie durchgeführt werden. Weiterhin sollen die intrazellulären/mitochondrialen ROS-Konzentrationen gemessen werden (1-4). Letztendlich soll eine Analyse der physikalischen Membraneigenschaften durch Fluoreszenz-Anisotropie und Neutronenstreuung durchgeführt sowie der Nachweis von Veränderungen in der Zusammensetzung der Lipidphase und dem Auftreten von Lipidmodifikationen mittels Dünnschichtchromatographie, HPLC und ESI-MS erbracht werden.

Dieses Projekt soll in Zusammenarbeit mit den Teilprojekten 3A Drossel (Modellierung strahlungsinduzierter Veränderungen von Protein-Protein-Wechselwirkungen und post-translationalen Modifikationen), 2B Fournier, 2F Rödel (Nachweis und Lokalisation von Survivin-Bindungspartner-Interaktionen) und 2E Schmidt durchgeführt werden. Insbesondere die bestehende erfolgreiche Zusammenarbeit mit den Teilprojekten 2B Fournier (humane Zellkulturmodelle, Nachweis strahleninduzierter Zellveränderungen, Bestrahlungstechnik; 3, 4, 5) und 2E Schmidt (Mechanismus und Angriffsort von Wirkstoffen/Leitstrukturen, Neutronenstreuung) soll weiter intensiviert werden.

Literatur:

(1) Reifschneider et al. (2006) J Proteome Res; 5:1117-32.

(2) Dani und Dencher (2008) Biotechnol J; 3:817-22.

(3) Colindres et al. (2008) GSI Report 2007.

(4) Frenzel et al. (2009) GSI Report 2008.

(5) Frenzel et al. (2010) GSI Report 2009

Prof. Dr. Paul G. Layer (Entwicklungsbiologie & Neurogenetik)

Background: Dividing cells are more sensitive to ionising radiation. Particularly endangered are tissues during their embryonic periods, and also adult lesioned or normal tissues, which regenerate through stem cell amplification. Thereby, it is possible that due to irradiation normal adult stem cells can be transformed into tumour stem cells (1). In this project three different types of tissues will be analysed from non- and irradiated mice: a) from small intestine, b) from central retina, and c) from the eye periphery (ciliary body). The small intestine represents a continuously regenerating tissue. Due to its high tumour incidences, it represents a classic model of cancer research. On one side, in this tissue ionising radiation can be employed for tumour therapy, on the other side it can induce tumours, thus representing unwanted side effects with vvv diagnostic imaging techniques (2). In contrast to the gut, in mouse retina almost no mitoses occur any longer after postnatal day 20. The retina is not tumour-stricken, and at the same time one of the most resistant tissues to irradiation (3). In its central part, the retina is a differentiated neural tissue “simply” constructed from six major cell types. Towards its periphery, the functional retina is transformed into the ciliary body. Although the mammalian retina cannot regenerate after lesioning or blinding (e.g. is not a regenerative tissue as the small intestine), stem cells have been detected in the eye periphery both of embryonic and adult retinae (4, 5). These adult stem cells are the focus for the artificial regeneration of retinal tissue (retinal tissue engineering, 5).

Aim of project: The effects of ionising radiation on three tissues (see above), whereby the intestinal tissue will be our base tissue (continuously regenerative, radiation-sensitive, tumor-afflicted). Changes in cell proliferation, apoptosis, double strand breaks, as well as expression of appropriate stem cell markers will be analysed; also, similar work on tissue explants will be performed.

Literature:

[1] Zhou BB et al. (2009). Tumour-initiating cells: challenges and opportunities for anticancer drug discovery. Nat Rev Drug Discov 8: 806-823.

[2] Oehler C, Ciernik IF (2006). Radiation therapy and combined modality treatment of gastrointestinal carcinomas. Cancer Treat Rev 32: 119-138.

[3] Krebs W et al. (1988). The effect of accelerated argon ions on the retina. Radiat Res 115: 192-201.

[4] Layer PG, Rothermel A, Willbold E (2001). From stem cells towards neural layers: a lesson from re-aggregated embryonic retinal cells. NeuroReport 12(7): A39-46.

[5] Ohta K, Ito A, Tanaka H (2008). Neuronal stem/progenitor cells in the vertebrate eye. Dev Growth Differ 50: 253-259.

Prof. Dr. Boris Schmidt (TU Darmstadt, Chemie)

Hintergrund: DNA-Schädigung durch Chemo- oder Radiotherapie ist ein zentrales Wirkprinzip der Tumortherapie. Die induzierte DNA-Reparatur führt jedoch häufig zu Therapieresistenz. Die simultane Chemotherapie in Kombination mit ionisierender Strahlung kann die induzierte DNA-Reparatur minimieren, die Zelltoxizität der Strahlung steigern und dadurch den Therapieerfolg erhöhen. Die potenziellen Ziele einer synergistischen Chemotherapie sind deshalb vor allem DNA-Reparatur (1,2) und Apoptosesignale.

Ziel des Projektes: Im Rahmen dieses Projektes sollen neue radiosensitivierende Leitstrukturen im in vitro -Strahlenassay identifiziert und eine Evaluation dieser Leitstrukturen im in vitro Assay mit dicht ionisierender Strahlung erbracht werden (Röntgenstrahlung: AG Löbrich, Schwerionenbestrahlung: GSI).

Arbeitsplan: Ein Hochdurchsatzscreen ist ungeeignet, um innerhalb der Projektdauer neue Leitstrukturen zu generieren und anschließend zu optimieren, da die typische Zeitdauer in der pharmazeutischen Industrie über 3 Jahre beträgt. Deshalb erfolgt die Auswahl bzw. Synthese der Screeningkandidaten aus verschiedenen Wirkstoffklassen anhand medizinal-chemischer Kriterien in iterativen Synthese/Assay-Zyklen. 50% der Ressourcen werden auf die Evaluation etablierter radiosensitivierender Leitstrukturen bzw. zugelassener Therapeutika verwendet, um nach spätestens 2 Jahren geeignete in vivo Kandidaten bereitstellen zu können: Histon-Deacetylase-Inhibitoren (HDACI), ATM-Kinase-, Polymerase-ŋ- und PARP-1-Inhibitoren. Die Hälfte der Ressourcen wird langfristigen high risk Ansätzen gewidmet: der Entwicklung selektiver 20S-Proteasom-, GSK3- bzw. Notch-Inhibitoren (3,4). Zwei dieser Ansätze werden durch rationales Wirkstoffdesign bzw. Ligandendocking an die proteolytische beta5-Untereinheit durch das Teilprojekt 3C Hamacher iterativ begleitet. Die unveröffentlichte Struktur des 20S-Proteasomkomplexes mit BSc2189 bietet exklusive Informationen zur S1’-Bindungstasche (3,5) und hierdurch rationalen Zugang zu optimierten, nicht-peptidischen und selektiven Enzym-Inhibitoren mit reduzierter Neurotoxizität.

Die Auswahl der privilegierten Testsubstanzen für die zellulären Assays erfolgt anhand etablierter medizinal-chemischer Kriterien (log D, Zellpermeabilität, Zelltoxizität, tPSA u.a.). Die Bestrahlung der Organismen bei gleichzeitiger Inhibition der DNA-Reparatur führt zu einer Radiosensibilisierung und damit zu einer deutlich reduzierten Zellproliferation und erhöhtem Zelltod. Als primärer Bioassay werden Zellproliferationstests eingesetzt. In Zebrafischlarven wird der DNA-Schaden und die Wirkstoff-Permeabilität bestimmt (6). Die detaillierte Analyse der DSB-Reparatur im Replikationsvorgang soll durch Checkpointanalysen (1D Deckbar), H2AX-Assay (3B Scholz), Foci-Analyse, Histon-Analyse (1C Rapp) und hochauflösende fluoreszenzmikroskopische Verfahren (u.a. konfokale Laser-Scanning-Mikroskopie) erfolgen (1A Löbrich). Hierfür werden fluoreszente radiosensitizer entwickelt und CDC25-Inhibitoren zur Kontrolle des G2/M-Arrests für das Teilprojekt 1D Deckbar bereitgestellt.

Als Sekundärassay wird im AG 2B Fournier ein Modell für die Epithelzellschicht und das angrenzende Bindegewebe etabliert, das mittels DIS-Mikrostrahl die gezielte Bestrahlung einzelner Zellen erlaubt. Gemeinsam mit der Arbeitsgruppe Laube (Projekt 2G) wird ein Zebrafischlarven-Assay aufgebaut in dem Permeabilität und Effektivität der Wirkstoffe unter -Strahlung untersucht wird (6). Die wirksamen Substanzen aus dem Zebrafisch-Assay werden anschließend in Maus und Ratte durch in vivo Untersuchung mit -Strahlung und DIS evaluiert. Diese Tiermodelle sind in der Arbeitsgruppe Roedel (Projekt 2F) etabliert.

Literatur:

(1) Adimoolam et al. (2007) Proc Natl Acad Sci USA; 104:19482-7.

(2) Chen et al. (2007) Cancer Res; 67:5318-27.

(3) Schmidt et al. (2005) J Biol Chem; 280:34441-6.

(4) Kukar et al. (2008) Nature; 453:925-9.

(5)Kloetzel et al. (2006) Cancer Res; 66:649-52.

(6)Jarvis et al. (2003) Mutat Res; 541:63-9.

PD Dr. Franz Rödel (GUF Frankfurt, Medicine)

Backgound: In recent years the ability of tumor cells to circumvent or evade apoptosis was recognized to be a major mechanism of therapy resistance. In this context, Survivin, the smallest member of the Inhibitor of Apoptosis (IAP) protein family deserves growing attention due to its universal over-expression in cancer cells. Functionally, Survivin is involved in a variety of molecular pathways and cellular networks, including cell division / proliferation, intracellular signal transduction and apoptosis regulation (1). In addition to its extraordinary ability to associate with a variety of protein partners, survivin localization in multiple cellular compartments, including the mitochondrion, is considered essential for its function. Thus, a nuclear association of Survivin with the proteins INCEP (Inner Centromere Protein), Borealin and AuroraB kinase at the kinetochore is essential of chromosomal segregation and cytokinesis, whereas cytoplasmic survivin displays cytoprotective properties due to the inhibition of caspases (1).

In a variety of human malignancies, over expression of Survivin is reported to be a marker of tumor progression and shortened survival. In colorectal cancer, for example, Survivin displays prognostic relevance for a decreased overall survival after preoperative radiochemotherapy. In vitro studies on colorectal cancer cell lines with different intrinsic radiation sensitivity further revealed a close correlation between the level of Survivin expression and radiation responsiveness (2). Moreover, a functional inhibition of Survivin by siRNA (3) or antisense oligonucleotides resulted in a significant radiosensitization, which was confirmed in animal models (4). On the basis of these properties Survivin is considered to be an attractive molecular target for selective tumor therapy. The radiosensitising effect of Survivin inhibition, however, involves caspase-dependent and caspase-independent pathways (3). In more recent studies, we demonstrated a direct interrelationship between survivin nuclear accumulation and the repair of radiation-induced DNA double strand breaks (DSBs), which is mediated by a significant co- localization of Survivin with members of the DNA repair machinery (Ku70, MDC1, 53BP1, DNA-PKcs, H2AX) (5).

The aim of the project: The project comprises research on the molecular mechanisms involved in survivin mediated radiation resistance and the impact and functionality of Survivin in DNA repair complexes and the DNA damage response following irradiation.

Work Plan: In selected tumor cell lines (colorectal cancer: SW480, HCT-15 and glioblastoma: LN229, U87MG) and in NHEJ-mutant cell lines (fibroblasts, knockout MEFs) Survivin expression will be modulated by RNAi or over-expression of survivin mutants to analyse its impact on the repair of radiation-induced DSBs. Most of the required constructs are already established. For these investigations, there is ideal interrelationship with other members of the consortium, especially with the project 1A Löbrich (DNA repair capacity, NHEJ), 1D Deckbar (Impact on G1/S-Checkpoint and cell cycle), 1F Meckel (Development of high resolution microscopy techniques), 2C Dencher (Radiation effect on the proteome and the activity of cells and mitochondria), 3A Drossel (Modeling of the recruitment of proteins involved in DNA-damage recognition) and 2G Laube (Ligand-regulated ion channels as target structures). As currently a variety of survivin antagonists were tested in preclinical or clinical studies, there is also synergism with the project 2E Schmidt (drug-induced modulation of radiation-response) to increase radiation responsiveness of tumor cells.

References:

(1) Altieri (2008) Nat Rev Cancer; 8:61-70.

(2) Rödel et al. (2003) Int J Radiat Oncol Biol Phys; 55:1341-7.

(3) Rödel et al. (2005) Cancer Res; 65:4881-7.

(4) Rödel et al. (2008) Int J Radiat Oncol Biol Phys; 71:247-55

(5) Capalbo et al. (2010) Int J Radiat Oncol Biol Phys; 77:226-34.

PD Dr. Bodo Laube (TU Darmstadt, Biology)

Background: The majority of brain tumors derives from glial cells and is collectively called gliomas. Although the genetics and cellular biology of malignant gliomas have been extensively studied, effective therapies are lacking because these cells seem to have developed adaptations to support a unique biology. Notably, glioma growth is physically restricted by the skull. Therefore, glioma cells release the neurotransmitter glutamate into adjacent brain tissue (1) at concentrations that are sufficient to cause neuronal cell death (2). The released glutamate also explains peritumoral seizures which are a common symptom early in the disease. In addition, glutamate serves as a trophic factor promoting glioma cell invasion and the formation of tumor metastasis (3). Therefore it is assumed that glutamate release protects tumor cells from endogenously produced reactive oxygen and nitrogen species and endows tumors with an enhanced resistance to radiation- and chemotherapy. Glutamate is the major excitatory neurotransmitter in the brain and is normally restricted to the synaptic and perisynaptic space of glutamatergic synapses. Here, the transmitter binds to post-synaptic neuronal ionotropic glutamate receptors and mediates fast signal transmission. The NMDA receptor, a prominent member of the ionotropic glutamate receptors assemble into tetramers from subsets of two subunits, NR1 and NR2 (4). The NR2 subunits A, B, C, and D determine spatial distribution and electrophysiological properties of the channel. Under physiological conditions, activation of glutamate NMDA receptors in neurons is translated to the nucleus by the extracellular signal-regulated kinase (ERK1/2)-signaling cascade leading to the phosphorylation of the cAMP-responsive element binding protein (CREB) promoting neuronal survival. However, prolonged activation of extra-synaptic NMDA receptors can trigger a sustained influx of Ca2+ which readily overwhelms intracellular Ca2+ regulatory processes ultimately leading to Ca2+-mediated cell death. This process, termed excitotoxicity, is believed to be a final common death pathway in many neurological diseases.

Aim of the project: Glutamate receptors contribute i) to the survival of cancer cells, their growth, and progression to the metastatic tumor phase and ii) to excitotoxic neuronal cell death. The underlying cellular mechanisms are not known so far. However, although there is much left to understand regarding the involvement of these receptors in “pro-survival” and “pro-death” signaling processes it is encouraging that modulating their function has clinical potential in terms of both improved diagnosis/prognosis and therapy.

Working strategy: Signaling pathways important during excitotoxic cell death, carcinogenesis, cell proliferation and the development of resistance to apoptosis that have been linked to glutamate receptor activation will be explored in selected tumor cell lines and upon heterologous expression of different NMDA receptor combinations in cultured cells. The topics will include analysis by electrophysiological, biophysical and biochemical methods and try to bridge ionizing radiation-induced foci and the role of glutamate receptors in cell survival and death (5).

References:

(1) Savaskan et al., (2008) Nature Medicine 14

(2) Stepulak et al., (2005) Proc Natl Acad Sci 102

(3) Sontheimer, (2008) J. Neurochem. 105

(4) Zhang et al., (2007) Neuron 53

(5) Crowe et al., (2006) Eur. J. Neurosci. 23