Patrick Maier

Cellular Pathways in Response to Ionizing Radiation and Their Targetability for Tumor Radiosensitization.

During the last few decades, improvements in the planning and application of radiotherapy in combination with surgery and chemotherapy resulted in increased survival rates of tumor patients. However, the success of radiotherapy is impaired by two reasons: firstly, the radioresistance of tumor cells and, secondly, the radiation-induced damage of normal tissue cells located in the field of ionizing radiation. These limitations demand the development of drugs for either radiosensitization of tumor cells or radioprotection of normal tissue cells. In order to identify potential targets, a detailed understanding of the cellular pathways involved in radiation response is an absolute requirement. This review describes the most important pathways of radioresponse and several key target proteins for radiosensitization.

Distinct role of endocytosis for Smad and non-Smad TGF-β signaling regulation in hepatocytes

BACKGROUND & AIMS In injured liver, TGF-β affects all hepatic cell types and participates in wound healing and fibrogenesis. TGF-β downstream signaling is highly complex and cell type dependent, involving Smad and non-Smad signaling cascades thus requiring tight regulation. Endocytosis has gained relevance as important mechanism to control signaling initiation and termination. In this study, we investigated endocytic mechanisms for TGF-β mediated Smad and non-Smad signaling in hepatocytes. METHODS Endocytosis in hepatocytes was elucidated using chemical inhibitors, RNAi, viral gene transfer and caveolin-1-/- mice. TGF-β signaling was monitored by Western blot, reporter assays and gene expression analysis. RESULTS In hepatocytes, Smad activation is to a large degree accomplished AP-2 complex dependent on the hepatocyte surface without the necessity of clathrin coated pit formation or an endocytic step. In contrast, non-Smad/AKT pathway activation required functional dynamin mediated endocytosis and the presence of caveolin-1, an essential protein for caveolae formation. Furthermore, these two TGF-β signaling initiation platforms discriminate distinct signaling routes that integrate at the transcriptional level as shown for TGF-β target genes, Id1, Smad7, and CTGF. Endocytosis inhibition increased canonical Smad signaling and culminated in a superinduction of Id1 and Smad7 expression, whereas caveolin-1 mediated AKT pathway activation was required for maximal CTGF induction. CONCLUSIONS Endocytosis is critical for TGF-β signaling regulation in hepatocytes and determines gene expression signature and (patho)physiological outcome.

Retroviral vectors for gene therapy

The present invention provides novel, improved retroviral vectors which can be used for gene therapy, more specifically, retroviral vectors which are not only safer, more versatile, and more convenient than any other existing vectors, but they also drive high levels of gene expression and high viral titer. In retroviral vectors of the present invention, gag and env coding sequences are deleted, and all or part of U3 can be readily substituted with heterologous, non-retroviral promoter elements. Furthermore, at least one internal ribosome entry site is employed to express more than one genes, and multicloning sites are placed in an insertion site for cloning of a heterologous promoter or a foreign gene.Since their first clinical trial 20 years ago, retroviral (gretroviral and lentiviral) vectors have now been used in more than 350 gene-therapy studies. Retroviral vectors are particularly suited for gene-correction of cells due to long-term and stable expression of the transferred transgene(s), and also because little effort is required for their cloning and production. Several monogenic inherited diseases, mostly immunodeficiencies, can now be successfully treated. The occurrence of insertional mutagenesis in some studies allowed extensive analysis of integration profiles of retroviral vectors, as well as the design of lentiviral vectors with increased safety properties. These new-generation vectors will enable us to continue the successful story of gene therapy, and treat more patients and even more complex diseases.

Good manufacturing practice-grade production of unrestricted somatic stem cell from fresh cord blood

BACKGROUND AIMS The discovery of unrestricted somatic stem cells (USSC), a non-hematopoietic stem cell population, brought cord blood (CB) to the attention of regenerative medicine for defining more protocols for non-hematopoietic indications. We demonstrate that a reliable and reproducible method for good manufacturing practice (GMP)-conforming generation of USSC is possible that fulfils safety requirements as well as criteria for clinical applications, such as adherence of strict regulations on cell isolation and expansion. METHODS In order to maintain GMP conformity, the automated cell processing system Sepax (Biosafe) was implemented for mononucleated cell (MNC) separation from fresh CB. After USSC generation, clinical-scale expansion was achieved by multi-layered CellSTACKs (Costar/Corning). Infectious disease markers, pyrogen and endotoxin levels, immunophenotype, potency, genetic stability and sterility of the cell product were evaluated. RESULTS The MNC isolation and cell cultivation methods used led to safe and reproducible GMP-conforming USSC production while maintaining somatic stem cell character. CONCLUSIONS Together with implemented in-process controls guaranteeing contamination-free products with adult stem cell character, USSC produced as suggested here may serve as a universal allogeneic stem cell source for future cell treatment and clinical settings.

The Disparate Twins: A Comparative Study of CXCR4 and CXCR7 in SDF-1α–Induced Gene Expression, Invasion and Chemosensitivity of Colon Cancer

Purpose: In colorectal cancer, increased expression of the CXC chemokine receptor 4 (CXCR4) has been shown to provoke metastatic disease due to the interaction with its ligand stromal cell-derived factor-1 (SDF-1). Recently, a second SDF-1 receptor, CXCR7, was found to enhance tumor growth in solid tumors. Albeit signaling cascades via SDF-1/CXCR4 have been intensively studied, the significance of the SDF-1/CXCR7–induced intracellular communication triggering malignancy is still only marginally understood. Experimental Design: In tumor tissue of 52 patients with colorectal cancer, we observed that expression of CXCR7 and CXCR4 increased with tumor stage and tumor size. Asking whether activation of CXCR4 or CXCR7 might result in a similar expression pattern, we performed microarray expression analyses using lentivirally CXCR4- and/or CXCR7-overexpressing SW480 colon cancer cell lines with and without stimulation by SDF-1α. Results: Gene regulation via SDF-1α/CXCR4 and SDF-1α/CXCR7 was completely different and partly antidromic. Differentially regulated genes were assigned by gene ontology to migration, proliferation, and lipid metabolic processes. Expressions of AKR1C3, AXL, C5, IGFBP7, IL24, RRAS, and TNNC1 were confirmed by quantitative real-time PCR. Using the in silico gene set enrichment analysis, we showed that expressions of miR-217 and miR-218 were increased in CXCR4 and reduced in CXCR7 cells after stimulation with SDF-1α. Functionally, exposure to SDF-1α increased invasiveness of CXCR4 and CXCR7 cells, AXL knockdown hampered invasion. Compared with controls, CXCR4 cells showed increased sensitivity against 5-FU, whereas CXCR7 cells were more chemoresistant. Conclusions: These opposing results for CXCR4- or CXCR7-overexpressing colon carcinoma cells demand an unexpected attention in the clinical application of chemokine receptor antagonists such as plerixafor. Clin Cancer Res; 20(3); 604–16. ©2013 AACR.

Localization Microscopy (SPDM) Reveals Clustered Formations of P-Glycoprotein in a Human Blood-Brain Barrier Model

P-glycoprotein (Pgp; also known as MDR1, ABCB1) is the most important and best studied efflux transporter at the blood-brain barrier (BBB); however, the organization of Pgp is unknown. The aim of this study was to employ the recently developed super-resolution fluorescence microscopy method spectral precision distance microscopy/spectral position determination microscopy (SPDM) to investigate the spatial distribution of Pgp in the luminal plasma membrane of brain capillary endothelial cells. Potential disturbing effects of cell membrane curvatures on the distribution analysis are addressed with computer simulations. Immortalized human cerebral microvascular endothelial cells (hCMEC/D3) served as a model of human BBB. hCMEC/D3 cells were transduced with a Pgp-green fluorescent protein (GFP) fusion protein incorporated in a lentivirus-derived vector. The expression and localization of the Pgp-GFP fusion protein was visualized by SPDM. The limited resolution of SPDM in the z-direction leads to a projection during the imaging process affecting the appeared spatial distribution of fluorescence molecules in the super-resolution images. Therefore, simulations of molecule distributions on differently curved cell membranes were performed and their projected spatial distribution was investigated. Function of the fusion protein was confirmed by FACS analysis after incubation of cells with the fluorescent probe eFluxx-ID Gold in absence and presence of verapamil. More than 112,000 single Pgp-GFP molecules (corresponding to approximately 5,600 Pgp-GFP molecules per cell) were detected by SPDM with an averaged spatial resolution of approximately 40 nm in hCMEC/D3 cells. We found that Pgp-GFP is distributed in clustered formations in hCMEC/D3 cells while the influence of present random cell membrane curvatures can be excluded based on the simulation results. Individual formations are distributed randomly over the cell membrane.

Overexpression of MDR1 Using a Retroviral Vector Differentially Regulates Genes Involved in Detoxification and Apoptosis and Confers Radioprotection

Abstract Maier, P., Fleckenstein, K., Li, L., Laufs, S., Zeller, W. J., Baum, C., Fruehauf, S., Herskind, C. and Wenz, F. Overexpression of MDR1 Using a Retroviral Vector Differentially Regulates Genes Involved in Detoxification and Apoptosis and Confers Radioprotection. Radiat. Res. 166, 463–473 (2006). Overexpression of P-glycoprotein (P-gp), the product of the MDR1 (multidrug resistance 1) gene, might complement chemotherapy and radiotherapy in the treatment of tumors. However, for safety and mechanistic reasons, it is important to know whether MDR1 overexpression influences the expression of other genes. Therefore, we analyzed differential gene expression in cells of the human lymphoblast cell line TK6 retrovirally transduced with MDR1 using the GeneChip Human Genome U133 Plus2.0 (Affymetrix). Sixty-one annotated genes showed a significant change in expression (P < 10−4) in MDR1-overexpressing cells compared to untransduced cells and cells transduced with a control virus expressing the neomycin phosphotransferase gene. Several genes coding for proteins involved in detoxification and exocytosis showed ∼1.4– 4-fold increases in transcript levels (e.g. ALDH1A, UNC13). Additionally, pro-apoptosis genes were down-regulated (e.g. twofold for CASP1, 2.5-fold for NALP7) with concomitant increased expression of the potential anti-apoptosis gene AKT3. In functional assays the influence of MDR1 overexpression on apoptosis signaling was further corroborated by showing reduced rates of apoptosis in response to irradiation in TK6 cells transduced with MDR1. In conclusion, the resistant phenotype of MDR1-mediated P-gp-overexpressing cells is associated with differential expression of genes coding for metabolic and apoptosis-related proteins. These results have important implications for understanding the mechanisms by which MDR1 gene therapy can protect normal tissues from radiation- or chemotherapy-induced damage during tumor treatment.

MDR1 Gene Transfer Using a Lentiviral SIN Vector Confers Radioprotection to Human CD34+ Hematopoietic Progenitor Cells

Abstract Maier, P., Herskind, C., Fleckenstein, K., Spier, I., Laufs, S., Jens Zeller, W., Fruehauf, S. and Wenz, F. MDR1 Gene Transfer Using a Lentiviral SIN Vector Confers Radioprotection to Human CD34+ Hematopoietic Progenitor Cells. Radiat. Res. 169, 301–310 (2008). Tumor radiotherapy with large-field irradiation results in an increase in apoptosis of the radiosensitive hematopoietic stem cells (CD34+). The aim of this study was to demonstrate the radioprotective potential of MDR1 overexpression in human CD34+ cells using a lentiviral self-inactivating vector. Transduced human undifferentiated CD34+ cells were irradiated with 0–8 Gy and held in liquid culture under myeloid-specific maturation conditions. After 12 days, MDR1 expression was determined by the rhodamine efflux assay. The proportion of MDR1-positive cells in cells from four human donors increased with increasing radiation dose (up to a 14-fold increase at 8 Gy). Determination of expression of myeloid-specific surface marker proteins revealed that myeloid differentiation was not affected by transduction and MDR1 overexpression. Irradiation after myeloid differentiation also led to an increase of MDR1-positive cells with escalating radiation doses (e.g. 12.5–16% from 0–8 Gy). Most importantly, fractionated irradiation (3 × 2 Gy; 24-h intervals) of MDR1-transduced CD34+ cells resulted in an increase in MDR1-positive cells (e.g. 3–8% from 0–3 × 2 Gy). Our results clearly support a radioprotective effect of lentiviral MDR1 overexpression in human CD34+ cells. Thus enhancing repopulation by surviving stem cells may increase the radiation tolerance of the hematopoietic system, which will contribute to widening the therapeutic index in radiotherapy.

SNAI2 as a novel radioprotector of normal tissue by gene transfer using a lentiviral bicistronic SIN vector.

Abstract Tumor radiotherapy with large-field irradiation results in an increase of p53-dependent apoptosis of the radiosensitive hematopoietic stem cells. Proapoptotic PUMA is a transcriptional target of p53. Thus suppression of PUMA expression by gene therapy with the transcription repressor SNAI2 as transgene might be a potential approach for normal tissue protection during radiotherapy. SNAI2 cDNA was cloned in a lentiviral SIN vector in a bicistronic expression cassette followed by a floxed IRES-EMCV linker and EGFP as selection gene. Wild-type p53 TK6 cells were used as the cellular model system. We could demonstrate the significant radioprotective effect of SNAI2 overexpression in a cytotoxicity assay after irradiation with 0–5 Gy compared with untransduced or control vector (inverse oriented SNAI2 cDNA)-transduced cells. Additionally, TK6-SNAI2 compared to TK6-SNAI2inv cells showed a survival advantage in a clonogenic assay after irradiation with 0–3 Gy. Determination of the proportion of sub-G1 cells in TK6-SNAI2 cells revealed an approximately 50% reduction in apoptosis compared with both control entities. In this study using a bicistronic lentiviral vector, we were able to provide proof of principle that lentiviral overexpression of SNAI2 might be used for radioprotective gene therapy to widen the therapeutic range in radiotherapy.

Overexpression of Caveolin-1 in Lymphoblastoid TK6 Cells Enhances Proliferation After Irradiation with Clinically Relevant Doses

Background and Purpose:The transmembrane protein caveolin-1 (CAV1) is an essential component of caveolae, small membrane invaginations involved in vesicle formation. CAV1 plays a role in signal transduction, tumor suppression and oncogene transformation. Previous studies with CAV1 knockout mice and CAV1 knockdown in pancreatic tumor cells implicated CAV1 in mediating radioresistance. The aim of this work was to test the effect of CAV1 overexpression after irradiation in human cells lacking endogenous CAV1 expression.Material and Methods:Human CAV1 was overexpressed in lymphoblastoid TK6 cells (TK6-wt) using a eukaryotic expression plasmid, pCI-CAV1, or a lentiviral SIN (self-inactivating) vector, HR’SIN-CAV1. CAV1 expression was verified in TK6 cells with Western blot analysis or intracellular FACS (fluorescence-activated cell sorting) staining. The effect of CAV1 on proliferation kinetics after irradiation of TK6 cells was measured with a growth assay.Results:TK6-wt showed no detectable endogenous CAV1 expression. Lentivirally mediated transduction with HR’SIN-CAV1 (TK6-CAV1) resulted in a considerably stronger CAV1 expression in comparison to TK6 cells electroporated with pCI-CAV1. Intracellular FACS analysis showed that 90% of transduced cells expressed CAV1. CAV1 enhanced early proliferation of TK6 cells after irradiation with a dose of 2 Gy, whereas proliferation of unirradiated cells was not affected. CAV1 also protected cells after irradiation with 4 Gy. This radioprotective effect was supported by a reduction of radiation-induced apoptosis.Conclusion:A model system for expression of exogenous CAV1 by stable lentiviral transduction of TK6 cells was established. Functional assays demonstrated enhanced early proliferation by CAV1 expression in TK6 cells after irradiation with clinically relevant doses supporting the role of CAV1 as a prosurvival factor.Hintergrund und Ziel:Das Transmembranprotein Caveolin-1 (CAV1) ist eine essentielle Komponente der Caveolae, kleiner Membraninvaginationen, die an der Vesikelbildung beteiligt sind. CAV1 spielt eine Rolle bei der Signaltransduktion, der Tumorunterdrückung und der onkogenen Transformation. Frühere Studien mit CAV1-Knock-out-Mäusen und CAV1-Knock-out in Pankreastumorzellen bringen CAV1 mit der Vermittlung von Radioresistenz in Zusammenhang. Ziel dieser Arbeit war es, die Effekte der CAV1-Überexpression nach Bestrahlung in humanen Zellen ohne endogene CAV1-Expression zu bestimmen.Material und Methodik:Überexpression von humanem CAV1 in lymphoblastiden TK6-Zellen (TK6-wt) wurde durch Transfektion mit einem eukaryotischen Expressionsplasmid (pCI-CAV1) oder Transduktion mit einem lentiviralen SIN-(self-inactivating-)Vektor (HR‘SIN-CAV1) vermittelt. Die CAV1-Expression wurde durch Western-Blot-Analyse bzw. durch intrazelluläre FACS-Analyse („fluorescence-activated cell sorting“) in TK6-Zellen nachgewiesen. Der Effekt von CAV1 auf die Wachstumskinetik nach Bestrahlung wurde mit einem Proliferationsassay analysiert.Ergebnisse:TK6-wt zeigten keine nachweisbare endogene Expression von CAV1. Die lentiviral vermittelte Transduktion mit HR‘SIN-CAV1 (TK6-CAV1) verursachte eine deutlich stärkere CAV1-Expression im Vergleich zu pCI-CAV1-transfizierten TK6-Zellen. Intrazelluläre FACS-Analysen zeigten, dass 90% der transduzierten Zellen CAV1 exprimierten. CAV1 verstärkte die frühe Proliferation nach Bestrahlung mit einer Dosis von 2 Gy, wobei die Wachstumsrate unbestrahlter Zellen nicht verändert war. Auch nach einer Dosis von 4 Gy schützte CAV1 TK6-Zellen. Diese radioprotektive Wirkung wurde durch eine Abnahme strahleninduzierter Apoptose unterstützt.Schlussfolgerung:Ein Modellsystem für die Expression von exogenem CAV1 durch stabile lentivirale Transduktion von TK6-Zellen wurde etabliert. Funktionelle Untersuchungen zeigten eine verstärkte frühe Proliferation durch die CAV1-Expression nach Bestrahlung von TK6-Zellen mit klinisch relevanten Dosen, wodurch die Rolle von CAV1 als Überlebensfaktor unterstützt wird.