Immunomodulatory properties and molecular effects in inflammatory diseases of low-dose X-irradiation

Inflammatory diseases are the result of complex and pathologically unbalanced multicellular interactions. For decades, low-dose X-irradiation therapy (LD-RT) has been clinically documented to exert an anti-inflammatory effect on benign diseases and chronic degenerative disorders. By contrast, experimental studies to confirm the effectiveness and to reveal underlying cellular and molecular mechanisms are still at their early stages. During the last decade, however, the modulation of a multitude of immunological processes by LD-RT has been explored in vitro and in vivo. These include leukocyte/endothelial cell adhesion, adhesion molecule and cytokine/chemokine expression, apoptosis induction, and mononuclear/polymorphonuclear cell metabolism and activity. Interestingly, these mechanisms display comparable dose dependences and dose-effect relationships with a maximum effect in the range between 0.3 and 0.7 Gy, already empirically identified to be most effective in the clinical routine. This review summarizes data and models exploring the mechanisms underlying the immunomodulatory properties of LD-RT that may serve as a prerequisite for further systematic analyses to optimize low-dose irradiation procedures in future clinical practice

YM155-Adapted Cancer Cell Lines Reveal Drug-Induced Heterogeneity and Enable the Identification of Biomarker Candidates for the Acquired Resistance Setting

Survivin is a drug target and its suppressant YM155 a drug candidate mainly investigated for high-risk neuroblastoma. Findings from one YM155-adapted subline of the neuroblastoma cell line UKF-NB-3 had suggested that increased ABCB1 (mediates YM155 efflux) levels, decreased SLC35F2 (mediates YM155 uptake) levels, decreased survivin levels, and TP53 mutations indicate YM155 resistance. Here, the investigation of 10 additional YM155-adapted UKF-NB-3 sublines only confirmed the roles of ABCB1 and SLC35F2. However, cellular ABCB1 and SLC35F2 levels did not indicate YM155 sensitivity in YM155-naïve cells, as indicated by drug response data derived from the Cancer Therapeutics Response Portal (CTRP) and the Genomics of Drug Sensitivity in Cancer (GDSC) databases. Moreover, the resistant sublines were characterized by a remarkable heterogeneity. Only seven sublines developed on-target resistance as indicated by resistance to RNAi-mediated survivin depletion. The sublines also varied in their response to other anti-cancer drugs. In conclusion, cancer cell populations of limited intrinsic heterogeneity can develop various resistance phenotypes in response to treatment. Therefore, individualized therapies will require monitoring of cancer cell evolution in response to treatment. Moreover, biomarkers can indicate resistance formation in the acquired resistance setting, even when they are not predictive in the intrinsic resistance setting

Genome-Wide Gene Expression Analysis in Cancer Cells Reveals 3D Growth to Affect ECM and Processes Associated with Cell Adhesion but Not DNA Repair

Cell morphology determines cell behavior, signal transduction, protein-protein interaction, and responsiveness to external stimuli. In cancer, these functions profoundly contribute to resistance mechanisms to radio- and chemotherapy. With regard to this aspect, this study compared the genome wide gene expression in exponentially growing cell lines from different tumor entities, lung carcinoma and squamous cell carcinoma, under more physiological three-dimensional (3D) versus monolayer cell culture conditions. Whole genome cDNA microarray analysis was accomplished using the Affymetrix HG U133 Plus 2.0 gene chip. Significance analysis of microarray (SAM) and t-test analysis revealed significant changes in gene expression profiles of 3D relative to 2D cell culture conditions. These changes affected the extracellular matrix and were mainly associated with biological processes like tissue development, cell adhesion, immune system and defense response in contrast to terms related to DNA repair, which lacked significant alterations. Selected genes were verified by semi-quantitative RT-PCR and Western blotting. Additionally, we show that 3D growth mediates a significant increase in tumor cell radio- and chemoresistance relative to 2D. Our findings show significant gene expression differences between 3D and 2D cell culture systems and indicate that cellular responsiveness to external stress such as ionizing radiation and chemotherapeutics is essentially influenced by differential expression of genes involved in the regulation of integrin signaling, cell shape and cell-cell contact

ROS- and Radiation Source-Dependent Modulation of Leukocyte Adhesion to Primary Microvascular Endothelial Cells

Anti-inflammatory effects of low-dose irradiation often follow a non-linear dose–effect relationship. These characteristics were also described for the modulation of leukocyte adhesion to endothelial cells. Previous results further revealed a contribution of reactive oxygen species (ROS) and anti-oxidative factors to a reduced leukocyte adhesion. Here, we evaluated the expression of anti-oxidative enzymes and the transcription factor Nrf2 (Nuclear factor-erythroid-2-related factor 2), intracellular ROS content, and leukocyte adhesion in primary human microvascular endothelial cells (HMVEC) upon low-dose irradiation under physiological laminar shear stress or static conditions after irradiation with X-ray or Carbon (C)-ions (0–2 Gy). Laminar conditions contributed to increased mRNA expression of anti-oxidative factors and reduced ROS in HMVEC following a 0.1 Gy X-ray and 0.5 Gy C-ion exposure, corresponding to reduced leukocyte adhesion and expression of adhesion molecules. By contrast, mRNA expression of anti-oxidative markers and adhesion molecules, ROS, and leukocyte adhesion were not altered by irradiation under static conditions. In conclusion, irradiation of endothelial cells with low doses under physiological laminar conditions modulates the mRNA expression of key factors of the anti-oxidative system, the intracellular ROS contents of which contribute at least in part to leucocyte adhesion, dependent on the radiation source

Immunomodulatory Properties and Molecular Effects in Inflammatory Diseases of Low-Dose X-Irradiation

Inflammatory diseases are the result of complex and pathologically unbalanced multicellular interactions. For decades, low-dose X-irradiation therapy (LD-RT) has been clinically documented to exert an anti-inflammatory effect on benign diseases and chronic degenerative disorders. By contrast, experimental studies to confirm the effectiveness and to reveal underlying cellular and molecular mechanisms are still at their early stages. During the last decade, however, the modulation of a multitude of immunological processes by LD-RT has been explored in vitro and in vivo. These include leukocyte/endothelial cell adhesion, adhesion molecule and cytokine/chemokine expression, apoptosis induction, and mononuclear/polymorphonuclear cell metabolism and activity. Interestingly, these mechanisms display comparable dose dependences and dose-effect relationships with a maximum effect in the range between 0.3 and 0.7 Gy, already empirically identified to be most effective in the clinical routine. This review summarizes data and models exploring the mechanisms underlying the immunomodulatory properties of LD-RT that may serve as a prerequisite for further systematic analyses to optimize low-dose irradiation procedures in future clinical practice

Basics of Radiation Biology When Treating Hyperproliferative Benign Diseases

For decades, low- and moderate-dose radiation therapy (RT) has been shown to exert a beneficial therapeutic effect in a multitude of non-malignant conditions including painful degenerative muscoloskeletal and hyperproliferative disorders. Dupuytren and Ledderhose diseases are benign fibroproliferative diseases of the hand/foot with fibrotic nodules and fascial cords, which determine debilitating contractures and deformities of fingers/toes, while keloids are exuberant scar formations following burn damage, surgery, and trauma. Although RT has become an established and effective option in the management of these diseases, experimental studies to illustrate cellular composites and factors involved remain to be elucidated. More recent findings, however, indicate the involvement of radiation-sensitive targets like mitotic fibroblasts/myofibroblasts as well as inflammatory cells. Radiation-related molecular mechanisms affecting these target cells include the production of free radicals to hamper proliferative activity and interference with growth factors and cytokines. Moreover, an impairment of activated immune cells involved in both myofibroblast proliferative and inflammatory processes may further contribute to the clinical effects. We here aim at briefly describing mechanisms contributing to a modulation of proliferative and inflammatory processes and to summarize current concepts of treating hyperproliferative diseases by low and moderate doses of ionizing radiation

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; gammaH2AX/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

Molecular analysis of the G20210A mutation in the 3' untranslated region of prothrombin

Der G20210A-Polymorphismus im humanen Prothrombingen liegt in der 3'-UTR der mRNA und betrifft das letzte Nukleotid. Die Mutation führt bei Patienten zu einer erhöhten Plasma-Prothrombinkonzentration und zu einer verstärkten Thromboseneigung. Das Thromboserisiko ist für diese Patienten 1,3- bis 6,6-fach erhöht. Die Mutation beeinflusst aufgrund ihrer Lage nicht die Struktur des Proteins. Deshalb wurde postuliert, dass die erhöhte Prothrombinkonzentration bei betroffenen Patienten durch eine verlängerte Halbwertszeit der Prothrombin-mRNA, durch eine verbesserte Transkriptions-, Translations- oder Polyadenylierungseffizienz verursacht wird. Die Aufklärung des zugrunde liegenden Mechanismus ist Voraussetzung für die Entwicklung von therapeutischen Strategien. Zur Untersuchung der Hypothese der mutationsbedingten höheren Stabilität der Prothrombin-mRNA wurden sowohl Wildtyp als auch 20210A 3'-Prothrombinbereich mit 97 bp 3'-UTR und 143 bp 3'-flankierende Region vor das 3'-Poly A-Signal eines beta-Globinreporterplasmids inseriert. Dieses Vektorsystem ermöglicht aufgrund der hohen Halbwertszeit der Globin-mRNA die Messung des Einflusses der 3'-Prothrombinregion auf die Stabilität der Reporter-mRNA. Die G20210A-Mutation an der Polyadenylierungsstelle des Prothrombingens führte in beta-Globinexpressionsstudien und bei rekombinanter Expression in verschiedenen Zelllinien nicht zu einer veränderten Halbwertszeit im Vergleich zur Wildtyp-mRNA. Sowohl die endogene Prothrombin-mRNA als auch beta-Globin-Prothrombin-Hybride wiesen eine hohe Halbwertszeit von 17 bis 50 h in primären Leberzellen und verschiedenen Zelllinien auf. Dabei war die Stabilität der mRNA abhängig von der Art der verwendeten Konstrukte und vom Differenzierungsstatus der Zellen. Die Polyadenylierung erfolgte bei den beta-Globinkonstrukten nicht an der Poly A-Stelle der Prothrombin 3'-Region, sondern ausschließlich an der stromabwärts gelegenen Poly A-Stelle des Reporterplasmids. Nach Exzision der Vektor-Poly A-Stelle war eine unvollständige Prozessierung zu beobachten. Diese Daten weisen auf ein schwaches Polyadenylierungssignal oder auf fehlende cis-regulatorische Elemente in der 3'-Prothrombinregion hin. Die Verlängerung der 3'-flankierenden Prothrombinregion führte zur Reduzierung der Transkriptzahl auf zwei Transkripte, von denen das kleinere vermutlich die korrekt an Position 20210 polyadenylierte mRNA, das größere dagegen die Prä-mRNA oder ein inkorrekt prozessiertes Transkript repräsentierten. Demnach enthält die erweiterte 3'-flankierende Region zusätzliche regulatorische Elemente, die jedoch nicht zu einer 100 %igen Polyadenylierung führen. Der Vergleich von Wildtyp- und 20210A-Transkriptmengen zeigte bei beta-Globinreportersystemen und bei rekombinanter mRNA einen erhöhten Anteil an polyadenylierter mRNA bei der Mutante. Diese Verschiebung zugunsten der korrekt prozessierten und damit für die anschließende Translation funktionellen mRNA führte bei Überexpression der mutierten Variante zu einer gegenüber dem Wildtyp 4-fach erhöhten Proteinkonzentration. Die Insertion der verlängerten 3'-Prothrombinregion in ein Luciferasereportersystem führte in Übereinstimmung mit den zuvor beschriebenen Befunden bei der Mutante zu einer erhöhten Luciferaseaktivität. Alle Daten sprechen dafür, dass der erhöhte Prothrombinplasmaspiegel bei Patienten mit der G20210A-Transition im Prothrombingen durch eine verbesserte Polyadenylierungseffizienz der Prä-mRNA hervorgerufen wird. Der damit verbundene Anstieg der Prothrombin-mRNA-Menge könnte bei Patienten zu einer vermehrten Translation und Proteinbildung führen. Diese Befunde stimmen mit dem bei Mutationsträgern erhöhten Plasmagehalt an Prothrombin überein. Die weitere Untersuchung der beteiligten cis-regulatorischen Sequenzelemente und der daran bindenden Faktoren könnte zu einem besseren Verständnis von Mutationen in der 3'-untranslatierten Region weiterer Gene führen, die eine Erhöhung der mRNA-Menge zur Folge haben und als Grundlage für zukünftige Therapien dienen.The phenotype of the 20210A allele of the prothrombin gene is associated with elevated plasma levels of prothrombin and an increased risk of developing venous thrombosis. Since this mutation is located within the 3' untranslated region of the prothrombin gene, it has been postulated that the mutation might increase the stability of the prothrombin mRNA, the efficiency of transcription or translation or the efficiency of polyadenylation. The half-life of the endogenous prothrombin mRNA was 50 h, measured in rat liver cells and in the human hepatocellular carcinoma cell line HepG2. To study the hypothesis whether the G to A transition at position 20210 of the prothrombin gene affects a regulatory sequence involved in turnover of prothrombin mRNA, the half-life of chimeric globin mRNA containing the 3' untranslated and 143 bp 3' flanking region of prothrombin wild-type or prothrombin mutant was compared. In stably transfected HepG2 and COS-1 cells there was no significant difference in mRNA half-life obtained from the wild-type or mutant prothrombin mRNA. The stability of the chimeric mRNA was dependent on the differentiation of the cells and varied between 17 h and over 50 h. The 3' end cleavage and polyadenylation of the chimeric mRNA didn t occur at the upstream poly (A) site of prothrombin but at the downstream poly (A) site of the reporter plasmid. Excision of the vector poly (A) site resulted in decreased 3' end cleavage efficiency and incorrectly processed mRNAs. This data suggest a weak poly (A) signal of prothrombin or the absence of cis-regulatory elements in the constructs. The extension of the prothrombin 3' flanking region resulted in a reduction of the incorrect processed transcripts, indicating the involvement of downstream cis-acting elements in 3' end cleavage and polyadenylation efficiency. The smaller transcript represented the correct 3' end processed mRNA, whereas the second transcript seemed to be 3' extended and probably arose from read-through at the prothrombin poly (A) site. The comparison of wild-type and G20210A transcript levels indicates increased amounts of correct polyadenylated mutant mRNA. This upregulation of 3' end processing efficiency and greater abundance of functional mRNA caused by the prothrombin G-> A transition led to a 4-fold increase in recombinant protein concentration in stably transfected COS-1 cells. The insertion of the mutant 3' untranslated region and prolonged 3' flanking region of prothrombin additionally contributes to higher luciferase activity levels, probably due to increased polyadenylation efficiency. In conclusion, the elevated plasma prothrombin level in carriers of the prothrombin 20210A allele is probably a result of the improved 3' end cleavage and polyadenylation efficiency and not the result of an increased stability of the prothrombin mRNA

Comprehensive analysis of signal transduction in three-dimensional ECM-based tumor cell cultures

Analysis of signal transduction and protein phosphorylation is fundamental to understand physiological and pathological cell behavior as well as identification of novel therapeutic targets. Despite the fact that more physiological three-dimensional cell culture assays are increasingly used, particularly proteomics and phosphoproteomics remain challenging due to easy, robust and reproducible sample preparation. Here, we present an easy-to-perform, reliable and time-efficient method for the production of 3D cell lysates without compromising cell adhesion before cell lysis. The samples can be used for Western blotting as well as phosphoproteome array technology. This technique would be of interest for researchers working in all fields of biology and drug development