Ina Patties

Influence of low dose irradiation on differentiation, maturation and T-cell activation of human dendritic cells

Ionizing irradiation could act directly on immune cells and may induce bystander effects mediated by soluble factors that are released by the irradiated cells. This is the first study analyzing both the direct effect of low dose ionizing radiation (LDIR) on the maturation and cytokine release of human dendritic cells (DCs) and the functional consequences for co-cultured T-cells. We showed that irradiation of DC-precursors in vitro does not influence surface marker expression or cytokine profile of immature DCs nor of mature DCs after LPS treatment. There was no difference of single dose irradiation versus fractionated irradiation protocols on the behavior of the mature DCs. Further, the low dose irradiation did not change the capacity of the DCs to stimulate T-cell proliferation. But the irradiation of the co-culture of DCs and T-cells revealed significantly lower proliferation of T-cells with higher doses. Summarizing the data from approx. 50 DC preparations there is no significant effect of low dose ionizing irradiation on the cytokine profile, surface marker expression and maturation of DCs in vitro although functional consequences cannot be excluded.

Additive effects of 5-aza-2'-deoxycytidine and irradiation on clonogenic survival of human medulloblastoma cell lines.

Background and Purpose:In recent years, epigenetic modulators were introduced into tumor therapy. Here, the authors investigated the antitumor effect of 5-aza-2’-deoxycytidine-(5-aza-dC-)induced demethylation combined with irradiation on human medulloblastoma (MB) cells, which form the most common malignant brain tumor in children.Material and Methods:Three MB cell lines were treated with 5-aza-dC in a low-dose (0.1 μM, 6 days) or high-dose (3/5 μM, 3 days) setting and irradiated with 2, 4, 6, or 8 Gy single dose on an X-ray unit. Methylation status and mRNA expression of three candidate genes were analyzed by methylation-specific PCR (polymerase chain reaction) and quantitative real-time RT-PCR. Cell survival and mortality were determined by trypan blue exclusion test. Proliferation was analyzed by BrdU incorporation assay, and long-term cell survival was assessed by clonogenic assay.Results:5-aza-dC treatment resulted in partial promoter demethylation and increased expression of hypermethylated candidate genes. A significant decrease of vital cell count, proliferation inhibition and increase of mortality was observed in 5-aza-dC-treated as well as in irradiated MB cells, whereby combination of both treatments led to additive effects. Although high-dose 5-aza-dC treatment was more effective in terms of demethylation, clonogenic assay revealed no differences between high- and low-dose settings indicating no relevance of 5-aza-dC-induced demethylation for decreased cell survival. MB cells pretreated with 5-aza-dC showed significantly lower plating efficiencies than untreated cells at all irradiation doses investigated. Analysis of surviving curves in irradiated MB cells, however, revealed no significant differences of α-, β-values and 2-Gy surviving fraction with or without 5-aza-dC treatment.Conclusion:5-aza-dC did not enhance radiation sensitivity of MB cells but significantly reduced the clonogenicity versus irradiation alone, which merits further investigation of its potential clinical application in MB possibly by combination with other chemotherapeutic agents.Hintergrund und Ziel:In den letzten Jahren wurden epigenetische Modulatoren in die Tumortherapie eingeführt. In dieser Arbeit untersuchten die Autoren den Antitumoreffekt der 5-Aza-2’-deoxycytidin-(5-aza-dC-)induzierten Demethylierung in Kombination mit Bestrahlung auf humane Medulloblastom-(MB-)Zellen, welche die häufigsten malignen Hirntumoren im Kindesalter bilden.Material und Methodik:Drei MB-Zell-Linien wurden mit 5-aza-dC in Niedrigdosis (0,1 μM, 6 Tage) oder Hochdosis (3/5 μM, 3 Tage) behandelt und mit 2, 4, 6 oder 8 Gy Einzeldosis bestrahlt. Die Untersuchung des Methylierungsstatus und der mRNA-Expression von drei Kandidatengenen erfolgte durch methylierungsspezifische PCR (Polymerase-Kettenreaktion) und quantitative Real-Time-RT-PCR. Lebendzellraten und Mortalität wurden durch Trypanblau-Ausschlusstest und die Proliferationsrate im BrdU-Assay bestimmt. Das Langzeitüberleben wurde durch einen klonogenen Assay ermittelt.Ergebnisse:Die 5-aza-dC-Behandlung führte zur partiellen Promotordemethylierung und zu einem Anstieg der Expression hypermethylierter Kandidatengene. Eine signifikante Verminderung der Lebendzellzahl und Proliferation bei Zunahme der Mortalität wurde sowohl in 5-aza-dC-behandelten als auch in bestrahlten MB-Zellen beobachtet. Bei kombinierter Behandlung summierten sich die Effekte der Einzelbehandlungen. Während die Inkubation mit 5-aza-dC in Hochdosis hinsichtlich der Demethylierung effektiver war als in Niedrigdosis, ergaben sich im klonogenen Assay keine Unterschiede zwischen beiden Behandlungsschemata, was darauf hinweist, dass die 5-aza-dC-induzierte Demethylierung nicht relevant für die Verminderung des Zellüberlebens ist. Mit 5-aza-dC vorbehandelte Zellen zeigten im untersuchten Bestrahlungsdosisbereich eine signifikant niedrigere „plating efficiency“ als unbehandelte Zellen. Die Überlebenskurven bestrahlter MB-Zellen wiesen jedoch keine signifikanten Unterschiede der α/β-Werte und der Überlebensfraktion nach 2 Gy in 5-aza-dC-behandelten gegenüber unbehandelten Zellen auf.Schlussfolgerung:5-aza-dC erhöhte die Strahlensensitivität von MB-Zellen nicht, reduzierte jedoch die Klonogenität signifikant gegenüber alleiniger Bestrahlung, was weitere Untersuchungen zur potentiellen klinischen Anwendung von 5-aza-dC bei MB, möglicherweise in Kombination mit anderen Chemotherapeutika, rechtfertigt.

The evolution of the histone methyltransferase gene Su(var)3-9 in metazoans includes a fusion with and a re-fission from a functionally unrelated gene

BackgroundIn eukaryotes, histone H3 lysine 9 (H3K9) methylation is a common mechanism involved in gene silencing and the establishment of heterochromatin. The loci of the major heterochromatic H3K9 methyltransferase Su(var)3-9 and the functionally unrelated γ subunit of the translation initiation factor eIF2 are fused in Drosophila melanogaster. Here we examined the phylogenetic distribution of this unusual gene fusion and the molecular evolution of the H3K9 HMTase Su(var)3-9.ResultsWe show that the gene fusion had taken place in the ancestral line of winged insects and silverfishs (Dicondylia) about 400 million years ago. We cloned Su(var)3-9 genes from a collembolan and a spider where both genes ancestrally exist as independent transcription units. In contrast, we found a Su(var)3-9-specific exon inside the conserved intron position 81-1 of the eIF2γ gene structure in species of eight different insect orders. Intriguinly, in the pea aphid Acyrthosiphon pisum, we detected only sequence remains of this Su(var)3-9 exon in the eIF2γ intron, along with an eIF2γ-independent Su(var)3-9 gene. This reveals an evolutionary re-fission of both genes in aphids. Su(var)3-9 chromo domains are similar to HP1 chromo domains, which points to a potential binding activity to methylated K9 of histone H3. SET domain comparisons suggest a weaker methyltransferase activity of Su(var)3-9 in comparison to other H3K9 HMTases. Astonishingly, 11 of 19 previously described, deleterious amino acid substitutions found in Drosophila Su(var)3-9 are seemingly compensable through accompanying substitutions during evolution.ConclusionExamination of the Su(var)3-9 evolution revealed strong evidence for the establishment of the Su(var)3-9/eIF2γ gene fusion in an ancestor of dicondylic insects and a re-fission of this fusion during the evolution of aphids. Our comparison of 65 selected chromo domains and 93 selected SET domains from Su(var)3-9 and related proteins offers functional predictions concerning both domains in Su(var)3-9 proteins.

Comparison of Hematopoietic Stem Cells Derived from Fresh and Cryopreserved Whole Cord Blood in the Generation of Humanized Mice

To study the function and maturation of the human hematopoietic and immune system without endangering individuals, translational human-like animal models are needed. We compare the efficiency of CD34+ stem cells isolated from cryopreserved cord blood from a blood bank (CCB) and fresh cord blood (FCB) in generating highly engrafted humanized mice in NOD-SCID IL2Rγnull (NSG) rodents. Interestingly, the isolation of CD34+ cells from CCB results in a lower yield and purity compared to FCB. The purity of CD34+ isolation from CCB decreases with an increasing number of mononuclear cells that is not evident in FCB. Despite the lower yield and purity of CD34+ stem cell isolation from CCB compared to FCB, the overall reconstitution with human immune cells (CD45) and the differentiation of its subpopulations e.g., B cells, T cells or monocytes is comparable between both sources. In addition, independent of the cord blood origin, human B cells are able to produce high amounts of human IgM antibodies and human T cells are able to proliferate after stimulation with anti-CD3 antibodies. Nevertheless, T cells generated from FCB showed increased response to restimulation with anti-CD3. Our study reveals that the application of CCB samples for the engraftment of humanized mice does not result in less engraftment or a loss of differentiation and function of its subpopulations. Therefore, CCB is a reasonable alternative to FCB and allows the selection of specific genotypes (or any other criteria), which allows scientists to be independent from the daily changing birth rate.

Inhibitory effects of epigenetic modulators and differentiation inducers on human medulloblastoma cell lines

BackgroundMedulloblastoma (MB) is the most common malignant brain tumor in childhood with a 5-year survival of approximately 60%. We have recently shown that treatment of human MB cells with 5-aza-2’-deoxycytidine (5-aza-dC) reduces the clonogenic survival significantly. Here, we tested combinatorial effects of 5-aza-dC with other epigenetic (valproic acid, SAHA) and differentiation-inducing drugs (resveratrol, abacavir, retinoic acid) on human MB cells in vitro to intensify the antitumor therapy further.MethodsThree human MB cell lines were treated with 5-aza-dC alone or in combination for three or six days. Metabolic activity was measured by WST-1 assay. To determine long-term reproductive survival, clonogenic assays were performed. Induction of DNA double-strand break (DSB) repair was measured by γH2AX assay.ResultsThe applied single drugs, except for ATRA, reduced the metabolic activity dose-dependently in all MB cell lines. Longer treatment times enhanced the reduction of metabolic activity by 5-aza-dC. Combinatorial treatments showed differential, cell line-dependent responses indicating an important impact of the genetic background. 5-Aza-dC together with resveratrol was found to exert the most significant inhibitory effects on metabolic activity in all cell lines. 5-aza-dC alone reduced the clonogenicity of MB cells significantly and induced DSB with no further changes after adjuvant administration of resveratrol.ConclusionThe observed significant decrease in metabolic activity by combinatorial treatment of MB cells with 5-aza-dC and resveratrol does not translate into long-term reproductive survival deficiency in vitro. Further studies in animal models are needed to clarify the resveratrol-mediated anticancer mechanisms in vivo.

Dose-dependent short- and long-term effects of ionizing irradiation on neural stem cells in murine hippocampal tissue cultures: neuroprotective potential of resveratrol.

Radiation therapy plays an essential role in the treatment of brain tumors, but neurocognitive deficits remain a significant risk, especially in pediatric patients. In recent trials, hippocampal sparing techniques are applied to reduce these adverse effects. Here, we investigate dose‐dependent effects of ionizing radiation (IR) on juvenile hippocampal neurogenesis. Additionally, we evaluate the radioprotective potential of resveratrol, a plant polyphenol recognized for its bifunctional tumor‐preventive and anticancer effects.

Efficient cell death induction in human glioblastoma cells by photodynamic treatment with Tetrahydroporphyrin-Tetratosylat (THPTS) and ionizing irradiation

Background So far, glioblastomas cannot be cured by standard therapy and have an extremely poor median survival of about 15 months. The photodynamic therapy (PDT) with next generation photosensitizers, reaching a higher therapeutic depth, might offer a new, adjuvant treatment strategy in brain cancer therapy. Here, we investigated the effect of THPTS-PDT combined with ionizing irradiation (IR) on glioblastoma cells in vitro and in vivo. Results THPTS colocalized to mitochondria and was not found in the nucleus. THPTS (2–20 μg/ml)-PDT significantly reduced the proliferation, metabolic activity and clonogenic survival and induced cell death mainly through apoptosis and autophagy. THPTS-PDT combined with IR decreased the clonogenicity significantly compared to single treatments. THPTS (≤ 300 μg/ml) alone showed no dark toxicity. The maximum therapeutic depth of THPTS-PDT in C6 glioblastomas was 13 mm. Materials and Methods Three human glioblastoma cell lines (U-87 MG, A-172, DBTRG-05MG) were incubated with THPTS (1–300 μg/ml) 3–24 hours before laser treatment (760 nm, 30 J/cm2). THPTS localization and effects on metabolic activity, proliferation, cell death mechanisms and long-term reproductive survival were assessed. IR was conducted on an X-ray unit (0.813 Gy/min). Results were verified in vivo on a subcutaneous C6 glioblastoma model in Wistar rats. Conclusions This study demonstrated efficient THPTS-PDT in glioblastoma cells, in vitro and in vivo. The combinatorial effects of THPTS-PDT and IR are of specific clinical interest as enhanced eradication of infiltrating glioblastoma cells in the tumor surrounding tissue might possibly reduce the commonly occurring local relapses.

Enhanced inhibition of clonogenic survival of human medulloblastoma cells by multimodal treatment with ionizing irradiation, epigenetic modifiers, and differentiation-inducing drugs

BackgroundMedulloblastoma (MB) is the most common pediatric brain tumor. Current treatment regimes consisting of primary surgery followed by radio- and chemotherapy, achieve 5-year overall survival rates of only about 60 %. Therapy-induced endocrine and neurocognitive deficits are common late adverse effects. Thus, improved antitumor strategies are urgently needed. In this study, we combined irradiation (IR) together with epigenetic modifiers and differentiation inducers in a multimodal approach to enhance the efficiency of tumor therapy in MB and also assessed possible late adverse effects on neurogenesis.MethodsIn three human MB cell lines (DAOY, MEB-Med8a, D283-Med) short-time survival (trypan blue exclusion assay), apoptosis, autophagy, cell cycle distribution, formation of gH2AX foci, and long-term reproductive survival (clonogenic assay) were analyzed after treatment with 5-aza-2′-deoxycytidine (5-azadC), valproic acid (VPA), suberanilohydroxamic acid (SAHA), abacavir (ABC), all-trans retinoic acid (ATRA) and resveratrol (RES) alone or combined with 5-aza-dC and/or IR. Effects of combinatorial treatments on neurogenesis were evaluated in cultured murine hippocampal slices from transgenic nestin-CFPnuc C57BL/J6 mice. Life imaging of nestin-positive neural stem cells was conducted at distinct time points for up to 28 days after treatment start.ResultsAll tested drugs showed a radiosynergistic action on overall clonogenic survival at least in two-outof-three MB cell lines. This effect was pronounced in multimodal treatments combining IR, 5-aza-dC and a second drug. Hereby, ABC and RES induced the strongest reduction of clongenic survival in all three MB cell lines and led to the induction of apoptosis (RES, ABC) and/or autophagy (ABC). Additionally, 5-aza-dC, RES, and ABC increased the S phase cell fraction and induced the formation of gH2AX foci at least in oneout-of-three cell lines. Thereby, the multimodal treatment with 5-aza-dC, IR, and RES or ABC did not change the number of normal neural progenitor cells in murine slice cultures.ConclusionIn conclusion, the radiosensitizing capacities of epigenetic and differentiation-inducing drugs presented here suggest that their adjuvant administration might improve MB therapy. Thereby, the combination of 5-aza-dC/IR with ABC and RES seemed to be the most promising to enhance tumor control without affecting the normal neural precursor cells.

Impact of X‐irradiation on microglia

Irradiation is widely used to treat brain tumors, and also to create bone marrow (BM) chimeras. BM chimeras are widely used to dissect functions and origin of microglia and blood‐derived mononuclear cells under homeostatic or pathological conditions. This is facilitated by the fact that microglia survive irradiation and are thus regarded radio‐resistant. In this study, we tested whether microglia are indeed radio‐resistant and looked for potential mechanisms that might explain this phenomenon. We analyzed the radio‐resistance of microglia independently of their physiological brain environment compared to other mononuclear cells from spleen and brain after X‐irradiation with 7 Gy or 30 Gy. Furthermore, we investigated long‐term effects of X‐irradiation on microglia using organotypic hippocampal slice cultures (OHSCs). We found a significant higher survival rate of isolated microglia 4 hr after X‐irradiation with 30 Gy accompanied by a decreased proliferation rate. Investigations of apoptosis‐related genes revealed no regulation of a specific antiapoptotic pathway but ataxia telangiectasia mutated (ATM), a DNA‐repair‐related gene, was significantly upregulated in isolated microglia 4 hr after 30 Gy. Irradiation of OHSCs with 7 and 30 Gy revealed a highly and significantly decreased cell number, morphological changes and an increase in migration velocity of microglia. Furthermore, cell loss, increased soma size and process length of microglia was also found in BM chimeras irradiated with 9.5 Gy 5 weeks after irradiation. Here, we present new evidence implying that microglia are not a homogeneous population of radio‐resistant cells and report on long‐term alterations of microglia that survived irradiation.