Katalin Lumniczky

Detailed characterization of the mouse glioma 261 tumor model for experimental glioblastoma therapy

Mouse glioma 261 (Gl261) cells are used frequently in experimental glioblastoma therapy; however, no detailed description of the Gl261 tumor model is available. Here we present that Gl261 cells carry point mutations in the K‐ras and p53 genes. Basal major histocompatibility complex (MHC)I, but not MHCII, expression was detected in Gl261 cells. The introduction of interferon‐γ‐encoding genes increased expression of both MHCI and MHCII. A low amount of B7‐1 and B7‐2 RNA was detected in wild‐type cells, but cytokine production did not change expression levels. Gl261 cells were transduced efficiently by adenoviral vectors; the infectivity of retroviral vectors was limited. Low numbers of transplanted Gl261 cells formed both subcutaneous and intracranial tumors in C57BL/6 mice. The cells were moderately immunogenic: prevaccination of mice with irradiated tumor cells 7 days before intracranial tumor challenge prevented tumor formation in approximately 90% of mice. When vaccination was carried out on the day or 3 days after tumor challenge, no surviving animals could be found. In vitro‐growing cells were radiosensitive: less than 2 Gy was required to achieve 50% cell mortality. Local tumor irradiation with 4 Gy X‐rays in brain tumor‐bearing mice slowed down tumor progression, but none of the mice were cured off the tumor. In conclusion, the Gl261 brain tumor model might be efficiently used to study the antitumor effects of various therapeutic modalities, but the moderate immunogenicity of the cells should be considered. (Cancer Sci 2006; 97: 546 – 553)

Effects of Low-Dose Radiation on the Immune System of Mice after Total-Body Irradiation

Abstract The effects of acute exposure to low- and high-dose radiation on the quantitative and functional parameters of the immune system were analyzed. C57BL/6 mice were irradiated with different doses of γ radiation (0.01, 0.05, 0.1, 0.5 and 2 Gy) and splenocytes were isolated at various times. Alterations in the distribution and surviving fraction of splenocyte subsets such as CD4+ and CD8+ T lymphocytes, regulatory T cells (Treg), natural killer (NK) cells, dendritic cells (DCs) and B lymphocytes were analyzed by flow cytometry. Apoptosis frequency was quantified by the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method 4 h after irradiation. Cytokine expression was investigated by real-time reverse transcription-polymerase chain reaction (RT-PCR). Low doses decreased apoptosis in the splenocyte subpopulations studied most prominently in NK cells and DCs. Exposure to 2 Gy increased apoptosis in all splenocyte subpopulations; B cells were the most sensitive and NK cells and DCs the least sensitive. The lowest cell numbers were measured 3 days after irradiation, with minor changes by day 7. CD8+ and B cells were rather resistant to low doses but were very sensitive to 2 Gy, while NK cells, DCs and Treg cells were much more resistant to high doses. Expression of the T-helper 1 (Th1)- and helper 2 (Th2)-type cytokines decreased after low doses and increased after high doses. Interleukin 6 (IL-6) reacted at early times and IL-10 at later times. IL-5 levels were consistently elevated. These data highlight the differences in the responses of different splenocyte subpopulations to low- and high-dose radiation.

Local tumor irradiation augments the antitumor effect of cytokine-producing autologous cancer cell vaccines in a murine glioma model

The combined therapeutic effect of cytokine-producing cancer cell vaccines and local radiotherapy was studied in a mouse glioma 261 (Gl261) brain tumor model. Brain tumor–bearing mice were treated with cytokine (IL-4, IL-6, IL-7, GM-CSF, TNF-α, LIF, LT) producing vaccines made by in vitro transduction of Gl261 cells with the corresponding adenoviral vectors. Vaccines producing either IL-4 or GM-CSF cured 20–40% of mice. The antitumor effect strongly depended on the secreted cytokine level. Vaccination therapy induced specific activation of cytotoxic T lymphocytes measured by cell-mediated cytotoxicity assay. Brain tumors were heavily infiltrated by CD4+ lymphocytes after treatment with IL-4– or GM-CSF–secreting cells. GM-CSF vaccination induced moderate CD8+ infiltration, as well. Depleting either CD4+ or CD8+ lymphocyte subsets abolished the anticancer effect of GM-CSF–expressing cells. Strong synergism was observed by combining cytokine vaccination (GM-CSF, IL-4, IL-12) with local tumor irradiation: about 80–100% of the glioma-bearing mice was cured. The high efficiency of combined treatment was maintained even under suboptimal conditions when neither of the modalities cured any of the mice alone. This suggests that vaccination therapy might open a new potential in the clinical treatment of high-grade gliomas when applied as adjuvant to existing treatment modalities.

The impact of radiation therapy on the antitumor immunity: local effects and systemic consequences.

The main antitumor efficacy of irradiation relies in its direct cytotoxic effect. Increasing evidence indicates a systemic effect of radiation though, mediated mainly by the immune system. In this review we wish to focus on the radiotherapy induced modifications of the soluble and cellular mediators of the antitumor immune response and summarize some of the mechanisms by which radiation driven local and systemic bystander effects can influence tumor immunogenicity. In different tumor types due to the intrinsic immunogenicity of the tumor cells and the immunological characteristics of the tumor microenvironment, different radiation induced immune modulatory mechanisms are predominant. Radiation most probably can only amplify or augment a pro-immunogenic phenotype and can hardly change by itself a net immune suppressing environment into an immune stimulating one. This immune modulatory potential of radiotherapy could be exploited in tumor treatment by developing combined radiotherapeutic and immunotherapeutic approaches. The last few years showed a dramatic increase in the knowledge of radiation induced out-of field and systemic effects, which foresees a rapid progress in the development and clinical application of these new, combined therapies for cancer cure.

Realising the European network of biodosimetry: RENEB—status quo

Creating a sustainable network in biological and retrospective dosimetry that involves a large number of experienced laboratories throughout the European Union (EU) will significantly improve the accident and emergency response capabilities in case of a large-scale radiological emergency. A well-organised cooperative action involving EU laboratories will offer the best chance for fast and trustworthy dose assessments that are urgently needed in an emergency situation. To this end, the EC supports the establishment of a European network in biological dosimetry (RENEB). The RENEB project started in January 2012 involving cooperation of 23 organisations from 16 European countries. The purpose of RENEB is to increase the biodosimetry capacities in case of large-scale radiological emergency scenarios. The progress of the project since its inception is presented, comprising the consolidation process of the network with its operational platform, intercomparison exercises, training activities, proceedings in quality assurance and horizon scanning for new methods and partners. Additionally, the benefit of the network for the radiation research community as a whole is addressed.

Cancer gene therapy: combination with radiation therapy and the role of bystander cell killing in the anti-tumor effect.

Current anti-cancer modalities such as surgery, chemo- and radiation therapies have only limited success in cancer treatment. Gene therapy is a promising new tool to improve outcomes. In this review, first we summarize the various strategies to kill tumor cells, and then focus on the bystander effect of gene therapy. A variety of strategies, such as gene-directed enzyme pro-drug therapy, activation of an anti-tumor immune attack, application of replication-competent and oncolytic viral vectors, tumor-specific as well as radiation and hypoxiainduced gene expression, might be applied to target tumor cells. We put special emphasis on the combination of these approaches with local tumor irradiation. Using the available vector systems, only a small portion of cancer cells contains the therapeutic genes under clinical situations. However, cells directly targeted by gene therapy will transfer death signals to neighboring cancer cells. This bystander cell killing improves the efficiency of cancer gene therapy. Death signals are delivered by cell-to-cell communication through gap junction intercellular contacts, release of toxic metabolites into the neighborhood or to larger distances, phagocytosis of apoptotic bodies, and the activation of the immune system. Bystander cell killing can be enhanced by the introduction of gap junction proteins into cells, by further activating the immune system with immune-stimulatory molecules, or by introducing genes that help the transfer of cytotoxic genes and/or metabolites into bystander cells. In conclusion, although bystander cell killing can improve therapeutic effects, there should be additional developments in cancer gene therapy for a more efficient clinical application.(Pathology Oncology Research Vol 12, No 2, 118–124)

The effect of ionizing radiation on regulatory T cells in health and disease

Treg cells are key elements of the immune system which are responsible for the immune suppressive phenotype of cancer patients. Interaction of Treg cells with conventional anticancer therapies might fundamentally influence cancer therapy response rates. Radiotherapy, apart from its direct tumor cell killing potential, has a contradictory effect on the antitumor immune response: it augments certain immune parameters, while it depresses others. Treg cells are intrinsically radioresistant due to reduced apoptosis and increased proliferation, which leads to their systemic and/or intratumoral enrichment. While physiologically Treg suppression is not enhanced by irradiation, this is not the case in a tumorous environment, where Tregs acquire a highly suppressive phenotype, which is further increased by radiotherapy. This is the reason why the interest for combined radiotherapy and immunotherapy approaches focusing on the abrogation of Treg suppression has increased in cancer therapy in the last few years. Here we summarize the basic mechanisms of Treg radiation response both in healthy and cancerous environments and discuss Treg-targeted pre-clinical and clinical immunotherapy approaches used in combination with radiotherapy. Finally, the discrepant findings regarding the predictive value of Tregs in therapy response are also reviewed.

Local tumour irradiation enhances the anti-tumour effect of a double-suicide gene therapy system in a murine glioma model.

Gliomas are invasive malignant tumours with poor prognosis. Combination of gene directed enzyme pro‐drug therapy with existing treatment modalities might open new therapeutic potentials.

Carcinogenic alterations in murine liver, lung, and uterine tumors induced by in utero exposure to ionizing radiation

The atomic bombing of Hiroshima and Nagasaki and the nuclear accident at Chernobyl raised the question of prenatal sensitivity to ionizing radiation–induced cancer. In this study, mice were exposed to single doses of γ‐radiation (0.2–2.0 Gy) at different embryonic stages. The tumor incidence increased with dose from 15% in control mice to 35% in mice irradiated with 2.0 Gy on 18 d of prenatal life. Various oncogenic events were investigated in lymphoid, liver, lung, and uterine tumors. We observed threefold to fivefold increases in myc expression in 25% of the lymphomas, and the expression of Ha‐ras and p53 genes decreased in 40% and 60% of the lung tumors by twofold to fivefold. Point mutations were tissue specific: Ha‐ras codon 61 mutations were found in about 40% of the liver adenocarcinomas, Ki‐ras codon 12 mutations in about 17% of lung tumors, and p53 mutations in about 15% of the lymphomas. Amplification and rearrangement of the p53, myc, and Ha‐, Ki‐ and N‐ras genes were not detected. Loss of heterozygosity on chromosome 4 at the multiple tumor suppressor 1 and 2 genes was observed in all types of malignancies. Allelic losses on chromosome 11 at the p53 locus were found in lymphoid, liver, and lung tumors, but they were absent from uterine tumors. Multiple oncogenic changes were often detected. The frequency of carcinogenic alterations was similar in spontaneous and radiation‐induced lymphoid, liver, and uterine tumors. In radiation‐induced lung adenocarcinomas, however, the incidences of many oncogenic changes were different from those found in their spontaneous counterparts. This suggests that different oncogenic pathways are activated during spontaneous and in utero γ‐radiation–induced murine lung carcinogenesis. Mol. Carcinog. 21:100–110, 1998.

RENEB intercomparisons applying the conventional Dicentric Chromosome Assay (DCA)

Abstract Purpose: Two quality controlled inter-laboratory exercises were organized within the EU project ‘Realizing the European Network of Biodosimetry (RENEB)’ to further optimize the dicentric chromosome assay (DCA) and to identify needs for training and harmonization activities within the RENEB network. Materials and methods: The general study design included blood shipment, sample processing, analysis of chromosome aberrations and radiation dose assessment. After manual scoring of dicentric chromosomes in different cell numbers dose estimations and corresponding 95% confidence intervals were submitted by the participants. Results: The shipment of blood samples to the partners in the European Community (EU) were performed successfully. Outside the EU unacceptable delays occurred. The results of the dose estimation demonstrate a very successful classification of the blood samples in medically relevant groups. In comparison to the 1st exercise the 2nd intercomparison showed an improvement in the accuracy of dose estimations especially for the high dose point. Conclusions: In case of a large-scale radiological incident, the pooling of ressources by networks can enhance the rapid classification of individuals in medically relevant treatment groups based on the DCA. The performance of the RENEB network as a whole has clearly benefited from harmonization processes and specific training activities for the network partners.