Imidazolyl Ethanamide Pentandioic Acid (IEPA) as Potential Radical Scavenger during Tumor Therapy in Human Hematopoietic Stem Cells.

Radiochemotherapy-associated leuco- or thrombocytopenia is a common complication, e.g., in head and neck cancer (HNSCC) and glioblastoma (GBM) patients, often compromising treatments and outcomes. Currently, no sufficient prophylaxis for hematological toxicities is available. The antiviral compound imidazolyl ethanamide pentandioic acid (IEPA) has been shown to induce maturation and differentiation of hematopoietic stem and progenitor cells (HSPCs), resulting in reduced chemotherapy-associated cytopenia. In order for it to be a potential prophylaxis for radiochemotherapy-related hematologic toxicity in cancer patients, the tumor-protective effects of IEPA should be precluded. In this study, we investigated the combinatorial effects of IEPA with radio- and/or chemotherapy in human HNSCC and GBM tumor cell lines and HSPCs. Treatment with IEPA was followed by irradiation (IR) or chemotherapy (ChT; cisplatin, CIS; lomustine, CCNU; temozolomide, TMZ). Metabolic activity, apoptosis, proliferation, reactive oxygen species (ROS) induction, long-term survival, differentiation capacity, cytokine release, and DNA double-strand breaks (DSBs) were measured. In tumor cells, IEPA dose-dependently diminished IR-induced ROS induction but did not affect the IR-induced changes in metabolic activity, proliferation, apoptosis, or cytokine release. In addition, IEPA showed no protective effect on the long-term survival of tumor cells after radio- or chemotherapy. In HSPCs, IEPA alone slightly enhanced CFU-GEMM and CFU-GM colony counts (2/2 donors). The IR- or ChT-induced decline of early progenitors could not be reversed by IEPA. Our data indicate that IEPA is a potential candidate for the prevention of hematologic toxicity in cancer treatment without affecting therapeutic benefits.

Immunomodulatory Effects by Photodynamic Treatment of Glioblastoma Cells In Vitro.

Multimodal treatment adding immunotherapy and photodynamic treatment (PDT) to standard therapy might improve the devastating therapeutic outcome of glioblastoma multiforme patients. As a first step, we provide investigations to optimize dendritic cell (DC) vaccination by using PDT and ionizing radiation (IR) to achieve maximal synergistic effects. In vitro experiments were conducted on murine glioblastoma GL261 cells, primary DCs differentiated from bone marrow and T cells, isolated from the spleen. Induction of cell death, reactive oxygen species, and inhibition of proliferation by tetrahydroporphyrin-tetratosylat (THPTS)-PDT and IR were confirmed by WST-1, LDH, ROS, and BrdU assay. Tumor cargo (lysate or cells) for DC load was treated with different combinations of THPTS-PDT, freeze/thaw cycles, and IR and immunogenicity analyzed by induction of T-cell activation. Cellular markers (CD11c, 83, 86, 40, 44, 69, 3, 4, 8, PD-L1) were quantified by flow cytometry. Cytotoxic T-cell response was evaluated by calcein AM assay. Immunogenicity of THPTS-PDT-treated GL261 cells lysate was superior to IR-treated lysate, or treated whole cells proven by increased DC phagocytosis, T-cell adhesion, proliferation, cytolytic activity, and cytokine release. These data strongly support the application of PDT together with IR for optimal immunogenic cell death induction in tumor cell lysate used to pulse DC vaccines.

Enhanced Survival of High-Risk Medulloblastoma-Bearing Mice after Multimodal Treatment with Radiotherapy, Decitabine, and Abacavir.

Children with high-risk SHH/TP53-mut and Group 3 medulloblastoma (MB) have a 5-year overall survival of only 40%. Innovative approaches to enhance survival while preventing adverse effects are urgently needed. We investigated an innovative therapy approach combining irradiation (RT), decitabine (DEC), and abacavir (ABC) in a patient-derived orthotopic SHH/TP53-mut and Group 3 MB mouse model. MB-bearing mice were treated with DEC, ABC and RT. Mouse survival, tumor growth (BLI, MRT) tumor histology (H/E), proliferation (Ki-67), and endothelial (CD31) staining were analyzed. Gene expression was examined by microarray and RT-PCR (Ki-67, VEGF, CD31, CD15, CD133, nestin, CD68, IBA). The RT/DEC/ABC therapy inhibited tumor growth and enhanced mouse survival. Ki-67 decreased in SHH/TP53-mut MBs after RT, DEC, RT/ABC, and RT/DEC/ABC therapy. CD31 was higher in SHH/TP53-mut compared to Group 3 MBs and decreased after RT/DEC/ABC. Microarray analyses showed a therapy-induced downregulation of cell cycle genes. By RT-PCR, no therapy-induced effect on stem cell fraction or immune cell invasion/activation could be shown. We showed for the first time that RT/DEC/ABC therapy improves survival of orthotopic SHH/TP53-mut and Group 3 MB-bearing mice without inducing adverse effects suggesting the potential for an adjuvant application of this multimodal therapy approach in the human clinic.

Radiotherapy in Medulloblastoma-Evolution of Treatment, Current Concepts and Future Perspectives.

Medulloblastoma is the most frequent malignant brain tumor in children. During the last decades, the therapeutic landscape has changed significantly with craniospinal irradiation as the backbone of treatment. Survival times have increased and treatments were stratified according to clinical and later molecular risk factors. In this review, current evidence regarding the efficacy and toxicity of radiotherapy in medulloblastoma is summarized and discussed mainly based on data of controlled trials. Current concepts and future perspectives based on current risk classification are outlined. With the introduction of CSI, medulloblastoma has become a curable disease. Due to combination with chemotherapy, survival rates have increased significantly, allowing for a reduction in radiation dose and a decrease of toxicity in low- and standard-risk patients. Furthermore, modern radiotherapy techniques are able to avoid side effects in a fragile patient population. However, high-risk patients remain with relevant mortality and many patients still suffer from treatment related toxicity. Treatment needs to be continually refined with regard to more efficacious combinatorial treatment in the future.

Dose and Dose Rate-Dependent Effects of Low-Dose Irradiation on Inflammatory Parameters in ApoE-Deficient and Wild Type Mice.

Anti-inflammatory low-dose therapy is well established, whereas the immunomodulatory impact of doses below 0.1 Gy is much less clear. In this study, we investigated dose, dose rate and time-dependent effects in a dose range of 0.005 to 2 Gy on immune parameters after whole body irradiation (IR) using a pro-inflammatory (ApoE-/-) and a wild type mouse model. Long-term effects on spleen function (proliferation, monocyte expression) were analyzed 3 months, and short-term effects on immune plasma parameters (IL6, IL10, IL12p70, KC, MCP1, INFγ, TGFß, fibrinogen, sICAM, sVCAM, sE-selectin/CD62) were analyzed 1, 7 and 28 days after Co60 γ-irradiation (IR) at low dose rate (LDR, 0.001 Gy/day) and at high dose rate (HDR). In vitro measurements of murine monocyte (WEHI-274.1) adhesion and cytokine release (KC, MCP1, IL6, TGFß) after low-dose IR (150 kV X-ray unit) of murine endothelial cell (EC) lines (H5V, mlEND1, bEND3) supplement the data. RT-PCR revealed significant reduction of Ki67 and CD68 expression in the spleen of ApoE-/- mice after 0.025 to 2 Gy exposure at HDR, but only after 2 Gy at LDR. Plasma levels in wild type mice, showed non-linear time-dependent induction of proinflammatory cytokines and reduction of TGFß at doses as low as 0.005 Gy at both dose rates, whereas sICAM and fibrinogen levels changed in a dose rate-specific manner. In ApoE-/- mice, levels of sICAM increased and fibrinogen decreased at both dose rates, whereas TGFß increased mainly at HDR. Non-irradiated plasma samples revealed significant age-related enhancement of cytokines and adhesion molecules except for sICAM. In vitro data indicate that endothelial cells may contribute to systemic IR effects and confirm changes of adhesion properties suggested by altered sICAM plasma levels. The differential immunomodulatory effects shown here provide insights in inflammatory changes occurring at doses far below standard anti-inflammatory therapy and are of particular importance after diagnostic and chronic environmental exposures.

Carnosine increases efficiency of temozolomide and irradiation treatment of isocitrate dehydrogenase-wildtype glioblastoma cells in culture.

Aim: The naturally occurring dipeptide carnosine (CAR) has been considered for glioblastoma therapy. As CAR also protects against ionizing irradiation (IR), we investigated whether it may counteract standard therapy consisting of postsurgery IR and treatment with temozolomide (TMZ). Materials & methods: Four isocitrate dehydrogenase-wildtype primary cell cultures were exposed to different doses of IR and different concentrations of TMZ and CAR. After exposure, viability under the different conditions and combinations of them was determined. Results: All cultures responded to treatment with TMZ and IR with reduced viability. CAR further decreased viability when TMZ and IR were combined. Conclusion: Treatment with CAR does not counteract glioblastoma standard therapy. As the dipeptide also protects nontumor cells from IR, it may reduce deleterious side effects of treatment.

The Chk1 inhibitor SAR-020106 sensitizes human glioblastoma cells to irradiation, to temozolomide, and to decitabine treatment.

BACKGROUND: Glioblastoma is the most common and aggressive brain tumour in adults with a median overall survival of only 14 months after standard therapy with radiation therapy (IR) and temozolomide (TMZ). In a novel multimodal treatment approach we combined the checkpoint kinase 1 (Chk1) inhibitor SAR-020106 (SAR), disrupting homologue recombination, with standard DNA damage inducers (IR, TMZ) and the epigenetic/cytotoxic drug decitabine (5-aza-2'-deoxycitidine, 5-aza-dC). Different in vitro glioblastoma models are monitored to evaluate if the impaired DNA damage repair may chemo/radiosensitize the tumour cells. METHODS: Human p53-mutated (p53-mut) and -wildtype (p53-wt) glioblastoma cell lines (p53-mut: LN405, T98G; p53-wt: A172, DBTRG) and primary glioblastoma cells (p53-mut: P0297; p53-wt: P0306) were treated with SAR combined with TMZ, 5-aza-dC, and/or IR and analysed for induction of apoptosis (AnnexinV and sub-G1 assay), cell cycle distribution (nuclear PI staining), DNA damage (alkaline comet or gH2A.X assay), proliferation inhibition (BrdU assay), reproductive survival (clonogenic assay), and potential tumour stem cells (nestinpos/GFAPneg fluorescence staining). Potential treatment-induced neurotoxicity was evaluated on nestin-positive neural progenitor cells in a murine entorhinal-hippocampal slice culture model. RESULTS: SAR showed radiosensitizing effects on the induction of apoptosis and on the reduction of long-term survival in p53-mut and p53-wt glioblastoma cell lines and primary cells. In p53-mut cells, this effect was accompanied by an abrogation of the IR-induced G2/M arrest and an enhancement of IR-induced DNA damage by SAR treatment. Also TMZ and 5-aza-dC acted radioadditively albeit to a lesser extent. The multimodal treatment achieved the most effective reduction of clonogenicity in all tested cell lines and did not affect the ratio of nestinpos/GFAPneg cells. No neurotoxic effects were detected when the number of nestin-positive neural progenitor cells remained unchanged after multimodal treatment. CONCLUSION: The Chk1 inhibitor SAR-020106 is a potent sensitizer for DNA damage-induced cell death in glioblastoma therapy strongly reducing clonogenicity of tumour cells. Selectively enhanced p53-mut cell death may provide stronger responses in tumours defective of non-homologous end joining (NHEJ). Our results suggest that a multimodal therapy involving DNA damage inducers and DNA repair inhibitors might be an effective anti-tumour strategy with a low risk of neurotoxicity.

D,L-Methadone does not improve radio- and chemotherapy in glioblastoma in vitro.

PURPOSE: Glioblastoma (GBM) is the most common malignant tumor of the central nervous system. Median survival of glioblastoma patients under standard therapy including radiotherapy and chemotherapy using temozolomide (TMZ) is 14.6 months. As cell culture experiments combining D,L-methadone with doxorubicin demonstrated an increased reduction of cell viability of glioblastoma cells, the opioid has been discussed as a drug for the treatment of GBM. Despite lack of clinical and experimental evidence that D,L-methadone in combination with standard therapy will be beneficial, an increasing number of tumor patients medicating themselves with D,L-methadone present to the hospitals in Germany. METHODS: As a first step towards understanding whether D,L-methadone may increase the efficacy of standard therapy, we used a cell culture model of primary GBM and fibroblast cell cultures derived from GBM patients. The cultures were treated with different concentrations of D,L-methadone in combination with X-irradiation, TMZ or both. Cell viability was determined by measuring ATP in cell lysates and dehydrogenase activity in living cells. RESULTS: When only treated with D,L-methadone, 1 µM of the opioid was sufficient to reduce viability of fibroblasts, whereas 10 µM was needed to significantly reduce glioblastoma cell viability. In addition, D,L-methadone did not improve the anti-neoplastic effects of X-irradiation, temozolomide or both. CONCLUSIONS: As D,L-methadone reduces glioblastoma cell viability only when concentrations are used that had been reported to be toxic to patients and as there were no interactions observable combining it with standard therapy, a recommendation for the use of D,L-methadone in glioblastoma therapy cannot be given.

Reduction of Muscle-Invasive Tumors by Photodynamic Therapy with Tetrahydroporphyrin-Tetratosylat in an Orthotopic Rat Bladder Cancer Model.

Photodynamic therapy (PDT) is a promising option for minimal-invasive treatment of bladder cancer. Efficacy of PDT in muscle-invasive urothelial cancer is still hampered by low tissue penetration of most photosensitizers due to short excitation wavelength. The novel light reactive agent tetrahydroporphyrin-tetratosylat (THPTS) is excitable at near-infrared (760 nm), allowing tissue penetration of up to 15 mm. Here, we established an orthotopic rat bladder cancer model and examined the effects of THPTS-PDT on tumor growth in vivo, and analyzed molecular mechanisms in vitro We examined pharmacokinetics and subcellular localization, and evoked cell death mode in cultured rat urothelial carcinoma cells (AY-27). We used female F344 Fischer rats for in vivo studies. Ten rats each were used for THPTS-PDT and light-only control. Bladders were evaluated by macroscopy and histology. Temperature-dependent THPTS uptake resulted in endosomal/lysosomal localization. PDT (0-50 µmol/L THPTS; 10 J/cm2) induced early onset of apoptosis leading to dose-dependent cytotoxicity in AY-27 cells. Single-time transurethral THPTS-PDT (100 µmol/L THPTS; 10 J/cm2) in F344 rats led to significant reduction of muscle-invasive tumor number (2/10 vs. 7/10 in controls) and total tumor volume (60% reduction) 2 weeks after PDT, while sparing healthy tissue. Here, we report for the first time effective tumor growth control by PDT in vivo THPTS is a promising new photosensitizer with the advantage of higher therapeutic depth and the potential of high-selective therapy in muscle-invasive urothelial cancer. This approach possibly allows minimal-invasive bladder preserving treatment of bladder cancer without systemic side 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.

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

INTRODUCTION: 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. METHODS: Organotypic entorhinal-hippocampal slice cultures from transgenic nestin-CFPnuc C57BL/J6 mice, postnatal days 3-6, were irradiated on a X-ray machine (4.5, 8, 12, and 16 Gy, single doses) after about 2 weeks. Nestin-positive neural stem cells were counted at a confocal live imaging microscope 0, 2, 4, 14, 25, and 42 days after IR. Resveratrol (15 µmol/L) was added 2 hr before and 24 hr after IR. Proliferation and cell death were assessed by BrdU pulse label, 48 hr after and by propidium iodide staining 96 hr after IR. GFAP- and NeuN-positive cells were counted 42 days after IR in cryosectioned immunofluorescence-stained slices. RESULTS: The observed age-related changes of nestin-positive stem cells in the organotypic slice culture model resembled the reduction of neural stem cells in vivo. IR (4.5-16 Gy) led to a dose-dependent damage of the neural stem cell pool in the dentate gyrus. No recovery was seen within 42 days after doses from 4.5 Gy onward. The decline of nestin-positive cells was paralleled by increased cell death and decreased proliferation. The number of GFAP-positive cells was significantly enhanced. No significant change was detected in the overall NeuN-positive cell population, whereas the number of newborn, NeuN/BrdU double-positive neurons was reduced. Resveratrol treatment reversed the irradiation-induced decline of neural stem cells. CONCLUSION: The neuroprotective action of resveratrol on irradiated hippocampal tissue warrants further investigation as a possible supplement to hippocampal sparing procedures.

Correction: Low-Dose Irradiation Affects Expression of Inflammatory Markers in the Heart of ApoE -/- Mice.

[This corrects the article DOI: 10.1371/journal.pone.0119661.].

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

BACKGROUND: Medulloblastoma (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. METHODS: In 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. RESULTS: All 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. CONCLUSION: In 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.

Low-dose irradiation affects expression of inflammatory markers in the heart of ApoE -/- mice.

Epidemiological studies indicate long-term risks of ionizing radiation on the heart, even at moderate doses. In this study, we investigated the inflammatory, thrombotic and fibrotic late responses of the heart after low-dose irradiation (IR) with specific emphasize on the dose rate. Hypercholesterolemic ApoE-deficient mice were sacrificed 3 and 6 months after total body irradiation (TBI) with 0.025, 0.05, 0.1, 0.5 or 2 Gy at low (1 mGy/min) or high dose rate (150 mGy/min). The expression of inflammatory and thrombotic markers was quantified in frozen heart sections (CD31, E-selectin, thrombomodulin, ICAM-1, VCAM-1, collagen IV, Thy-1, and CD45) and in plasma samples (IL6, KC, MCP-1, TNFα, INFγ, IL-1ß, TGFß, INFγ, IL-10, sICAM-1, sE-selectin, sVCAM-1 and fibrinogen) by fluorescence analysis and ELISA. We found that even very low irradiation doses induced adaptive late responses, such as increases of capillary density and changes in collagen IV and Thy-1 levels indicating compensatory regulation. Slight decreases of ICAM-1 levels and reduction of Thy 1 expression at 0.025-0.5 Gy indicate anti-inflammatory effects, whereas at the highest dose (2 Gy) increased VCAM-1 levels on the endocardium may represent a switch to a pro-inflammatory response. Plasma samples partially confirmed this pattern, showing a decrease of proinflammatory markers (sVCAM, sICAM) at 0.025-2.0 Gy. In contrast, an enhancement of MCP-1, TNFα and fibrinogen at 0.05-2.0 Gy indicated a proinflammatory and prothrombotic systemic response. Multivariate analysis also revealed significant age-dependent increases (KC, MCP-1, fibrinogen) and decreases (sICAM, sVCAM, sE-selectin) of plasma markers. This paper represents local and systemic effects of low-dose irradiation, including also age- and dose rate-dependent responses in the ApoE-/- mouse model. These insights in the multiple inflammatory/thrombotic effects caused by low-dose irradiation might facilitate an individual evaluation and intervention of radiation related, long-term side effects but also give important implications for low dose anti-inflammatory radiotherapy.

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

BACKGROUND: Medulloblastoma (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. METHODS: Three 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. RESULTS: The 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. CONCLUSION: The 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.

Effects of ionizing radiation on the immune system with special emphasis on the interaction of dendritic and T cells.

Dendritic cells (DCs), as professional antigen-presenting cells, are members of the innate immune system and function as key players during the induction phase of adaptive immune responses. Uptake, processing, and presentation of antigens direct the outcome toward either tolerance or immunity. The cells of the immune system are among the most highly radiosensitive cells in the body. For high doses of ionizing radiation (HD-IR) both immune-suppressive effects after whole body irradiation and possible immune activation during tumor therapy were observed. On the other hand, the effects of low doses of ionizing radiation (LD-IR) on the immune system are controversial and seem to show high variability among different individuals and species. There are reports revealing that protracted LD-IR can result in radioresistance. But immune-suppressive effects of chronic LD-IR are also reported, including the killing or sensitizing of certain cell types. This article shall review the current knowledge of radiation-induced effects on the immune system, paying special attention to the interaction of DCs and T cells.

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 microM, 6 days) or high-dose (3/5 microM, 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 alpha-, beta-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.

DNA methylation-independent loss of RARA gene expression in acute myeloid leukemia.

The retinoic acid receptor (RAR) alpha gene (RARA) encodes 2 major isoforms and mediates positive effects of all-trans retinoic acid (ATRA) on myelomonocytic differentiation. Expression of the ATRA-inducible (RARalpha2) isoform increases with myelomonocytic differentiation and appears to be down-regulated in many acute myeloid leukemia (AML) cell lines. Here, we demonstrate that relative to normal myeloid stem/progenitor cells, RARalpha2 expression is dramatically reduced in primary AML blasts. Expression of the RARalpha1 isoform is also significantly reduced in primary AML cells, but not in AML cell lines. Although the promoters directing expression of RARalpha1 and RARalpha2 are respectively unmethylated and methylated in AML cell lines, these regulatory regions are unmethylated in all the AML patient cell samples analyzed. Moreover, in primary AML cells, histones associated with the RARalpha2 promoter possessed diminished levels of H3 acetylation and lysine 4 methylation. These results underscore the complexities of the mechanisms responsible for deregulation of gene expression in AML and support the notion that diminished RARA expression contributes to leukemogenesis.

Benzodithiophenes potentiate differentiation of acute promyelocytic leukemia cells by lowering the threshold for ligand-mediated corepressor/coactivator exchange with retinoic acid receptor alpha and enhancing changes in all-trans-retinoic acid-regulated gene expression.

Differentiation induction is an effective therapy for acute promyelocytic leukemia (APL), which dramatically responds to all-trans-retinoic acid (ATRA). Recent studies have indicated that combinatorial use of retinoid and nonretinoid compounds, such as histone deacetylase inhibitors, arsenics, and PKA agonists, has higher therapeutic value in this disease and potentially in other malignancies. In a screen of 370 compounds, we identified benzodithiophene analogues as potent enhancers of ATRA-induced APL cell differentiation. These effects were not associated with changes in global histone acetylation and, for the most potent compounds, were exerted at very low nanomolar concentrations, and were paralleled by enhancement of some, but not all, ATRA-modulated gene expressions. Investigating the mechanism underlying the effects of these drugs on ATRA-induced APL cell differentiation, we have shown that benzodithiophenes enhance ATRA-mediated dissociation and association of corepressor N-CoR and coactivator p300 acetyltransferase, respectively, with retinoic acid receptor (RAR) alpha proteins. These data suggest that benzodithiophenes act at the level of receptor activation, possibly by affecting posttranslational modification of the receptor (and/or coregulators), thus leading to an enhancement in ATRA-mediated effects on gene expression and APL cell differentiation. Given the specificities of these low benzodithiophene concentrations for PML-RARalpha and RARalpha, these drugs may be useful for combinatorial differentiation therapy of APL and possibly other acute myelogenous leukemia subtypes in which the overall ATRA signaling is suppressed.