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ABSTRACT: Dysregulation of microRNAs (miRs) has been linked to several types of cancer. In the present study, we investigated the expression of miR-181a in different leukemia cell lines and healthy hematopoietic cells, as well as its influence on cell proliferation, metabolic activity and potential targets. Expression of miR-181a differed between various leukemia cell lines and mature blood cells. Inhibition of miR 181a expression in T- and B-Acute Lymphoblastic Leukemia (ALL) cells revealed an influence on the potential targets High-Mobility-Group-Protein B1 (HMGB1) and Cluster of Differentiation 4 (CD4). Overexpression of miR-181a in AML cells led to a significant decrease in cell proliferation and metabolic activity. The present data indicate a possible role of this specific miRNA in immunogenicity.
Anticancer research 02/2013; 33(2):445-52. · 1.73 Impact Factor
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ABSTRACT: Inhibition of signal transduction pathways has been successfully introduced into cancer treatment. The dual phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitor NVP-BEZ235 has antitumor activity in vitro against solid tumors. Here, we examined the activity of NVP-BEZ235 in acute lymphoblastic leukemia (ALL) cells and the best modalities for combination approaches.
ALL cell lines (SEM, RS4;11, Jurkat and MOLT4) were treated with NVP-BEZ235 alone, or in combination with cytarabine (AraC), doxorubicin (Doxo) or dexamethasone (Dexa).
NVP-BEZ235 potently inhibited the proliferation and metabolic activity of ALL cells. Antiproliferative effects were associated with G(0)/G(1) arrest and reduced levels of cyclin-dependent kinase 4 (CDK4) and cyclin D3. Inhibition of PI3K and mTOR activity was detected at 10 and 100 nM. NVP-BEZ235 combined with AraC, Doxo or Dexa synergistically enhanced the cytotoxicity compared to single-drug treatment, even in glucocorticoid-resistant cells.
NVP-BEZ235 displays pronounced antiproliferative effects in ALL cells and might therefore be a useful drug in the treatment of ALL.
Anticancer research 02/2012; 32(2):463-74. · 1.73 Impact Factor
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ABSTRACT: Everolimus (RAD001) is an mTOR inhibitor that has been successfully used as an immunosuppressant in solid-organ transplantation. Data in allogeneic hematopoietic stem cell transplantation (HSCT) is limited. This study aimed to investigate pharmacokinetics, safety, and efficacy of RAD001 in a canine allogeneic HSCT model. First, pharmacokinetics of RAD001 were performed in healthy dogs in order to determine the appropriate dosing. Doses of 0.25 mg RAD001 twice daily in combination with 15 mg/kg cyclosporin A (CsA) twice daily were identified as appropriate starting doses to achieve the targeted range of RAD001 (3-8 μg/L) when orally administered. Subsequently, 10 dogs were transplanted using 2 Gy total body irradiation (TBI) for conditioning and 0.25 mg RAD001 twice daily plus 15 mg/kg CsA twice daily for pre- and posttransplantation immunosuppression. Seven of the 10 transplanted dogs were maintained at the starting RAD001 dose throughout the study. For the remaining 3 dogs, dose adjustments were necessary. RAD001 accumulation over time did not occur. All dogs initially engrafted. Five dogs eventually rejected the graft (weeks 10, 10, 13, 27, and 56). Two dogs died of pneumonia (weeks 8 and 72) but were chimeric until then. Total cholesterol rose from median 4.1 mmol/L (3.5-5.7 mmol/L) before HSCT to 6.0 mmol/l (5.0-8.5 mmol/l) at day 21 after HSCT, but remained always within normal range. Changes in creatinine and triglyceride values were not observed. Long-term engraftment rates were inferior to sirolimus/CsA and mycophenolate mofetil (MMF)/CsA regimen, respectively. RAD001/CsA caused a more pronounced reduction of platelet counts to median 2 × 10(9)/L (range: 0-21 × 10(9)/L) and longer time to platelet recovery of 21 days (range: 14-24 days) compared with MMF/CsA. CsA c(2h) levels were significantly enhanced in the RAD001/CsA regimen, but c(0h) and area under the curve from 0 to 12 hours (AUC(0-12h)) values did not differ compared with an MMF/CsA immunosuppression. In summary, immunosuppression consisting of RAD001 and CsA is well tolerated but not as efficient as with other established immunosuppressants in a canine nonmyeloablative HSCT regimen. Hence, our study does not support the application of RAD001/CsA as standard practice in this setting.
Biology of blood and marrow transplantation: journal of the American Society for Blood and Marrow Transplantation 12/2011; 18(7):1061-8. · 3.15 Impact Factor
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ABSTRACT: Mammary neoplasias are one of the most frequent and spontaneously occurring malignancies in dogs and humans. Due to the similar anatomy of the mammary gland in both species, the dog has become an important animal model for this cancer entity. In human breast carcinomas, the overexpression of a protein named high-mobility group box 1 (HMGB1) was reported. Cells of the immune system were described to release HMGB1 actively exerting cytokine function. Thereby it is involved in the immune system activation, tissue repair, and cell migration. Passive release of HMGB1 by necrotic cells at sites of tissue damage or in necrotic hypoxic regions of tumors induces cellular responses e.g. release of proinflammatory cytokines leading to elevated inflammatory response and neo-vascularization of necrotic tumor areas. Herein we investigated if a time-dependent stimulation with the separately applied proinflammatory cytokines TNF-α and IFN-γ can cause secretion of HMGB1 in a non-immune related HMGB1-non-secreting epithelial canine mammary cell line (MTH53A) derived from non-neoplastic tissue.
The canine cell line was transfected with recombinant HMGB1 bicistronic expression vectors and stimulated after transfection with the respective cytokine independently for 6, 24 and 48 h. HMGB1 protein detection was performed by Western blot analysis and quantified a by enzyme-linked immunosorbent assay. Live cell laser scanning multiphoton microscopy of MTH53A cells expressing a HMGB1-GFP fusion protein was performed in order to examine, if secretion of HMGB1 under cytokine stimulating conditions is also visible by fluorescence imaging.
The observed HMGB1 release kinetics showed a clearly time-dependent manner with a peak release 24h after TNF-α stimulation, while stimulation with IFN-γ had only small effects on the HMGB1 release. Multiphoton HMGB1 live cell microscopy showed diffuse cell membrane structure changes 29 h after cytokine-stimulation but no clear secretion of HMGB1-GFP after TNF-α stimulation was visible.
Our results demonstrate that non-immune HMGB1-non-secreting cells of epithelial origin derived from mammary non-neoplastic tissue can be induced to release HMGB1 by single cytokine application. This indicates that tumor and surrounding tissue can be stimulated by tumor present inflammatory and necrotic cytokines to release HMGB1 acting as neo-vascularizing factor thus promoting tumor growth.
Cytokine 12/2011; 57(2):210-20. · 3.02 Impact Factor
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ABSTRACT: Denileukin Diftitox (ONTAK(®), DAB(389) IL-2) is a recombinant DNA-derived fusion protein depleting cells that express high-affinity IL-2 receptor. Important cell targets are CD4(+)CD25(+)Foxp3(+) regulatory T cells (T(reg)). Elimination of immunosuppressive T(reg) by Denileukin Diftitox may provide a way to modulate immune tolerance following stem cell transplantation. Here, we combined T(reg) depletion with a vaccination approach to induce donor-specific immune reactions. To investigate this approach we chose the mixed chimerism canine stem cell transplantation model which represents a high state of tolerance between two hematopoietic systems. The aim was therefore to induce a graft versus hematopoiesis effect thereby converting mixed to full donor chimerism. Dog leukocyte antigen identical siblings that had developed a stable mixed chimerism after non-myeloablative stem cell transplantation received a single dose of Denileukin Diftitox (18μg/kg, i.v.) followed by several cell-lysate vaccinations. Host peripheral blood mononuclear cell lysates combined with CpG-ODN, and Montanide(®) ISA 51 were locally applied. In vitro studies demonstrated that canine T(reg) are a target of Denileukin Diftitox. The suppression of T-cell proliferation by T(reg) was abolished by addition of Denileukin Diftitox (10nM). An increase of proliferation of median 300% (range: 200%-425%) was observed. No change in donor chimerism was observed after administration of Denileukin Diftitox and vaccination. This study highlights that application of Denileukin Diftitox resulted in a depletion of T(reg) followed by an increase of immune response in vitro. This effect could not be confirmed in vivo even if the immune system was stimulated by vaccinations.
Veterinary Immunology and Immunopathology 11/2011; 145(1-2):233-40. · 2.08 Impact Factor
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ABSTRACT: Regulatory T cells (T(reg)) are CD4(+) T lymphocytes with constitutive expression of CD25 and FOXP3, as well as the ability to modulate cellular immune responses. In this study, the phenotypic characteristics, function and feasibility of enrichment and expansion of canine T(reg) were examined. Canine peripheral blood mononuclear cells were isolated and enriched by labelling of CD25, and expansion of T(reg) was achieved by adding interleukin (IL)-2 for 1 week. Phenotypic and functional analyses of T(reg) were performed prior to and after expansion. Canine T(reg) could be phenotypically characterized by CD4, CD25, and FOXP3 expression. Isolation and enrichment of canine T(reg) is possible, but high purities are difficult to achieve without significant cell loss. Expansion of canine T(reg) was possible by adding IL-2 without other growth factors. Higher initial cell numbers seeded allow more substantial T(reg) expansion in vitro. Canine T(reg) have the potential to suppress proliferation of effector T cells (T(eff)). By adding expanded T(reg), a higher capability for suppressing T(eff) could be shown in comparison with freshly isolated T(reg). Enrichment and expansion of canine T(reg) is feasible, and canine T(reg) had similar characteristics to T(reg) from other species.
Experimental Animals 01/2011; 60(5):471-9. · 0.92 Impact Factor
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ABSTRACT: Canine lymphoma is a commonly occurring, spontaneously developing neoplasia similar to human non-Hodgkin's lymphoma and, thus, is used as a valuable model for human malignancy. HMGB1 and RAGE are strongly associated with tumour progression and vascularisation. Consequently, deregulated RAGE and HMGB1 may play an important role in the mechanisms involved in lymphoma progression.
Expression patterns of HMGB1 and RAGE were analysed in 22 canine lymphoma and three canine non-neoplastic control samples via real time PCR and canine beta-glucuronidase gene (GUSB) as endogenous control.
HMGB1 was up-regulated in the neoplastic samples, while RAGE expression remained inconspicuous.
This study demonstrated similar mechanisms in lymphoma progression in humans and dogs due to overexpression of HMGB1, which was described in human lymphomas. RAGE remained stable in terms of expression indicating that the extracellular HMGB1-induced effects are regulated by HMGB1 itself.
Anticancer research 12/2010; 30(12):5043-8. · 1.73 Impact Factor
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Catrin Schult,
Meike Dahlhaus,
Sabine Ruck,
Mandy Sawitzky,
Francesca Amoroso, Sandra Lange,
Daniela Etro,
Aenne Glass,
Georg Fuellen,
Sonja Boldt,
Olaf Wolkenhauer,
Luca Maria Neri,
Mathias Freund,
Christian Junghanss
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ABSTRACT: Targeted therapy approaches have been successfully introduced into the treatment of several cancers. The multikinase inhibitor Sorafenib has antitumor activity in solid tumors and its effects on acute lymphoblastic leukemia (ALL) cells are still unclear.
ALL cell lines (SEM, RS4;11 and Jurkat) were treated with Sorafenib alone or in combination with cytarabine, doxorubicin or RAD001. Cell count, apoptosis and necrosis rates, cell cycle distribution, protein phosphorylation and metabolic activity were determined.
Sorafenib inhibited the proliferation of ALL cells by cell cycle arrest accompanied by down-regulation of CyclinD3 and CDK4. Furthermore, Sorafenib initiated apoptosis by cleavage of caspases 3, 7 and PARP. Apoptosis and necrosis rates increased significantly with most pronounced effects after 96 h. Antiproliferative effects of Sorafenib were associated with a decreased phosphorylation of Akt (Ser473 and Thr308), FoxO3A (Thr32) and 4EBP-1 (Ser65 and Thr70) as early as 0.5 h after treatment. Synergistic effects were seen when Sorafenib was combined with other cytotoxic drugs or a mTOR inhibitor emphasizing the Sorafenib effect.
Sorafenib displays significant antileukemic activity in vitro by inducing cell cycle arrest and apoptosis. Furthermore, it influences PI3K/Akt/mTOR signaling in ALL cells.
BMC Cancer 10/2010; 10:560. · 3.01 Impact Factor
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Sandra Lange,
Simone Altmann,
Bettina Brandt,
Carsten Adam,
Franziska Riebau,
Heike Vogel,
Volker Weirich,
Inken Hilgendorf,
Rainer Storb,
Mathias Freund,
Christian Junghanss
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ABSTRACT: Stable mixed hematopoietic chimerism can be established in a canine stem cell transplantation model using a conditioning consisting of total body irradiation (TBI; 2 Gy) and postgrafting immunosuppression with mycophenolate mofetil (MMF) and cyclosporin (CSA). Reduction of TBI had resulted previously in graft rejection in this model. We investigated whether postgrafting stimulation of donor T cells against recipient's hematopoietic antigens or graft augmentation with donor monocyte-derived dendritic cells (MoDC) promote engraftment following 1 Gy TBI.
All dogs received dog leukocyte-antigen-identical bone marrow transplantation. Dogs were conditioned with either 2 Gy TBI (group 1) or 1 Gy TBI, followed by repetitive recipient hematopoietic cell lysate vaccinations (group 2) or graft augmentation with MoDC (group 3). Immunosuppression consisted of CSA and MMF.
In group 1, four animals remained stable chimeras for >110 weeks, and three rejected their grafts (week 10, week 14, week 16). All dogs in groups 2 and 3 rejected their graft (median: week 10 and 11, respectively). Peak chimerism and engraftment duration was shorter in the 1-Gy groups (p < 0.05) compared to group 1.
Neither postgrafting vaccination nor graft augmentation with MoDC were effective in supporting durable engraftment. Additional modifications are necessary to improve potential strategies aimed at establishment of early tissue specific graft-vs-host reactions.
Experimental hematology 01/2009; 37(1):143-50. · 3.11 Impact Factor
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ABSTRACT: The effect of exposure to 50 Hz, 1 mT magnetic fields (MF) on the cell cycle in general, on the DNA synthesis in S-phase, and on the G1-phase regulating proteins Cdk4, cyclin D1, p16INK4a, and p21CIP1 was investigated in human amniotic fluid cells. The BrdU-incorporation assay revealed a significant diminution of S-phase cells in MF-exposed cultures. The protein level of Cdk4 did not change, but MF induced a decreased expression of cyclin D1 after 24 h and 30 h exposures. The level of p16INK4a increased at 1 h and 12 h after exposure, whereas the expression of p21CIP1 was enhanced at 6 h and 12 h after exposure. Reduced levels of both Cdk inhibitors were observed at longer exposure times (24 h, 30 h). Our results suggest an inhibitory effect of MF on the G1-phase induced by altered expression of p16INK4a and p21CIP1.
Biophysik 07/2002; 41(2):131-7. · 1.70 Impact Factor
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ABSTRACT: It still is an unsolved issue whether exposure to power-line frequency electromagnetic fields (EMF) may promote carcinogenesis and if so whether it does so by influencing the proliferation, the survival, and the differentiation of cells. Since the family of protein kinases C (PKC) takes part in these processes by interacting with signal transduction pathways at several levels including the activation of transcription factors, we evaluated in the present study the effects of exposure of human amniotic fluid cells (AFC) to 50 Hz, 1 mT electromagnetic fields (EMF) alone and in combination with the tumour promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) on the subcellular localization of PKC protein, on PKC enzyme activity, and on the cell cycle distribution. Quantitative analyses of the PKC expression pattern demonstrated the translocation of PKC from the cytosolic to the membrane fraction after exposure to 10, 50, 100 nM, and 1 microM TPA. EMF exposure alone showed no effect on PKC translocation. Co-exposure to 10, 50, and 100 nM TPA and I mT EMF revealed a significant additive effect (25 +/- 50, 66 +/- 29, 22 +/- 50%, respectively) with the most prominent increase at the concentration of 50 nM TPA. At the highest concentration of TPA used (1 microM) no additive effect of EMF could be observed. Data on enzymatic activity indicate that EMF modulate the PKC activity, showing a significant increase of 10 +/- 16% in total PKC activity after co-exposure to 50 nM TPA and 1 mT EMF when compared to 50 nM TPA alone. Flow cytometric analyses showed a transient cell cycle arrest in G0/G1-phase followed by a delayed transit through S-phase in response to TPA, which was, however, not enhanced by co-exposure with EMF. We conclude that in AFC cells TPA at lower concentrations (< or = 100 nM) induces a less than maximum effect on the PKC pathway, which can be enhanced by the applied EMF.
Molecular and Cellular Biochemistry 03/2002; 232(1-2):133-41. · 2.06 Impact Factor
