64Cu antibody-targeting of the T-cell receptor and subsequent internalization enables in vivo tracking of lymphocytes by PET.

T cells are key players in inflammation, autoimmune diseases, and immunotherapy. Thus, holistic and noninvasive in vivo characterizations of the temporal distribution and homing dynamics of lymphocytes in mammals are of special interest. Herein, we show that PET-based T-cell labeling facilitates quantitative, highly sensitive, and holistic monitoring of T-cell homing patterns in vivo. We developed a new T-cell receptor (TCR)-specific labeling approach for the intracellular labeling of mouse T cells. We found that continuous TCR plasma membrane turnover and the endocytosis of the specific (64)Cu-monoclonal antibody (mAb)-TCR complex enables a stable labeling of T cells. The TCR-mAb complex was internalized within 24 h, whereas antigen recognition was not impaired. Harmful effects of the label on the viability, DNA-damage and apoptosis-necrosis induction, could be minimized while yielding a high contrast in in vivo PET images. We were able to follow and quantify the specific homing of systemically applied (64)Cu-labeled chicken ovalbumin (cOVA)-TCR transgenic T cells into the pulmonary and perithymic lymph nodes (LNs) of mice with cOVA-induced airway delayed-type hypersensitivity reaction (DTHR) but not into pulmonary and perithymic LNs of naïve control mice or mice diseased from turkey or pheasant OVA-induced DTHR. Our protocol provides consequent advancements in the detection of small accumulations of immune cells in single LNs and specific homing to the sites of inflammation by PET using the internalization of TCR-specific mAbs as a specific label of T cells. Thus, our labeling approach is applicable to other cells with constant membrane receptor turnover.

In vivo tracking of Th1 cells by PET reveals quantitative and temporal distribution and specific homing in lymphatic tissue.

UNLABELLED: Although T cells can be labeled for noninvasive in vivo imaging, little is known about the impact of such labeling on T-cell function, and most imaging methods do not provide holistic information about trafficking kinetics, homing sites, or quantification. METHODS: We developed protocols that minimize the inhibitory effects of (64)Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) ((64)Cu-PTSM) labeling on T-cell function and permit the homing patterns of T cells to be followed by PET. Thus, we labeled ovalbumin (OVA) T-cell receptor transgenic interferon (IFN)-γ-producing CD4(+) T (Th1) cells with 0.7-2.2 MBq of (64)Cu-PTSM and analyzed cell viability, IFN-γ production, proliferation, apoptosis, and DNA double-strand breaks and identified intracellular (64)Cu accumulation sites by energy dispersive x-ray analysis. To elucidate the fate of Th1 cell homing by PET, 10(7 64)Cu-OVA-Th1 cells were injected intraperitoneally or intravenously into healthy mice. To test the functional capacities of (64)Cu-OVA-Th1 cells during experimental OVA-induced airway hyperreactivity, we injected 10(7 64)Cu-OVA-Th1 cells intraperitoneally into OVA-immunized or nonimmunized healthy mice, which were challenged with OVA peptide or phosphate-buffered saline or remained untreated. In vivo PET investigations were followed by biodistribution, autoradiography, and fluorescence-activated cell sorting analysis. RESULTS: PET revealed unexpected homing patterns depending on the mode of T-cell administration. Within 20 min after intraperitoneal administration, (64)Cu-OVA-Th1 cells homed to the perithymic lymph nodes (LNs) of naive mice. Interestingly, intravenously administered (64)Cu-OVA-Th1 cells homed predominantly into the lung and spleen but not into the perithymic LNs. The accumulation of (64)Cu-OVA-Th1 cells in the pulmonary LNs (6.8 ± 1.1 percentage injected dose per cubic centimeter [%ID/cm(3)]) 24 h after injection was highest in the OVA-immunized and OVA-challenged OVA airway hyperreactivity-diseased littermates 24 h after intraperitoneal administration and lowest in the untreated littermates (3.7 ± 0.4 %ID/cm(3)). As expected, (64)Cu-OVA-Th1 cells also accumulated significantly in the pulmonary LNs of nonimmunized OVA-challenged animals (6.1 ± 0.5 %ID/cm(3)) when compared with phosphate-buffered saline-challenged animals (4.6 ± 0.5 %ID/cm(3)). CONCLUSION: Our protocol permits the detection of Th1 cells in single LNs and enables temporal in vivo monitoring of T-cell homing over 48 h. This work enables future applications for (64)Cu-PTSM-labeled T cells in clinical trials and novel therapy concepts focusing on T-cell-based immunotherapies of autoimmune diseases or cancer.

Balloon coating with rapamycin using an on-site coating device.

PURPOSE: The efficacy of drug-eluting balloons has been demonstrated in clinical trials. The drug predominantly used is paclitaxel because of its lipophilic properties and the rapid onset of action. The aim of the investigation was to evaluate the feasibility and efficacy of an alternative balloon coating with rapamycin that can be applied on site. METHODS: The balloon coating (3.0/18 and 3.0/12 mm, Cathy No. 4, Translumina GmbH) with rapamycin was conducted with a coating machine (Translumina GmbH). Concentrations were 2, 2 × 2, 3, and 4 %. Measurements regarding the amount of substance released to the vessel wall were carried out on explanted porcine coronaries by means of ultraviolet and visible-light spectroscopy. Inflation time varied between 30 and 120 s. The biological effect of the coating was evaluated in a porcine peripheral overstretch and stent implantation model. RESULTS: The amount of rapamycin on the balloon surface ranged from 558 ± 108 µg for the 2 % solution to 1,441 ± 228 µg in the 4 % solution. An amount of 95 ± 63-193 ± 113 µg was released into the vessel wall. The quantitative measurements of the angiographic examinations 4 weeks after treatment revealed a reduction of diameter stenosis from 20.6 ± 17.4 % in the control group to 11.6 ± 5.5 % in the drug-eluting balloon group. CONCLUSION: A balloon coating with rapamycin omitting an excipient is possible with a dose-adjustable coating machine. However, the biological effects are moderate, which make further optimization of the coating process and evaluation of appropriate excipients necessary.

Utilizing echo-shifts in k-space for generation of positive contrast in areas with marked susceptibility alterations.

A technique for generation of positive contrast near susceptibility alterations utilizing echo-shifts in k-space is introduced, based on altered Larmor-frequencies and resulting phase-shifts accumulating during the echo-time at the site of local magnetic field gradients. 3D gradient-echo raw-data is acquired and weighted with an inverse Hanning filter. The filter partly suppresses central raw-data points, while maintaining outer areas. Reconstruction of the filtered raw-data results in images where pixels with apparent magnetic field gradients are highlighted against homogeneous pixels. Further processing steps are introduced to remove remaining intensities in the homogeneous parts of the filtered image. Feasibility is shown by an agar phantom containing magnetically labeled cells, with concentrations of 25, 50, 100, and 250 cells/µL, and by images of the human head. The technique allows detection of echo-shifted pixels with automatic suppression of magnetically homogeneous parts while keeping post-processing time short. Fewer than four labeled cells per pixel were clearly displayed with positive contrast. Application to the human head shows bright veins and complete suppression of homogeneous regions. The presented technique has high potential for specific detection of low concentrations of labeled cells or susceptibility altered regions in vivo with positive contrast, whereas areas with low spin density are not highlighted.

Decreased neointimal extracellular matrix formation in RAGE-knockout mice after microvascular denudation.

PURPOSE: To evaluate in vivo the role of RAGE (receptor for advanced glycated end products) in the development of restenosis and neointimal proliferation in RAGE-deficient knockout (KO) mice compared with wild-type (WT) mice in an animal model. MATERIALS AND METHODS: Sixteen WT and 15 RAGE-deficient mice underwent microvascular denudation of the common femoral artery under general anaesthesia. Contralateral arteries underwent a sham operation and served as controls. Four weeks after the intervention, all animals were killed, and paraformaldehyde-fixed specimens of the femoral artery were analysed with different stains (hematoxylin and eosin and Elastica van Gieson) and several different types of immunostaining (proliferating cell nuclear antigen, α-actin, collagen, von Willebrand factor, RAGE). Luminal area, area of the neointima, and area of the media were measured in all specimens. In addition, colony-formation assays were performed, and collagen production by WT smooth muscle cells (SMCs) and RAGE-KO SMCs was determined. For statistical analysis, P < 0.05 was considered statistically significant. RESULTS: Four weeks after denudation, WT mice showed a 49.6% loss of luminal area compared with 14.9% loss of luminal area in RAGE-deficient mice (sham = 0% loss) (P < 0.001). The neointima was 18.2 (*1000 µm(2) [n = 15) in the WT group compared with only 8.4 (*1000 µm(2) [n = 16]) in the RAGE-KO group. RAGE-KO SMCs showed significantly decreased proliferation activity and production of extracellular matrix protein. CONCLUSION: RAGE may be shown to play a considerable role in the formation of neointima leading to restenosis after vascular injury.

K-RAS(V12) induces autocrine production of EGFR ligands and mediates radioresistance through EGFR-dependent Akt signaling and activation of DNA-PKcs.

PURPOSE: It is known that postirradiation survival of tumor cells presenting mutated K-RAS is mediated through autocrine activation of epidermal growth factor receptor (EGFR). In this study the molecular mechanism of radioresistance of cells overexpressing mutated K-RAS(V12) was investigated. METHODS AND MATERIALS: Head-and-neck cancer cells (FaDu) presenting wild-type K-RAS were transfected with empty vector or vector expressing mutated K-RAS(V12). The effect of K-RAS(V12) on autocrine production of EGFR ligands, activation of EGFR downstream pathways, DNA damage repair, and postirradiation survival was analyzed. RESULTS: Conditioned medium collected from K-RAS(V12)-transfected cells enhanced activation of the phosphatidylinositol-3-kinase-Akt pathway and increased postirradiation survival of wild-type K-RAS parental cells when compared with controls. These effects were reversed by amphiregulin (AREG)-neutralizing antibody. In addition, secretion of the EGFR ligands AREG and transforming growth factor α was significantly increased upon overexpression of K-RAS(V12). Expression of mutated K-RAS(V12) resulted in an increase in radiation-induced DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation at S2056. This increase was accompanied by increased repair of DNA double-strand breaks. Abrogation of DNA-PKcs phosphorylation by serum depletion or AREG-neutralizing antibody underscored the role of autocrine production of EGFR ligands, namely, AREG, in regulating DNA-PKcs activation in K-RAS mutated cells. CONCLUSIONS: These data indicate that radioresistance of K-RAS mutated tumor cells is at least in part due to constitutive production of EGFR ligands, which mediate enhanced repair of DNA double-strand breaks through the EGFR-phosphatidylinositol-3-kinase-Akt cascade.

Autophagy contributes to resistance of tumor cells to ionizing radiation.

BACKGROUND AND PURPOSE: Autophagy signaling is a novel important target to improve anticancer therapy. To study the role of autophagy on resistance of tumor cells to ionizing radiation (IR), breast cancer cell lines differing in their intrinsic radiosensitivity were used. MATERIALS AND METHODS: Breast cancer cell lines MDA-MB-231 and HBL-100 were examined with respect to clonogenic cell survival and induction of autophagy after radiation exposure and pharmacological interference of the autophagic process. As marker for autophagy the appearance of LC3-I and LC3-II proteins was analyzed by SDS-PAGE and Western blotting. Formation of autophagic vacuoles was monitored by immunofluorescence staining of LC3. RESULTS: LC3-I and LC3-II formation differs markedly in radioresistant MDA-MB-231 versus radiosensitive HBL-100 cells. Western blot analyses of LC3-II/LC3-I ratio indicated marked induction of autophagy by IR in radioresistant MDA-MB-231 cells, but not in radiosensitive HBL-100 cells. Indirect immunofluorescence analysis of LC3-II positive vacuoles confirmed this differential effect. Pre-treatment with 3-methyladenine (3-MA) antagonized IR-induced autophagy. Likewise, pretreatment of radioresistant MDA-231 cells with autophagy inhibitors 3-MA or chloroquine (CQ) significantly reduced clonogenic survival of irradiated cells. CONCLUSION: Our data clearly indicate that radioresistant breast tumor cells show a strong post-irradiation induction of autophagy, which thus serves as a protective and pro-survival mechanism in radioresistance.

Nuclear epidermal growth factor receptor modulates cellular radio-sensitivity by regulation of chromatin access.

PURPOSE: Nuclear EGFR is involved in cellular stress management and regulation of cellular radio-sensitivity. The aim of this study was to elucidate the molecular mode of nuclear EGFR action. METHODS: Radiation induced nuclear EGFR-shuttling and EGFR-foci formation was analyzed with immunohistochemistry and confocal microscopy. Composition of γH(2)AX-protein complexes was analyzed by western-blotting after immuno-precipitation. Functional relevance of nuclear EGFR was analyzed after siRNA mediated depletion of EGFR with respect to activation of ATM, histone H3 acetylation, residual DNA-damage and cell survival after irradiation. RESULTS: Following radiation nuclear EGFR was localized in foci similar to γH(2)AX. EGFR co-localized in a sub-fraction of γH(2)AX-foci. Analysis of composition of γH(2)AX-complexes revealed presence of EGFR, ATM, promyelocytic leukemia protein (PML), histone H3 and hetero-chromatin binding protein (HP1) in response to radiation. Depletion of EGFR protein inhibited ATM activation due to inhibition of acetylase TIP60 activity following irradiation. Consequently, histone H3 acetylation and phosphorylation was blocked and chromatin could not be opened for repair. Thus, residual DNA-damage was increased 24 h after irradiation and cells were radio-sensitized. Comparable results were obtained when cells were treated with EGFR-NLS-peptide, which blocks EGFR nuclear shuttling specifically. CONCLUSIONS: Nuclear EGFR is part of DNA-damage repair complex and is involved in regulation of TIP60-acetylase activity. TIP60 is essential for ATM activation and chromatin relaxation which is a prerequisite for DNA-repair in heterochromatic DNA. Thus interventional EGFR strategies during tumor treatment may also interact with DNA-repair by blocking access to damaged DNA.

Selection of radioresistant tumor cells and presence of ALDH1 activity in vitro.

BACKGROUND: Tumor resistance to radiotherapy has been hypothesized to be mediated by a tumor subpopulation, called cancer stem cells (CSCs). Based on the proposed function of CSCs in radioresistance, we explored the cancer stem cell properties of cells selected for radioresistance phenotype. MATERIALS AND METHODS: A549 and SK-BR-3 cells were radioselected with four single doses of 4 or 3 Gy in intervals of 10-12 days and used for colony formation assay and γ-H2AX foci formation assay. Expression of putative stem cell markers, i.e. Sox2, Oct4, ALDH1, and CD133 were analyzed using Western blotting. A549 and SK-BR-3 cells sorted based on their ALDH1 activity were analyzed in clonogenic survival assays. RESULTS: Radioselected A549 and SK-BR-3 cells (A549-R, SK-BR-3-R) showed increased radioresistance and A549-R cells presented enhanced repair of DNA-double strand breaks. PI3K inhibition significantly reduced radioresistance of A549-R cells. Cell line specific differences in the expression of the putative CSC markers Sox2 and Oct4 were observed when parental and radioselected cells were compared but could not be directly correlated to the radioresistant phenotype. However, enzyme activity of the putative stem cell marker ALDH1 showed a correlation to radioresistance. CONCLUSIONS: Subpopulations of pooled radioresistant colonies, selected by various radiation exposures were analyzed for the presence of putative stem cell markers. Although the pattern of Sox2, Oct4, and CD133 expression was not generally associated with radioresistance, presence of ALDH1 seems to be indicative for subpopulations with increased radioresistance.

A dual-inhibition study on vascular smooth muscle cells with meclofenamic acid and ß-irradiation for the prevention of restenosis.

PURPOSE: Restenosis is still one of the major limitations after angioplasty. A therapeutic treatment combining ß-irradiation and pharmacologic cyclooxygenase-2 inhibition was employed to study the impact on vascular smooth muscle cells (SMCs). MATERIALS AND METHODS: The effects of meclofenamic acid in combination with yttrium-90 ((90)Y) on cell growth, clonogenic activity, cell migration, and cell cycle distribution of human aortic SMCs were investigated. Treatment was sustained over a period of 4 days and recovery of cells was determined until day 20 after initiation. The hypothesis was that there is no difference between control and treated groups. RESULTS: A dose-dependent growth inhibition was observed in single and combined treatment groups for meclofenamic acid and ß-irradiation. Cumulative radiation dosage of 8 Gy completely inhibited colony formation. This was also observed for 200 µM meclofenamic acid alone or in combination with minor ß-irradiation dosages. Results of the migration tests showed also a dose dependency with additive effects of combined therapy. Meclofenamic acid 200 µM alone and with cumulative ß-irradiation dosages resulted in an increased G2/M-phase share. CONCLUSIONS: Incubating human SMCs with meclofenamic acid and (90)Y for a period of 4 d (ie, 1.5 half-life times) resulted in an effective inhibition of smooth muscle cell proliferation, colony formation, and migration.

Impact of oncogenic K-RAS on YB-1 phosphorylation induced by ionizing radiation.

INTRODUCTION: Expression of Y-box binding protein-1 (YB-1) is associated with tumor progression and drug resistance. Phosphorylation of YB-1 at serine residue 102 (S102) in response to growth factors is required for its transcriptional activity and is thought to be regulated by cytoplasmic signaling phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathways. These pathways can be activated by growth factors and by exposure to ionizing radiation (IR). So far, however, no studies have been conducted on IR-induced YB-1 phosphorylation. METHODS: IR-induced YB-1 phosphorylation in K-RAS wild-type (K-RASwt) and K-RAS-mutated (K-RASmt) breast cancer cell lines was investigated. Using pharmacological inhibitors, small interfering RNA (siRNA) and plasmid-based overexpression approaches, we analyzed pathways involved in YB-1 phosphorylation by IR. Using γ-H2AX foci and standard colony formation assays, we investigated the function of YB-1 in repair of IR-induced DNA double-stranded breaks (DNA-DSB) and postirradiation survival was investigated. RESULTS: The average level of phosphorylation of YB-1 in the breast cancer cell lines SKBr3, MCF-7, HBL100 and MDA-MB-231 was significantly higher than that in normal cells. Exposure to IR and stimulation with erbB1 ligands resulted in phosphorylation of YB-1 in K-RASwt SKBr3, MCF-7 and HBL100 cells, which was shown to be K-Ras-independent. In contrast, lack of YB-1 phosphorylation after stimulation with either IR or erbB1 ligands was observed in K-RASmt MDA-MB-231 cells. Similarly to MDA-MB-231 cells, YB-1 became constitutively phosphorylated in K-RASwt cells following the overexpression of mutated K-RAS, and its phosphorylation was not further enhanced by IR. Phosphorylation of YB-1 as a result of irradiation or K-RAS mutation was dependent on erbB1 and its downstream pathways, PI3K and MAPK/ERK. In K-RASmt cells K-RAS siRNA as well as YB-1 siRNA blocked repair of DNA-DSB. Likewise, YB-1 siRNA increased radiation sensitivity. CONCLUSIONS: IR induces YB-1 phosphorylation. YB-1 phosphorylation induced by oncogenic K-Ras or IR enhances repair of DNA-DSB and postirradiation survival via erbB1 downstream PI3K/Akt and MAPK/ERK signaling pathways.

Effects of magnetic resonance imaging contrast agents on human umbilical vein endothelial cells and evaluation of magnetic resonance imaging contrast media-triggered transforming growth factor-beta induction in dermal fibroblasts (HSF) as a model for nephrogenic systemic fibrosis.

RATIONALE AND OBJECTIVES: The objective of this study was to evaluate effects of 6 commercially available magnetic resonance contrast media (CM) on human umbilical vein endothelial cells (HUVEC) and the induction of transforming growth factor-beta (TGF-ß) in dermal fibroblasts (HSF) as a possible model for the pathogenesis of nephrogenic systemic fibrosis. METHODS: HUVECs were incubated with 10× and 20× of the molar standard blood concentration achieved with CM applications for magnetic resonance imaging examinations (10× and 20× concentration) for 24 hours using gadolinium-based CM Gadovist, Magnevist, Multihance, and Omniscan, as well as Teslascan (Manganese-based), and Resovist (Iron-based). Proliferation kinetics (PK), colony formation, and viability assays were performed. Additionally, human dermal fibroblasts (HSF) were incubated for 24 hours with 1× and 20× concentration in all 6 CM, and TGF-ß levels were assessed directly after the incubation period as well as on days 3 and 8 postincubation. RESULTS: HUVEC PK data show similar gains in cell numbers for all 6 CM in both concentration groups over the 17-day assessment period. Only cells incubated with Omniscan and Teslascan differed from the other groups on days 3 and 7 postincubation (P < 0.05). After day 7, a cell regain occurred in the Omniscan and Teslascan groups reaching the numbers of the other groups in sequel. Differences in colony formation were consistent with PK results with a statistically significant reduction in clonogenic activity for Teslascan and Omniscan in HUVEC cells, P < 0.05. No reduction in viability was seen for all groups and conditions. TGF-ß expression of HSF cells incubated with 1× concentration and all CM did not differ significantly from control cells for any point in time investigated. At 20× concentration directly after incubation, TGF-ß was significantly reduced for the Teslascan and Resovist group as 3 compared with control and all other CM groups, P < 0.05. On day 3 postincubation, only Resovist-incubated HSF cells showed a significant reduction of TGF-ß (1.614, standard deviations: 89) as compared with the control group (2.883, standard deviations: 30) and the other CM. TGF-ß was slightly reduced for all CM groups 8 days after incubation (not statistically significant, P > 0.05). CONCLUSIONS: After 24 hours of incubation with Omniscan and Teslascan (10× and 20× concentration), considerable short-term antiproliferative effects in HUVECs were observed. HSF cells (20× concentration) showed a reduction of TGF-ß for Resovist and Teslascan directly after incubation, whereas TGF-ß levels in HSF cells were slightly reduced for all CM 8 days after incubation. Therefore, TGF-ß-mediated proliferative effects on fibroblasts or on collagen synthesis potentially leading to nephrogenic systemic fibrosis may mainly be triggered by tissue monocytes and macrophages in the peripheral blood instead of dermal fibroblasts.

In vitro comparison of the antiproliferative effects of rhenium-186 and rhenium-188 on human aortic endothelial cells.

PURPOSE: Rhenium-186 ((186)Re) and rhenium-188 ((188)Re) are promising radionuclides for the inhibition of restenosis after percutaneous transluminal angioplasty or other vascular interventions. Until now the maximal dose tolerance of endothelial cells has not been clearly known. MATERIALS AND METHODS: To characterize the effects of local irradiation treatment, human aortic endothelial cells (ECs) were incubated with different doses of (186)Re and (188)Re. Two days after plating, ECs received treatment for a period of 5 days. The total radiation doses applied were 1, 4, 8, 16, and 32 Gy. On days 1, 3, 5, 7, and 12 after initial rhenium incubation, cell growth, clonogenic activity, cell-cycle distribution, and cytoskeletal architecture were evaluated. RESULTS: From the first day on, a dose-dependent growth inhibition was observed. Cumulative doses of ≥32 Gy caused a weak colony formation and significant alterations in the cytoskeletal architecture. An increased fraction of cells in G2/M phase was seen for cumulative radiation doses of ≥16 Gy. Interestingly, there were no significant differences between (186)Re and (188)Re. CONCLUSION: Even for low dose rates of ß particles a dose-dependent proliferation inhibition of ECs is seen. Doses beyond 32 Gy alter the cytoskeletal architecture with possibly endothelial dysfunction and late thrombosis.

Nuclear EGFR shuttling induced by ionizing radiation is regulated by phosphorylation at residue Thr654.

Nuclear localisation of EGFR is associated with treatment resistance of tumor cells. The aim of this study was to identify molecular targets to block nuclear shuttling of EGFR. Mutation of Thr654, located within the putative EGFR NLS demonstrated that phosphorylation of this residue is essential for nuclear EGFR shuttling following irradiation. Deletion of Thr654 blocked nuclear transport of EGFR, whereas mutation to Glu increased shuttling. Treatment with a peptide, corresponding to the phosphorylated NLS, abolished nuclear EGFR transport and reduced radiation-induced activation of DNA-PK, essential for DNA-repair. In accordance with that, lack of nuclear EGFR increased residual DNA damage in tumor cells and reduced cellular survival following irradiation. Blockage of nuclear EGFR shuttling may be a new strategy to fight treatment resistance.

Effects of MRI contrast agents on human embryonic lung fibroblasts.

RATIONALE AND OBJECTIVES: The objective of this investigation was to evaluate 6 magnetic resonance contrast media (CM) with regard to their different effects on human embryonic lung fibroblasts (HEL-299). METHODS: Human embryonic fibroblasts (HEL-299) were incubated with 1x, 5x, 10x, and 20x of the normal molar blood concentration (1x, 5x, 10x, 20x conc.) reached through routine contrast media applications for MRI examinations. Four gadolinium-based CM, ie, Gadovist, Magnevist, Multihance, Omniscan, Teslascan (Manganese-based), and Resovist (Iron-based), with incubation periods over 4 hours and 24 hours were investigated. Proliferation kinetics, colony formation, and viability assays were performed after 4 and 24 hours of treatment. Apoptotic cells were quantified after tetramethylrhodamine ethyl ester staining following 24 hours of CM media incubation (20x conc.) by fluorescence activated cell sorting cytometry. Furthermore, immunofluorescence images with vimentin staining were obtained (20x conc., 24 hours treatment). Cell cycle analysis was performed after 24 hours of incubation and 20x conc. directly after incubation and 24 hours later (fluorescence activated cell sorting cytometry). RESULTS: The proliferation kinetics performed with 20x conc. revealed a persistent increase in cell numbers until day 11 for all CM without significant differences after 4 hours of incubation. A significant reduction in initial cell numbers was recorded in the 24-hours-group after 4 days of CM incubation with Magnevist, Multihance, Omniscan, and Teslascan. Solely cells incubated with Resovist and Gadovist failed to show decreased cell numbers when compared with the control group. However, a considerable cell regain occurred afterward reaching control-group levels on day 21. Colony numbers were significantly reduced (about 20%, respectively) with Magnevist at 10x and 20x conc., as well as Omniscan and Multihance at 20x conc. when compared with all other groups, P < 0.05. Cell-cycle distribution showed a reduction of S-phase cells for Magnevist, Omniscan, and Multihance (2.9%-10.5%) when compared with Gadovist, Resovist and Teslascan (16.7%-21.0%). Twenty-four hours after incubation, the percentiles of cells in S-phase were significantly increased for Magnevist, Omniscan, and Multihance (31.4%-38.5%) when compared with Gadovist, Resovist, and Teslascan (18.6%-26.8%), P < 0.05. Viability was not impaired by administration of any CM and no apoptosis was seen after tetramethylrhodamine ethyl ester staining at 24 hours of incubation. Cell morphology remained unchanged in vimentin-staining for all CM and conditioning regimens. CONCLUSIONS: No toxic effects on embryonic fetal lung fibroblasts were detectable after 4 and 24 hours of incubation in 6 MRI CM and 10x to 20x conc. in our setting. Antiproliferative effects, initially detected with Magnevist, Omniscan and Multihance, were rapidly compensated for.

Positive contrast imaging of iron oxide nanoparticles with susceptibility-weighted imaging.

Superparamagnetic iron oxide particles can be utilized to label cells for immune cell and stem cell therapy. The labeled cells cause significant field distortions induced in their vicinity, which can be detected with magnetic resonance imaging (MRI). In conventional imaging, the signal voids arising from the field distortions lead to negative contrast, which is not desirable, as detection of the cells can be masked by native low signal tissue. In this work, a new method for visualizing magnetically labeled cells with positive contrast is proposed and described. The technique presented is based on the susceptibility-weighted imaging (SWI) post-processing algorithm. Phase images from gradient-echo sequences are evaluated pixel by pixel, and a mask is created with values ranging from 0 to 1, depending on the phase value of the pixel. The magnitude image is then multiplied by the mask. With an appropriate mask function, positive contrast in the vicinity of the labeled cells is created. The feasibility of this technique is proved using an agar phantom containing superparamagnetic iron oxide particles-labeled cells and an ex vivo bovine liver. The results show high potential for detecting even small labeled cell concentrations in structurally inhomogeneous tissue types.

Functional investigations on human mesenchymal stem cells exposed to magnetic fields and labeled with clinically approved iron nanoparticles.

BACKGROUND: For clinical applications of mesenchymal stem cells (MSCs), labeling and tracking is crucial to evaluate cell distribution and homing. Magnetic resonance imaging (MRI) has been successfully established detecting MSCs labeled with superparamagnetic particles of iron oxide (SPIO). Despite initial reports that labeling of MSCs with SPIO is safe without affecting the MSC's biology, recent studies report on influences of SPIO-labeling on metabolism and function of MSCs. Exposition of cells and tissues to high magnetic fields is the functional principle of MRI. In this study we established innovative labeling protocols for human MSCs using clinically established SPIO in combination with magnetic fields and investigated on functional effects (migration assays, quantification of colony forming units, analyses of gene and protein expression and analyses on the proliferation capacity, the viability and the differentiation potential) of magnetic fields on unlabeled and labeled human MSCs. To evaluate the imaging properties, quantification of the total iron load per cell (TIL), electron microscopy, and MRI at 3.0 T were performed. RESULTS: Human MSCs labeled with SPIO permanently exposed to magnetic fields arranged and grew according to the magnetic flux lines. Exposure of MSCs to magnetic fields after labeling with SPIO significantly enhanced the TIL compared to SPIO labeled MSCs without exposure to magnetic fields resulting in optimized imaging properties (detection limit: 1,000 MSCs). Concerning the TIL and the imaging properties, immediate exposition to magnetic fields after labeling was superior to exposition after 24 h. On functional level, exposition to magnetic fields inhibited the ability of colony formation of labeled MSCs and led to an enhanced expression of lipoprotein lipase and peroxisome proliferator-activated receptor-gamma in labeled MSCs under adipogenic differentiation, and to a reduced expression of alkaline phosphatase in unlabeled MSCs under osteogenic differentiation as detected by qRT-PCR. Moreover, microarray analyses revealed that exposition of labeled MSCs to magnetic fields led to an up regulation of CD93 mRNA and cadherin 7 mRNA and to a down regulation of Zinc finger FYVE domain mRNA. Exposition of unlabeled MSCs to magnetic fields led to an up regulation of CD93 mRNA, lipocalin 6 mRNA, sialic acid acetylesterase mRNA, and olfactory receptor mRNA and to a down regulation of ubiquilin 1 mRNA. No influence of the exposition to magnetic fields could be observed on the migration capacity, the viability, the proliferation rate and the chondrogenic differentiation capacity of labeled or unlabeled MSCs. CONCLUSIONS: In our study an innovative labeling protocol for tracking MSCs by MRI using SPIO in combination with magnetic fields was established. Both, SPIO and the static magnetic field were identified as independent factors which affect the functional biology of human MSCs. Further in vivo investigations are needed to elucidate the molecular mechanisms of the interaction of magnetic fields with stem cell biology.

ErbB2 expression through heterodimerization with erbB1 is necessary for ionizing radiation- but not EGF-induced activation of Akt survival pathway.

PURPOSE: ErbB1-dependent Akt phosphorylation improves post-irradiation cellular survival. In the present study, we investigated the contribution of erbB2 as a heterodimerization partner of erbB1 in activation of Akt survival signaling after irradiation or EGF treatment. MATERIALS AND METHODS: Pattern of receptor dimerization and protein phosphorylation were investigated by Western and immunoblotting as well as immunoprecipitation techniques. Residual DNA double-strand breaks (DNA-DSB) and clonogenic activity were analyzed by γH2AX and standard clonogenic assay. To knocked erbB2 expression siRNA was used. RESULTS: In lung carcinoma cell lines A549 and H661, the erbB1-tyrosine kinase (TK) inhibitor erlotinib blocked EGF as well as ionizing radiation (IR)-induced Akt and DNA-PKcs phosphorylation. Targeting Akt and erbB1 induced cellular radiation sensitivity while, the erbB2-TK inhibitor AG825 neither affected phosphorylation of Akt and DNA-PKcs nor induced radiosensitization. ErbB2-siRNA and the anti-erbB2 antibody trastuzumab blocked IR-induced, but not EGF-stimulated Akt phosphorylation and impaired the repair of DNA-DSB. Likewise, IR but not EGF enhanced erbB1/erbB2 heterodimerization and resulted in the release of phosphorylated erbB2 cleavage products p135 and p95. Trastuzumab prevented radiation-induced formation of an active erbB1/erbB2 heterodimer and increased cellular radiation sensitivity. ErbB1- but not erbB2-TK inhibition stabilized erbB2 (p185) through preventing its cleavage. CONCLUSIONS: The data indicates that ErbB2 through heterodimerization with erbB1 is necessary for the activation of Akt signaling following irradiation but not following EGF treatment.

Radiocontrast media affect radiation-induced DNA damage repair in vitro and in vivo by affecting Akt signalling.

PURPOSE: The study was performed to investigate cytogenetic effects of ionic and non-ionic radiocontrast media (RCM) meglumine, iohexol alone and in combination with irradiation in mouse bone marrow cells in vivo and in vitro. MATERIALS AND METHODS: Micronuclei assay was performed in bone marrow cells (BMC) of Balb/C mice intraperitoneally injected with RCM in the presence or absence of whole-body irradiation of 50 mGy. DNA repair (NHEJ) signalling and efficiency were analyzed by Western blot and gammaH2AX-foci assay in normal fibroblast HSF-7 and HUVEC cells. RESULTS: Both compounds reduced proliferation of BMC significantly. Concentrations of 0.5, 1 and 2 ml/kg meglumine or iohexol significantly enhanced the frequency of micronucleated polychromatic erythrocytes (MnPCEs) at all doses of meglumine (p<0.01) and 2 ml/kg of iohexol (p<0.05). Combined with irradiation meglumine at 0.5 and 1 ml/kg led to a higher frequency of MnPCEs than iohexol/IR (p<0.05). Meglumine induced DNA-double strand breaks (DNA-DSB) in non-irradiated HSF and strongly increased residual DNA-DSB within 10 min to 24h after irradiation with 200 or 400 mGy (p<0.001). Iohexol did not induce DNA-DSB but blocked repair of radiation-induced DNA-DSB significantly (p<0.05). Meglumine blocked IR-induced Akt phosphorylation, phosphorylation of DNA-PKcs (S2056, T2609) and ATM (S1981). Iohexol only blocked phosphorylation of Akt and DNA-PKcs at S2056. CONCLUSION: RCM result in clastogenic effects through interference intracellular signalling cascades involved in the regulation of non-homologous end-joining repair of DNA-DSB.

2-Methoxyestradiol-induced radiosensitization is independent of SOD but depends on inhibition of Akt and DNA-PKcs activities.

BACKGROUND AND PURPOSE: 2-Methoxyestradiol (2-ME) is described as an inhibitor of the superoxide dismutase (SOD) enzyme activity. However, it attenuates PI3K/Akt pathway and induces radiosensitization in human tumor cells as well. Since the activation of catalytic subunit of DNA-protein kinase (DNA-PKcs) is partially regulated by Akt activity, in the present study we investigated whether 2-ME-induced radiosensitization is dependent on inhibition of Akt and DNA-PKcs activities or on SOD targeting. MATERIALS AND METHODS: This study was performed using the lung carcinoma cell line A549. Ionizing radiation-induced SOD activity was analyzed by superoxide dismutase activity assay. Applying Western blotting, the pattern of radiation-induced SOD expression and activation of Akt as well as DNA-PKcs was analyzed. Colony formation assay and gammaH2AX foci assay were performed to measure radiosensitization and DNA-double strand break (DNA-DSB) repair. To downregulate SOD expression small interfering RNA (siRNA) was used. RESULTS: Irradiation with 4Gy stimulated SOD enzyme activity as early as 1min after radiation exposure. Expression of Cu/Zn-SOD (SOD1) as well as Mn-SOD (SOD2) was increased by single doses of 1-4Gy within 24-36h. 2-ME blocked radiation-induced SOD enzyme activity but not protein expression and enhanced radiation sensitivity. Pretreatment with 2-ME blocked IR-induced Akt as well as DNA-PKcs phosphorylation and impaired the repair of DNA-DSB. SiRNA targeting of SOD1 and SOD2 affected neither DNA-PKcs phosphorylation nor post-irradiation survival while inhibition of Akt by specific inhibitor abrogated 2-ME-induced radiosensitization. CONCLUSION: These results may indicate that 2-ME-induced radiosensitization is independent of SOD inhibition but mainly depends on inhibition of Akt and DNA-PKcs activities.