Circulating microRNAs as prognostic therapy biomarkers in head and neck cancer patients.

BACKGROUND: The prediction of therapy response in head and neck squamous cell cancer (HNSCC) requires biomarkers, which are also a prerequisite for personalised therapy concepts. The current study aimed to identify therapy-responsive microRNAs (miRNAs) in the circulation that can serve as minimally invasive prognostic markers for HNSCC patients undergoing radiotherapy. METHODS: We screened plasma miRNAs in a discovery cohort of HNSCC patients before therapy and after treatment. We further compared the plasma miRNAs of the patients to age- and sex-matched healthy controls. All miRNAs identified as biomarker candidates were then confirmed in an independent validation cohort of HNSCC patients and tested for correlation with the clinical outcome. RESULTS: We identified a signature of eight plasma miRNAs that differentiated significantly (P=0.003) between HNSCC patients and healthy donors. MiR-186-5p demonstrated the highest sensitivity and specificity to classify HNSCC patients and healthy individuals. All therapy-responsive and patient-specific miRNAs in plasma were also detectable in tumour tissues derived from the same patients. High expression of miR-142-3p, miR-186-5p, miR-195-5p, miR-374b-5p and miR-574-3p in the plasma correlated with worse prognosis. CONCLUSIONS: Circulating miR-142-3p, miR-186-5p, miR-195-5p, miR-374b-5p and miR-574-3p represent the most promising markers for prognosis and therapy monitoring in the plasma of HNSCC patients. We found strong evidence that the circulating therapy-responsive miRNAs are tumour related and were able to validate them in an independent cohort of HNSCC patients.

Three-dimensional imaging of whole mouse models: comparing nondestructive X-ray phase-contrast micro-CT with cryotome-based planar epi-illumination imaging.

In this study, we compare two evolving techniques for obtaining high-resolution 3D anatomical data of a mouse specimen. On the one hand, we investigate cryotome-based planar epi-illumination imaging (cryo-imaging). On the other hand, we examine X-ray phase-contrast micro-computed tomography (micro-CT) using synchrotron radiation. Cryo-imaging is a technique in which an electron multiplying charge coupled camera takes images of a cryo-frozen specimen during the sectioning process. Subsequent image alignment and virtual stacking result in volumetric data. X-ray phase-contrast imaging is based on the minute refraction of X-rays inside the specimen and features higher soft-tissue contrast than conventional, attenuation-based micro-CT. To explore the potential of both techniques for studying whole mouse disease models, one mouse specimen was imaged using both techniques. Obtained data are compared visually and quantitatively, specifically with regard to the visibility of fine anatomical details. Internal structure of the mouse specimen is visible in great detail with both techniques and the study shows in particular that soft-tissue contrast is strongly enhanced in the X-ray phase images compared to the attenuation-based images. This identifies phase-contrast micro-CT as a powerful tool for the study of small animal disease models.

Extracellular heat shock protein 70 levels in tumour-bearing dogs and cats treated with radiation therapy and hyperthermia.

Hyperthermia is a form of a cancer treatment which is frequently applied in combination with radiotherapy (RT) to improve therapy responses and radiosensitivity. The mode of action of hyperthermia is multifactorial; the one hand by altering the amount of the blood circulation in the treated tissue, on the other hand by modulating molecular pathways involved in cell survival processes and immunogenic interactions. One of the most dominant proteins induced by hyperthermia is the major stress-inducible heat shock protein 70 (Hsp70). Hsp70 can be found in the blood either as a free-protein (free HSP70) derived from necrotic cells, or lipid-bound (liposomal Hsp70) when it is actively released in extracellular vesicles (EVs) by living cells. The aim of the study was to evaluate the levels of free and liposomal Hsp70 before and after treatment with RT alone or hyperthermia combined with radiotherapy (HTRT) in dogs and cats to evaluate therapy responses. Peripheral blood was collected from feline and canine patients before and at 2, 4, 6 and 24 h after treatment with RT or HTRT. Hsp70 enzyme-linked immunosorbent assays (ELISAs) were performed to determine the free and liposomal Hsp70 concentrations in the serum. The levels were analysed after the first fraction of radiation to study immediate effects and after all applied fractions to study cumulative effects. The levels of free and liposomal Hsp70 levels in the circulation were not affected by the first singular treatment and cumulative effects of RT in cats however, after finalizing all treatment cycles with HTRT free and liposomal Hsp70 levels significantly increased. In dogs, HTRT, but not treatment with RT alone, significantly affected liposomal Hsp70 levels during the first fraction. Free Hsp70 levels were significantly increased after RT, but not HTRT, during the first fraction in dogs. In dogs, on the other hand, RT alone resulted in a significant increase in liposomal Hsp70, but HTRT did not significantly affect the liposomal Hsp70 when cumulative effects were analysed. Free Hsp70 was significantly induced in dogs after both, RT and HTRT when cumulative effects were analysed. RT and HTRT treatments differentially affect the levels of free and liposomal Hsp70 in dogs and cats. Both forms of Hsp70 could potentially be further investigated as potential liquid biopsy markers to study responses to RT and HTRT treatment in companion animals.

Basal HIF-1α expression levels are not predictive for radiosensitivity of human cancer cell lines.

BACKGROUND AND PURPOSE: High levels of hypoxia inducible factor (HIF)-1α in tumors are reported to be associated with tumor progression and resistance to therapy. To examine the impact of HIF-1α on radioresistance under normoxia, the sensitivity towards irradiation was measured in human tumor cell lines that differ significantly in their basal HIF-1α levels. MATERIAL AND METHODS: HIF-1α levels were quantified in lysates of H1339, EPLC-272H, A549, SAS, XF354, FaDu, BHY, and CX- tumor cell lines by ELISA. Protein levels of HIF-1α, HIF-2α, carbonic anhydrase IX (CA IX), and GAPDH were assessed by Western blot analysis. Knock-down experiments were performed using HIF-1α siRNA. Clonogenic survival after irradiation was determined by the colony forming assay. RESULTS: According to their basal HIF-1α status, the tumor cell lines were divided into low (SAS, XF354, FaDu, A549, CX-), intermediate (EPLC-272H, BHY), and high (H1339) HIF-1α expressors. The functionality of the high basal HIF-1α expression in H1339 cells was proven by reduced CA IX expression after knocking-down HIF-1α. Linear regression analysis revealed no correlation between basal HIF-1α levels and the survival fraction at either 2 or 4 Gy in all tumor cell lines investigated. CONCLUSION: Our data suggest that basal HIF-1α levels in human tumor cell lines do not predict their radiosensitivity under normoxia.

Induction and repair of DNA double-strand breaks assessed by gamma-H2AX foci after irradiation with pulsed or continuous proton beams.

In particle tumor therapy including beam scanning at accelerators, the dose per voxel is delivered within about 100 ms. In contrast, the new technology of laser plasma acceleration will produce ultimately shorter particle packages that deliver the dose within a nanosecond. Here, possible differences for relative biological effectiveness in creating DNA double-strand breaks in pulsed or continuous irradiation mode are studied. HeLa cells were irradiated with 1 or 5 Gy of 20-MeV protons at the Munich tandem accelerator, either at continuous mode (100 ms), or applying a single pulse of 1-ns duration. Cells were fixed 1 h after 1-Gy irradiation and 24 h after 5-Gy irradiation, respectively. A dose-effect curve based on five doses of X-rays was taken as reference. The total number of phosphorylated histone H2AX (gamma-H2AX) foci per cell was determined using a custom-made software macro for gamma-H2AX foci counting. For 1 h after 1-Gy 20-MeV proton exposures, values for the relative biological effectiveness (RBE) of 0.97 ± 0.19 for pulsed and 1.13 ± 0.21 for continuous irradiations were obtained in the first experiment 1.13 ± 0.09 and 1.16 ± 0.09 in the second experiment. After 5 Gy and 24 h, RBE values of 0.99 ± 0.29 and 0.91 ± 0.23 were calculated, respectively. Based on the gamma-H2AX foci numbers obtained, no significant differences in RBE between pulsed and continuous proton irradiation in HeLa cells were detected. These results are well in line with our data on micronucleus induction in HeLa cells.

Interleukin-10 promotes NK cell killing of autologous macrophages by stimulating expression of NKG2D ligands.

Under inflammatory conditions, the pleiotropic cytokine interleukin-10 (IL-10) is released in many tissues. It mediates anti-inflammatory effects in particular by inhibiting the release of T helper type 1 (Th1) cytokines. In contrast, we show here that NK cell cytotoxicity against autologous macrophages is elevated if both cell types are cultured with IL-10. The expression of most activatory NK receptors is increased after culture in the presence of IL-10. On the other hand, macrophages cultured in the presence of IL-10 show elevated expression of the NKG2D ligands major histocompatibility complex (MHC) class 1-like molecules (MIC) - A and - B, as well as UL-16 binding proteins (ULBP) - ULBP-1, ULBP-2 and ULBP-3. By masking the interaction of NK cells with macrophages through interruption of the NKG2D receptor with its ligands, we could reverse the IL-10-induced lysis of macrophages. Our data therefore reveal that IL-10 may exert a novel immunomodulatory role by stimulating NKG2D ligand expression on macrophages, thereby rendering them susceptible to NK cell elimination. This suggests that NK cells would delete macrophages and potentially other immature antigen-presenting cells (APC) or their precursors under inflammatory conditions as a feedback mechanism to shut off uncontrolled immune responses.

Noradrenaline increases the expression and release of Hsp72 by human neutrophils.

UNLABELLED: The blood concentration of extracellular 72kDa heat shock protein (eHsp72) increases under conditions of stress, including intense exercise. However, the signal(s), source(s), and secretory pathways in its release into the bloodstream have yet to be clarified. The aim of the present study was to evaluate the role of noradrenaline (NA) as a stress signal on the expression and release of Hsp72 by circulating neutrophils (as a source), all within a context of the immunophysiological regulation during exercise-induced stress in sedentary and healthy young (21-26years) women. The expression of Hsp72 on the surface of isolated neutrophils was determined by flow cytometry, and its release by cultured isolated neutrophils was determined by ELISA. Incubation with cmHsp70-FITC showed that neutrophils express Hsp72 on their surface under basal conditions. In addition, cultured isolated neutrophils (37 degrees C and 5% CO(2)) also released Hsp72 under basal conditions, with this release increasing from 10min to 24h in the absence of cell damage. NA at 10(-9)-10(-5)M doubled the percentage of neutrophils expressing Hsp72 after 60min and 24h incubation. NA also stimulated (by about 20%) the release of Hsp72 after 10min of incubation. IN CONCLUSION: (1) Hsp72 is expressed on the surface of isolated neutrophils under basal conditions, and this expression is augmented by NA. (2) Isolated neutrophils can also release Hsp72 under cultured basal conditions in the absence of cell death, and NA can increase this release. These results may contribute to confirming the hypothesis that NA can act as a "stress signal" for the increased eHsp72 in the context of exercise stress, with a role for neutrophils as a source for the expression and, to a lesser degree, the release of Hsp72 after activation by NA.

Intercomparison of dose enhancement ratio and secondary electron spectra for gold nanoparticles irradiated by X-rays calculated using multiple Monte Carlo simulation codes.

PURPOSE: Targeted radiation therapy has seen an increased interest in the past decade. In vitro and in vivo experiments showed enhanced radiation doses due to gold nanoparticles (GNPs) to tumors in mice and demonstrated a high potential for clinical application. However, finding a functionalized molecular formulation for actively targeting GNPs in tumor cells is challenging. Furthermore, the enhanced energy deposition by secondary electrons around GNPs, particularly by short-ranged Auger electrons is difficult to measure. Computational models, such as Monte Carlo (MC) radiation transport codes, have been used to estimate the physical quantities and effects of GNPs. However, as these codes differ from one to another, the reliability of physical and dosimetric quantities needs to be established at cellular and molecular levels, so that the subsequent biological effects can be assessed quantitatively. METHODS: In this work, irradiation of single GNPs of 50 nm and 100 nm diameter by X-ray spectra generated by 50 and 100 peak kilovoltages was simulated for a defined geometry setup, by applying multiple MC codes in the EURADOS framework. RESULTS: The mean dose enhancement ratio of the first 10 nm-thick water shell around a 100 nm GNP ranges from 400 for 100 kVp X-rays to 600 for 50 kVp X-rays with large uncertainty factors up to 2.3. CONCLUSIONS: It is concluded that the absolute dose enhancement effects have large uncertainties and need an inter-code intercomparison for a high quality assurance; relative properties may be a better measure until more experimental data is available to constrain the models.

NKG2D-dependent effector function of bronchial epithelium-activated alloreactive T-cells.

Allogeneic haematopoietic stem cell transplantation (SCT) has emerged as a curative therapeutic option. However, the role of graft-versus-host disease in lung injury after SCT has yet to be determined. In the present study, primary bronchial epithelial cells and the bronchial epithelial cell line BEAS-2B were used to investigate immune responses of allogeneic CD8+ T-cells directed against respiratory epithelial cells. Following stimulation with irradiated bronchial epithelial cells, CD8+ T-cells produced significant amounts of interferon-gamma, upregulated alloantigen activation markers and proliferated highly compared with T-cells stimulated with interleukin-2 alone. Furthermore, cytotoxicity assays demonstrated that bronchial epithelial cell-specific and granzyme B-mediated cytolytic activity was induced in CD8+ T-cells. Generation of natural killer (NK) T-cells, NK-like T-cells, cytokine-induced killer cells or lymphokine-activated killer cells could be excluded by phenotyping, culture conditions and neglectable lytic activity following stimulation with interleukin-2 alone. Inhibition experiments showed that lysis of bronchial epithelial cells was not major histocompatibility complex-I restricted, but depended on NK group 2 member D signalling; a stimulatory receptor initially shown to be expressed on NK cells. The present data imply that the respiratory epithelium has an antigen presenting function and directly alloactivates cytotoxic CD8+ T-cells that show nonclassical effector function.

Intracellular and extracellular functions of heat shock proteins: repercussions in cancer therapy.

Stress or heat shock proteins (HSPs) are the most conserved proteins present in both prokaryotes and eukaryotes. Their expression is induced in response to a wide variety of physiological and environmental insults. These proteins play an essential role as molecular chaperones by assisting the correct folding of nascent and stress-accumulated misfolded proteins, and preventing their aggregation. HSPs have a dual function depending on their intracellular or extracellular location. Intracellular HSPs have a protective function. They allow the cells to survive lethal conditions. Various mechanisms have been proposed to account for the cytoprotective functions of HSPs. Several HSPs have also been demonstrated to directly interact with various components of the tightly regulated programmed cell death machinery, upstream and downstream of the mitochondrial events. On the other hand, extracellular located or membrane-bound HSPs mediate immunological functions. They can elicit an immune response modulated either by the adaptive or innate immune system. This review will focus on HSP27, HSP70, and HSP90. We will discuss the dual role of these HSPs, protective vs. immunogenic properties, making a special emphasis in their utility as targets in cancer therapy.

Dual function of membrane-bound heat shock protein 70 (Hsp70), Bag-4, and Hsp40: protection against radiation-induced effects and target structure for natural killer cells.

CX+/CX- and Colo+/Colo- tumor sublines with stable heat shock protein 70 (Hsp70) high and low membrane expression were generated by fluorescence activated cell sorting of the parental human colon (CX2) and pancreas (Colo357) carcinoma cell lines, using an Hsp70-specific antibody. Two-parameter flow cytometry revealed that Hsp70 colocalizes with Bag-4, also termed silencer of death domain, not only in the cytosol but also on the plasma membrane. After nonlethal gamma-irradiation, the percentage of membrane-positive cells and the protein density of Hsp70 and Bag-4 were found to be strongly upregulated in carcinoma sublines with initially low expression levels (CX-, Colo-). Membrane expression of Hsp70 was also elevated in Bag-4 overexpressing HeLa cervix carcinoma cells when compared to neo-transfected cells. In response to gamma-irradiation, neo-transfected HeLa cells behaved like Hsp70/Bag-4 low-expressing CX- and Colo-, and Bag-4-transfected HeLa cells like Hsp70/Bag-4 high-expressing carcinoma sublines CX+ and Colo+. Immunoprecipitation studies further confirmed colocalization of Hsp70 and Bag-4 but also point to an association of Hsp70 and Hsp40 on the plasma membrane of CX+ and Colo+ cells; on CX- and Colo- tumor sublines, Hsp40 was detectable in the absence of Hsp70 and Bag-4. Other co-chaperones including Hsp60 and Hsp90 were neither found on the cell surface of CX+/CX-, Colo+/Colo- nor on HeLa neo-/HeLa Bag-4-transfected tumor cells. Functionally, Hsp70/Bag-4 and Hsp70/Hsp40 membrane-positive tumor cells appeared to be better protected against radiation-induced effects, including G2/M arrest and growth inhibition, on the one hand. On the other hand, membrane-bound Hsp70, but neither Bag-4 nor Hsp40, served as a recognition site for the cytolytic attack mediated by natural killer cells.

Heat shock proteins in immunity.

This chapter focuses on immunological effects of eukaryotic and microbial heat shock proteins (HSPs), with molecular weights of about 60, 70, and 90 kDa. The search for tumor-specific antigens resulted in the identification of HSPs. They have been found to elicit a potent anti-cancer immune response mediated by the adoptive and innate immune system. Following receptor-mediated uptake of HSP (HSP70 and gp96) peptide complexes by antigen-presenting cells and representation of HSP-chaperoned peptides by MHC class I molecules, a CD8-specific T cell response is induced. Apart from chaperoning immunogenic peptides derived from tumors, bacterial and virally infected cells, they by themselves provide activatory signals for antigen-presenting cells and natural killer (NK) cells. After binding of peptide-free HSP70 to Toll-like receptors, the secretion of pro-inflammatory cytokines is initiated by antigen-presenting cells and thus results in a nonspecific stimulation of the immune system. Moreover, soluble as well as cell membrane-bound HSP70 on tumor cells can directly activate the cytolytic and migratory capacity of NK cells. Apart form cancer, HSPs of different origins, with a molecular weight of about 60, 70, and 90 kDa, also play a pivotal role in viral infections, including human and simian immunodeficiency virus (HIV, SIV), measles, and choriomeningitis. Moreover, HSPs have been found to induce tolerance against autoimmune diseases. In summary, depending on their mode of induction, intracellular/extracellular location, cellular origin (eukaryote/prokaryote), peptide loading status, intracellular ADP/ATP content, concentration, and route of application, HSPs either exert immune activation as danger signals in cancer immunity and mediate protection against infectious diseases or exhibit regulatory activities in controlling and preventing autoimmunity.

Zero-valent Fe confined mesoporous silica nanocarriers (Fe(0) @ MCM-41) for targeting experimental orthotopic glioma in rats.

Mesoporous silica nanoparticles (MSNs) impregnated with zero-valent Fe (Fe(0) @ MCM-41) represent an attractive nanocarrier system for drug delivery into tumor cells. The major goal of this work was to assess whether MSNs can penetrate the blood-brain barrier in a glioblastoma rat model. Synthesized MSNs nanomaterials were characterized by energy dispersive X-ray spectroscopy, measurements of X-ray diffraction, scanning electron microscopy and Mössbauer spectroscopy. For the detection of the MSNs by MR and for biodistribution studies MSNs were labeled with zero-valent Fe. Subsequent magnetometry and nonlinear-longitudinal-response-M2 (NLR-M2) measurements confirmed the MR negative contrast enhancement properties of the nanoparticles. After incubation of different tumor (C6 glioma, U87 glioma, K562 erythroleukemia, HeLa cervix carcinoma) and normal cells such as fibroblasts and peripheral blood mononuclear cells (PBMCs) MSNs rapidly get internalized into the cytosol. Intracellular residing MSNs result in an enhanced cytotoxicity as Fe(0) @ MCM-41 promote the reactive oxygen species production. MRI and histological studies indicated an accumulation of intravenously injected Fe(0) @ MCM-41 MSNs in orthotopic C6 glioma model. Biodistribution studies with measurements of second harmonic of magnetization demonstrated an increased and dose-dependent retention of MSNs in tumor tissues. Taken together, this study demonstrates that MSNs can enter the blood-brain barrier and accumulate in tumorous tissues.

A comparison of the effects of ifosfamide vs. mafosfamide treatment on intracellular glutathione levels and immunological functions of immunocompetent lymphocyte subsets.

This study compares the effects of ifosfamide treatment with those of mafosfamide treatment with respect to important immunological functions and intracellular glutathione (GSH) levels of immunocompetent lymphocyte subsets such as cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. The proliferative and cytotoxic capacity of human peripheral blood lymphocyte (PBL) subsets was measured by a standard [3H] thymidine uptake assay and a [51Cr] release assay; the intracellular glutathione levels were determined by using an established HPLC method described by Reed. Following incubation of human PBLs with the activated forms of ifosfamide (4-OH-IF) and mafosfamide (4-OH-CP), the proliferative capacity of recombinant interleukin 2 (rIL-2)-stimulated PBLs was reduced by both drugs in a dose-dependent manner. However, a three-fold higher concentration of ifosfamide compared with mafosfamide is needed to achieve a comparable inhibition rate in the proliferative capacity of theses lymphocytes. Separation of PBLs into a CD3+ CTL and a CD3- NK subpopulation revealed that proliferative activity was reduced in both subpopulations in a dose-dependent manner by ifosfamide and mafosfamide. However, growth inhibition was much more pronounced in the CD3+ CTL compared with the CD3- NK cells. The intracellular GSH level in CTL, and to a lower extent in NK cells, was reduced more substantially following an ifosfamide treatment compared with a mafosfamide treatment. With respect to our previous finding that an ifosfamide-induced reduction of intracellular GSH levels correlates with decreased cytotoxic function, in this study we compared the effects of ifosfamide treatment with those of mafosfamide treatment on the cytolytic activity of lymphocyte subpopulations. The cytotoxic activity of CD3+ CTL against allogeneic target cells (B-lymphoblastoid cells) was significantly reduced after preincubation with either activated ifosfamide or mafosfamide. In contrast, the lysis of NK-sensitive tumor target cells (K562), mediated by CD3- NK cells is only affected if the effector cells are exposed to high concentrations (100 microM) of activated ifosfamide. The cytotoxic activity of NK cells pretreated with high concentrations of activated mafosfamide (33 microM) had no significant inhibitory effect on the cytotoxic function. Taken together, our findings were as follows: 1) A three-fold higher concentration of activated ifosfamide compared with mafosfamide results in a comparable inhibition of the proliferative activity, in vitro. 2) The intracellular GSH levels of unseparated rIL-2 activated lymphocytes were reduced by ifosfamide and mafosfamide at concentrations above 16 microM. 3) Separated NK cells compared with CTLs are more resistant to treatment with ifosfamide with respect to their intracellular GSH levels. This phenomenon is even more pronounced after treatment with mafosfamide. 4) The reduction in intracellular GSH levels after treatment with ifosfamide and mafosfamide could be correlated with a reduction in the cytotoxic activity.

Synergistic effects of heat and ET-18-OCH3 on membrane expression of hsp70 and lysis of leukemic K562 cells.

We previously reported that cell surface expression of hsp70, the major stress inducible member of the 70-kDa heat shock protein family, is inducible by nonlethal heat as well as by treatment with the membrane-interactive compound alkyl-lysophospholipid 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) selectively on human tumor cell lines. Plasma membrane expression of hsp70 increases selectively the sensitivity of tumor cells to lysis and, therefore, might play an important role in the antitumor immune response. Here, we demonstrate that a combined treatment consisting of sublethal heat (41.8 degrees C) and a noncytotoxic concentration of ET-18-OCH3 (25 micrograms/mL) results in a synergistic increase in the amount of cell membrane-bound hsp70 on leukemic K562 cells and on freshly isolated bone marrow of a chronic myelogeneous leukemia (CML) patient, but not on peripheral blood lymphocytes or CD34+ hematopoietic progenitor cells of healthy human individuals. Under these conditions the repopulating capacity of progenitor cells was not influenced. The increased hsp70 membrane expression on leukemic K562 cells results in a significantly increased sensitivity to lysis mediated by natural killer cells. In contrast to leukemic cells, the lysis of peripheral blood lymphocytes and CD34+ progenitor cells that lack expression of hsp70 on their plasma membrane was not negatively influenced by this treatment. A nonspecific disruption of the plasma membrane could be excluded, because treatment with a nontoxic concentration of the detergent Tween20 did not have an influence on hsp70 cell surface expression or on the sensitivity to lysis. Our findings might have further clinical implications with respect to purging of bone marrow from patients suffering from leukemia at sublethal conditions to induce a tumor-selective immune response.

Heat shock protein 70 (Hsp70) stimulates proliferation and cytolytic activity of natural killer cells.

We previously demonstrated that lysis of tumor cells that express Hsp70, the highly stress-inducible member of the HSP70 family, on their plasma membrane is mediated by natural killer (NK) cells. Here, we studied the effects of different proteins of the HSP70 family in combination with interleukin 2 (IL-2) on the proliferation and cytotoxic activity of human NK cells in vitro. Proliferation of NK cells was significantly enhanced by human recombinant Hsp70 (rHsp70) and to a lesser extent by rHsp70homC, the recombinant C-terminal peptide-binding domain derived from Hsp70hom, but not by the constitutive Hsc70 or DnaK, the Escherichia coli analogue of human Hsp70. Even rHsp70 protein alone moderately enhances proliferation and cytolytic activity of NK cells, thus indicating that the stimulatory effect is not strictly dependent on IL-2. NK cells stimulated with rHsp70 protein also exhibit an increased secretion of interferon gamma (IFN-gamma). The phenotypic characterization of NK cells with specificity for Hsp70-expressing tumor cells revealed a CD16dim/CD56bright and increased CD57 and CD94 expression. The cytolytic activity of NK cells also was significantly reduced when a CD94-specific antibody or rHsp70 was added directly before the cytotoxicity assay, whereas other antibodies directed against CD57 and major histocompatibility complex class I molecules or Hsp70 proteins, including Hsc70 and DnaK, did not affect the NK-mediated killing. However, long-term incubation of NK cells with rHsp70 protein enhances not only the proliferative but also the cytolytic response against Hsp70-expressing tumor cells. Our results indicate that the C-terminal domain of Hsp70 protein affects not only the proliferative but also the cytolytic activity of a phenotypically distinct NK cell population with specificity for Hsp70-expressing tumor cells. 1999 International Society for Experimental Hematology.

Sub-micrometer 20MeV protons or 45MeV lithium spot irradiation enhances yields of dicentric chromosomes due to clustering of DNA double-strand breaks.

In conventional experiments on biological effects of radiation types of diverse quality, micrometer-scale double-strand break (DSB) clustering is inherently interlinked with clustering of energy deposition events on nanometer scale relevant for DSB induction. Due to this limitation, the role of the micrometer and nanometer scales in diverse biological endpoints cannot be fully separated. To address this issue, hybrid human-hamster AL cells have been irradiated with 45MeV (60keV/µm) lithium ions or 20MeV (2.6keV/µm) protons quasi-homogeneously distributed or focused to 0.5×1µm(2) spots on regular matrix patterns (point distances up to 10.6×10.6µm), with pre-defined particle numbers per spot to provide the same mean dose of 1.7Gy. The yields of dicentrics and their distribution among cells have been scored. In parallel, track-structure based simulations of DSB induction and chromosome aberration formation with PARTRAC have been performed. The results show that the sub-micrometer beam focusing does not enhance DSB yields, but significantly affects the DSB distribution within the nucleus and increases the chance to form DSB pairs in close proximity, which may lead to increased yields of chromosome aberrations. Indeed, the experiments show that focusing 20 lithium ions or 451 protons per spot on a 10.6µm grid induces two or three times more dicentrics, respectively, than a quasi-homogenous irradiation. The simulations reproduce the data in part, but in part suggest more complex behavior such as saturation or overkill not seen in the experiments. The direct experimental demonstration that sub-micrometer clustering of DSB plays a critical role in the induction of dicentrics improves the knowledge on the mechanisms by which these lethal lesions arise, and indicates how the assumptions of the biophysical model could be improved. It also provides a better understanding of the increased biological effectiveness of high-LET radiation.