Influence of osteopontin silencing on survival and migration of lung cancer cells.

BACKGROUND AND PURPOSE: Osteopontin (OPN) is a multifunctional protein overexpressed in many cancers and is involved in tumor progression and metastasis. In lung cancer, elevated OPN expression is associated with an unfavorable prognosis. Therefore, inhibition of OPN is an attractive approach for improving survival. MATERIALS AND METHODS: We used siRNA to specifically downregulate OPN expression in A549 lung cancer cells. OPN silencing was evaluated with quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for mRNA levels and with Western blotting for protein levels. Effects on cell proliferation were measured by cell counting. The influence on tumor cell migration was detected using a modified Boyden chamber. Changes in cell cycle distribution were assessed by flow cytometry. Using the colony formation assay, we determined changes in radiosensitivity. RESULTS: A specific and effective downregulation of OPN expression was detected in both RNA and protein levels. Cell proliferation and cell migration were significantly reduced by OPN silencing after 24 h and the effects were further increased by the addition of irradiation. The cell cycle distribution showed a reduction in S phase and an increase in cells arrested in both G(0)/G(1) and G(2)/M phases. Specific enhancement of radiosensitivity was clearly shown after OPN knockdown. CONCLUSION: The combination of OPN silencing and irradiation showed a synergistic effect leading to reduced cell survival.

Micronucleus formation kinetics in buccal mucosa cells of head and neck cancer patients undergoing radiotherapy.

In head and neck cancer, radiotherapy is one of the main treatment modalities besides surgery and chemotherapy either in a primary or an adjuvant setting. Radiation kills tumor cells by damaging the DNA within these cells. One of the methods to assess the degree of genomic damage is the micronucleus (MN) test. The effect of radiation therapy on the MN frequency in buccal mucosa cells has only been investigated in small studies looking at single time points or including a limited number of patients. In the present study, normal tissue buccal mucosa cells from 17 patients were analyzed for genomic damage at four different time points during radiation therapy. A clear increase was observed for every time point. Additionally, buccal mucosa cells of a cohort of 16 patients were analyzed after the end of the therapy and compared to samples from 25 patients sampled before the therapy. 10 healthy controls were included, of which 5 were sampled once, and 5 were sampled four times similar to the patients. Also, the influence of additional chemotherapy was investigated. No difference was observed between radiation-only patients and patients receiving additional chemotherapy. Age, gender, and tumor stage did not have an influence on the MN formation kinetics.

Radiosensitizing effect of the novel Hsp90 inhibitor NVP-AUY922 in human tumour cell lines silenced for Hsp90α.

BACKGROUND: Hsp90 inhibitors can enhance the tumour sensitivity to ionising radiation (IR). However, Hsp90 inhibition leads to the up-regulation of anti-apoptotic Hsp90 and Hsp70, which might diminish the radiosensitizing effects of the inhibitors. Therefore, inhibition of the up-regulation of Hsp90 by siRNA might be a promising strategy to enhance drug-mediated radiosensitization. MATERIALS AND METHODS: The expression of Hsp90α was silenced in A549 and GaMG tumour cell lines by siRNA treatment. Pre-silenced for Hsp90α cells were treated with NVP-AUY922, a novel Hsp90 inhibitor, for 24 h and then irradiated. Radiation response was determined by colony-forming ability. The expression of several marker proteins was analysed by Western blot. DNA damage and repair were assessed by histone γH2AX measurements. RESULTS: We found that transfection with siRNA against Hsp90α reduced Hsp90α at mRNA and protein levels. Pre-silencing of Hsp90α reduced NVP-AUY922-mediated up-regulation of Hsp90α but it did not increase drug-mediated radiosensitization in both tumour cell lines. As revealed by Western blot, pre-silencing of Hsp90α followed by NVP-AUY922 did not change the expression of Hsp90 client proteins (Akt, Raf-1, Cdk1 and Cdk4) compared with drug treatment alone, suggesting unchanged chaperone function in transfected cells. CONCLUSION: Pre-silencing of Hsp90α followed by Hsp90 inhibition did not enhance the radiosensitizing effect of NVP-AUY922 in both tested tumour cell lines. Future work will be done on stable transfection with shRNA against Hsp90α or simultaneous silencing of both Hsp90 isoforms, Hsp90α and Hsp90ß, in order to optimize tumour cell killing.

Novel HSP90 inhibitors, NVP-AUY922 and NVP-BEP800, radiosensitise tumour cells through cell-cycle impairment, increased DNA damage and repair protraction.

BACKGROUND: Heat-shock protein 90 (Hsp90) has a crucial role in both the stabilisation and regulation of various proteins, including those related to radioresistance. Inhibition of Hsp90 may therefore provide a strategy for enhancing the radiosensitivity of tumour cells. This study explores the responses of four tumour cell lines (A549, GaMG, HT 1080 and SNB19) to combined treatment with ionising radiation (IR) and two novel inhibitors of Hsp90, NVP-AUY922 and NVP-BEP800. The techniques used included cell and colony counts, expression of Hsp90, Hsp70, Akt, survivin, cleaved caspase 3, p53, cell-cycle progression and associated proteins. DNA damage was analysed by histone gammaH2AX and Comet assays. RESULTS: We found that NVP-AUY922 and NVP-BEP800 enhanced radiosensitivity in all tested cell lines. In contrast, only two cell lines (HT 1080 and GaMG) exhibited an increased rate of apoptosis after drug pretreatment, as revealed by western blot. In all tested cell lines, the expression of histone gammaH2AX, a marker of DNA double-strand breaks, after combined drug-IR treatment was higher and its decay rate was slower than those after each single treatment modality. Drug-IR treatment also resulted in impaired cell-cycle progression, as indicated by S-phase depletion and G2/M arrest. In addition, the cell cycle-associated proteins, Cdk1 and Cdk4, were downregulated after Hsp90 inhibition. INTERPRETATION: These findings show that the novel inhibitors of Hsp90 can radiosensitise tumour cell lines of different entities through destabilisation and depletion of several Hsp90 client proteins, thus causing the depletion of S phase and G2/M arrest, increased DNA damage and repair protraction and, to some extent, apoptosis. The results might have important implications for the radiotherapy of solid tumours.

Radiosensitivity in breast cancer assessed by the Comet and micronucleus assays.

Spontaneous and radiation-induced genetic instability of peripheral blood mononuclear cells derived from unselected breast cancer (BC) patients (n=50) was examined using the single-cell gel electrophoresis (Comet) assay and a modified G2 micronucleus (MN) test. Cells from apparently healthy donors (n=16) and from cancer patients (n=9) with an adverse early skin reaction to radiotherapy (RT) served as references. Nonirradiated cells from the three tested groups exhibited similar baseline levels of DNA fragmentation assessed by the Comet assay. Likewise, the Comet analysis of in vitro irradiated (5 Gy) cells did not reveal any significant differences among the three groups with respect to the initial and residual DNA fragmentation, as well as the DNA repair kinetics. The G2 MN test showed that cells from cancer patients with an adverse skin reaction to RT displayed increased frequencies of both spontaneous and radiation-induced MN compared to healthy control or the group of unselected BC patients. Two patients from the latter group developed an increased early skin reaction to RT, which was associated with an increased initial DNA fragmentation in vitro only in one of them. Cells from the other BC patient exhibited a striking slope in the dose-response curve detected by the G2 MN test. We also found that previous RT strongly increased both spontaneous and in vitro radiation-induced MN levels, and to a lesser extent, the radiation-induced DNA damage assessed by the Comet assay. These data suggest that clinical radiation may provoke genetic instability and/or induce persistent DNA damage in normal cells of cancer patients, thus leading to increased levels of MN induction and DNA fragmentation after irradiation in vitro. Therefore, care has to be taken when blood samples collected postradiotherapeutically are used to assess the radiosensitivity of cancer patients.

Normal expression of DNA repair proteins, hMre11, Rad50 and Rad51 but protracted formation of Rad50 containing foci in X-irradiated skin fibroblasts from radiosensitive cancer patients.

About 5% of oncology patients treated by radiation therapy develop acute or late radiotoxic effects whose molecular mechanisms remain poorly understood. In this study, we evaluated the potential role of DNA repair proteins in the hypersensitivity of cancer patients to radiation therapy. The expression levels and focal nuclear distribution of DNA repair proteins, hMre11, Rad50 and Rad51 were investigated in skin fibroblasts strains derived from cancer patients with adverse early skin reaction to radiotherapy using Western blot and foci immunofluorescence techniques, respectively. Cells from cancer patients with normal reaction to radiotherapy as well as cells from apparently healthy subjects served as controls. Cellular radiosensitivity after in vitro irradiation was assessed by the clonogenic survival assay. The clonogenic survival assay and Western blot analysis of the DNA repair proteins did not reveal any abnormalities in cellular radiosensitivity in vitro and in protein expression levels or their migration patterns in the fibroblasts derived from cancer patients with hypersensitive reaction to radiotherapy. In contrast, in vitro irradiated cells from radiosensitive patients exhibited a significantly higher number of nuclei with focally concentrated Rad50 protein than in both control groups. The observed alteration of the distribution of radiation-induced Rad50 foci in cells derived from cancer patients with acute side reactions to radiotherapy might contribute to their radiation therapy outcome. These data suggest the usefulness of the Rad50 foci analysis for predicting clinical response of cancer patients to radiotherapy.

Light scatter and DNA accessibility to propidium iodide of ataxia telangiectasia and fanconi anemia cells.

Cells from individuals with genetic diseases ataxia telangiectasia (AT) and Fanconi anemia (FA) exhibit hypersensitivity to ionizing radiation (AT) or DNA cross-linking agents (FA) which may be caused by multiple factors including defects in chromatin structure and DNA repair. In this study, a combination of cytometric techniques was employed to study the chromatin conformation of AT and FA cells. Nuclei of peripheral blood mononuclear cells (PBMCs) and of skin fibroblasts established from AT and FA patients were analyzed by light scattering and fluorimetric titration with the DNA-intercalating dye propidium iodide. The light scatter measurements revealed the presence of small-sized nuclei with reduced granularity in PBMCs and fibroblasts from both AT and FA patients. The fluorometric titration data could be interpreted by assuming two classes of propidium iodide binding sites with different affinities. The number of high-affinity sites in AT and FA fibroblasts was significantly larger (by 20%) than in control cells. Our findings show the applicability of cytometric techniques for the rapid assessment of chromatin conformation and also suggest the possibility to identify AT and FA carriers.

Response to X-irradiation of Fanconi anemia homozygous and heterozygous cells assessed by the single-cell gel electrophoresis (comet) assay.

Fanconi anemia (FA) is an autosomal recessive disorder characterized by bone marrow failure and cancer susceptibility. Patient cells are sensitive to a variety of clastogens, most prominently cross-linking agents. Although there is the long-standing clinical impression of radiosensitivity, in vitro studies have yielded conflicting results. We exposed peripheral blood mononuclear cells from FA patients and carriers to x-rays and determined their DNA damage and repair profiles using the alkaline single-cell gel electrophoresis (comet) assay. Studies were carried out in two independent series of experiments by two laboratories using different protocols. The cells of both FA patients and carriers showed uniformly high initial DNA damage rates as assessed by the total initial tail moment. In addition, the average residual tail moment at 30 to 50 minutes and the repair half-time parameters were significantly elevated. These findings suggest an increased release of fragmented DNA following x-ray exposure in cells that carry one or two mutations in one of the FA genes. The comet assay may be a useful adjunct for heterozygote detection in families of FA patients.

Identification of ataxia telangiectasia heterozygotes, a cancer-prone population, using the single-cell gel electrophoresis (Comet) assay.

Heterozygotes of ataxia telangiectasia (AT) may comprise up to 1% of the general population. Because these individuals have no clinical expression of AT but may be highly radiosensitive and strongly predisposed for several forms of cancer, identification of AT carriers represents a considerable interest in cancer epidemiology and radiotherapy. We report a new approach for the in vitro identification of AT-heterozygotes based on the evaluation of the radiosensitivity and DNA damage repair ability of peripheral blood mononuclear cells using the single-cell gel electrophoresis (Comet) assay. The assay was performed on cells isolated from four different groups of individuals: (1) apparently healthy donors (n = 10); (2) patients with breast cancer showing a normal reaction to radiotherapy (n = 10); (3) a group of obligate AT carriers (parents of AT-homozygotes, n = 20); and (4) AT-homozygotes (n = 4). Cells irradiated with 3 Gy of x-rays were assayed for three parameters: (1) the initial and (2) residual DNA damage and (3) the kinetics of DNA damage repair. Both AT-heterozygotes' and AT-homozygotes' cells were found to be highly sensitive to x-irradiation. Quantitative evaluation of the single-cell electrophoregrams revealed that the average initial DNA damage in AT-heterozygous and AT-homozygous cells was almost three times higher than that in control non-AT cells. In addition, the DNA repair process in irradiated AT carrier cells was almost three times slower, and the extent of irreparable DNA damage in these cells was three times greater than in controls. Simultaneous assessment of the three parameters enabled correct identification of all tested AT carriers. This method seems to be a sensitive and useful tool for populational studies as a rapid prescreening test for a mutated AT status. The approach can also be extended for prediction of the in vivo radiosensitivity, which would enable optimization of individual radiotherapy schedules.

Effect of medium conductivity and composition on the uptake of propidium iodide into electropermeabilized myeloma cells.

The effects of ionic composition and conductivity of the medium on electropermeabilization of the plasma membrane of mammalian cells were studied. Temporal and spatial uptake of propidium iodide (PI) into field-treated cells was measured by means of flow cytometry, spectrofluorimetry and confocal laser scanning microscopy. Murine myeloma cells were electropulsed in iso-osmolar solutions. These contained 10-100 micrograms ml-1 PI at different conductivities (0.8-14 mS cm-1) and ionic strengths, adjusted by varying concentrations of K+, Na+, Cl- and SO4(2-). Field-induced incorporation of PI into reversibly permeabilized cells was (almost) independent of ionic composition and strength (at a fixed medium conductivity), but increased dramatically with decreasing medium conductivity at a fixed field strength. The time-course of PI uptake (which apparently reflected the resealing process of the membrane) could be fitted by single-exponential curve (relaxation time about 2 min in the absence of Ca2+) and was independent of medium conductivity and composition. These and other data suggested that the expansion of the 'electroleaks' during the breakdown process is field-controlled and depends, therefore, on the (conductivity-dependent) discharging process of the permeabilized membrane. The threshold field strength for dye uptake increased with increasing K+ concentration (about 0.6 kV cm-1 in K(+)-free, NaCl-containing medium and about 0.9 kV cm-1 in 30 mM KCl-containing medium). Also, the spatial uptake pattern of PI shifted from an asymmetric permeation through the cell hemisphere facing the anode to a more symmetric uptake through both hemispheres. These results suggested that the generated potential is superimposed on the (K(+)-dependent) resting membrane potential. Taking this into account, the breakdown voltage of the membrane was estimated to be about 1 V.

Electropermeabilization and fluorescent tracer exchange: the role of whole-cell capacitance.

Transmembrane crossing of charged fluorescent tracers such as propidium iodide (PI) and carboxyfluorescein+ (CF) can be used to quantitate membrane permeabilization. Murine myeloma Sp2/0-Ag14 cells were loaded with CF (0.1 fmol/cell) before electropulsation (0.5-3.0 kV/cm, 40 microseconds) in medium containing 25-50 micrograms/ml PI at 21-23 degrees C. Cytograms of PI vs. CF fluorescence showed three readily distinguishable subpopulations: 1) intact living cells with CF but without PI (these form > 95% of the prepulsed population), 2) transiently electropermeabilized but resealed cells showing both CF and low-level PI fluorescence, and 3) permanently permeabilized cells without CF but with very high PI fluorescence. Despite the ready influx of PI, the efflux of CF from transiently permeabilized cells was negligible and was insensitive to pulse parameters; however, electrically killed cells (subpopulation 3) lost all CF fluorescence and probably lost their cytoplasm. This difference in transmembrane passage of the dyes is best explained by binding of intracellular CF to macromolecules (and/or organelles). In isotonic "pulse medium," the membranes resealed after electropulsing with a time constant (tau R) of about 2 min. In 150 mOsm medium, resealing was faster (typically tau R approximately 0.5 min). The population distribution of PI uptake [coefficient of variation (CV) > 40%] was very broad and could not be accounted for by the radius dependence of pulse-induced voltage (CVradius approximately 10%). The variability in PI uptake could be explained if the electrical energy of the charged membrane, which depends on the whole-cell capacitance (Cc), was taken into account. Evaluation of the Cc values with single-cell resolution was based on measurement of the electrical charging time constant of the plasma membrane by electrorotation.

DNA, protein, and plasma-membrane incorporation by arrested mammalian cells.

Incorporation of DNA, protein, and plasma membrane during blockage by aphidicolin or by doxorubicin was studied by flow cytometry and electrorotation of three cell lines (mouse-myeloma Sp2/0-Ag14, hybridoma H73C11, and fibroblast-like L929 cells). Drug-mediated arrest at the G1-S boundary (aphidicolin) or in G2/M (doxorubicin) did not arrest synthesis of either protein or total membrane area, the increases in which outstripped growth in cell volume and apparent cell area, respectively. Measurements of membrane capacity in normal and hypo-osmotic media showed that the drugs had not changed the fundamental bilayer, but that an increase in the number or size of microvilli must have occurred. Aphidicolin-arrested cells withstood hypo-osmotic stress better than untreated cells could, indicating that the membrane excess can be utilized as a reserve during rapid cell expansion. Hypo-osmotically treated cell populations exhibited only about half the coefficient of variance (CV) in membrane properties of cells at physiological osmolality. Populations of arrested cells exhibited the same high CV as asynchronous cells, indicating that chemical arrest does not give uniformly villated cell populations. However, the lowest CV values were given by some synchronized (aphidicolin-blocked, then released) populations. Removal of aphidicolin allowed most cells to progress through S and G2, and then divide. During these processes, the membrane excess was reduced. After removal of doxorubicin, the cells did not divide: some continued protein synthesis, grew abnormally large, and further increased their membrane excess. Membrane breakdown by electric pulsing (3 x 5kV/cm, 40 microseconds decay time) of aphidicolin-synchronized L cells in G2/M led to a 22% loss of plasma membrane (both the area-specific and the whole-cell capacitance were reduced), presumably via endocytosis-like vesiculation.

Effect of electric field pulses on the viability and on the membrane-bound immunoglobulins of LPS-activated murine B-lymphocytes: correlation with the cell cycle.

The effects of microsecond electropulses (1-5 kV/cm) on the viability of murine B lymphocytes and on their binding of antibodies by surface immunoglobulin (Ig) were studied in relation to the cell cycle. Before electropulsing, cultures given 48 h mitogenic stimulation showed at least two cell subpopulations, which were distinguishable by their levels of surface-Ig expression as assessed with FITC-labelled antibodies against mouse Ig. The immunofluorescence intensity of cells in S and G2/M phases was higher than that of G0/G1 cells. After exposure of the mitogen-stimulated lymphocytes to three exponentially decaying (time constant tau = 5-40 microseconds) electric field pulses, dye exclusion assay showed that pulsing at 1 or 2 kV/cm (at 4 degrees C or 20 degrees C) did not cause permeabilization. Field strengths of 3, 4, or 5 kV/cm resulted in 20%, 45%, or 70% of dye-permeable cells, respectively, if the pulsed cells were transferred to phosphate-buffered saline on ice for 30 min. Incubation in full medium at 37 degrees C for 30 min ("resealing") significantly decreased the percentage of permeabilized cells. Electropulsed G0/G1 cells were not only more resistant to direct electric exposure (tolerated higher field strengths) than S + G2/M cells but also responded better to resealing. The surface Ig of lymphocytes pulsed at higher fields and low temperature (4 or 5 kV/cm, tau = 5 microseconds, three pulses, 4 degrees C) was less easily immunostained than in controls or in cells pulsed at 2 kV/cm or less. At 5 kV/cm those cells that were not permeabilized showed a greater reduction in immunostaining, especially if resealed.