Tob1 enhances radiosensitivity of breast cancer cells involving the JNK and p38 pathways.

The aim of the present study was to evaluate the effect of Tob1 on the radiosensitivity of breast cancer cells. The results showed that overexpression of Tob1 reduced the clonogenic growth of 231 cells and induced the rate of apoptosis. Tob1 caused an accumulation of cells in the G0 /G1 phase and decreased the percentage of cells in S phase. We also found that overexpression of Tob1 significantly reduced the phosphorylation of JNK and p38. The activator of JNK and p38, anisomycin, attenuated the blockage of Tob1 on the cell cycle and reversed the effect of Tob1 on apoptosis. Taken together, Tob1 enhanced radiosensitivity of breast cancer cells through regulation of the JNK and p38 pathways. The results indicated that Tob1 might be a promising molecular in gene therapy for the treatment of breast cancer.

Comparison of UVB and UVC effects on the DNA damage-response protein 53BP1 in human pancreatic cancer.

We have previously demonstrated that ultraviolet (UV) light is effective against a variety of cancer cells expressing fluorescent proteins in vivo as well as in vitro. In the present report, we compared the DNA damage repair (DDR) response of pancreatic cancer cells after UVB or UVC irradiation. The UV-induced DNA damage repair was imaged with green fluorescent protein (GFP) fused to the DDR-related chromatin-binding protein 53BP1 in MiaPaCa-2 human pancreatic cancer cells growing in 3D Gelfoam® histoculture and in superficial tumors grown in nude mice. 53BP1-GFP forms foci during DNA damage repair. A clonogenic assay in 2D monolayer culture initially showed that UVC and UVB inhibited MiaPaCa-2 cell proliferation in a dose-dependent manner, with UVC having more efficacy. Three-dimensional Gelfoam® histocultures and confocal imaging enabled 53BP1-GFP foci to be observed within 1 h after UV irradiation, indicating the onset of DDR response. UVB-induced 53BP1-GFP focus formation was observed up to a depth of 120 µm in MiaPaCa-2 cells on Gelfoam® compared to 80 µm for UVC. UVB-induced 53BP1-GFP focus formation was observed up to a depth of 80 µm in MiaPaCa-2 cells, implanted within skin flaps in mice, at a significantly greater extent than UVC. MiaPaCa-2 cells irradiated by UVB or UVC in the skin-flap mouse model had a significant decrease in tumor growth compared to untreated controls with UVB having more efficacy than UVC. Our results demonstrate that UVB has greater tissue penetration than UVC because of its longer wavelength and has clinical potential for eradicating superficial cancer.

[DNA double-strand breaks in human lymphocytes after single irradiation by low doses of pulsed X-rays: non-linear dose-response relationship].

Effects of ionizing radiation registered in cells after low dose irradiation are still poorly understood. A pulsed mode of irradiation is even more problematic in terms of predicting the radiation-induced response in cells. Thus, the aim of this paper was to study and analyze the effects of dose and frequency of pulsed X-rays on the frequency of radiation-induced DNA double-strand breaks and their repair kinetics in human peripheral blood lymphocytes in vitro. Analysis of radiation-induced gammaH2AX and 53BP1 repair foci was used to assess the DNA damage in these cells. The dose-response curve of radiation-induced foci of both proteins has shown deviations from linearity to a higher effect in the 12-32 mGy dose range and a lower effect at 72 mGy. The dose-response curve was linear at doses higher than 100 mGy. The number of radiation-induced gammaH2AX and 53BP1 foci depended on the frequency of X-ray pulses: the highest effect was registered at 13 pulses per second. Moreover, slower repair kinetics was observed for those foci induced by very low doses with a nonlinear dose-response relationship.

Development of the light sensitivity of the clock genes Period1 and Period2, and immediate-early gene c-fos within the rat suprachiasmatic nucleus.

The molecular mechanism underlying circadian rhythmicity within the suprachiasmatic nuclei (SCN) of the hypothalamus has two light-sensitive components, namely the clock genes Per1 and Per2. Besides, light induces the immediate-early gene c-fos. In adult rats, expression of all three genes is induced by light administered during the subjective night but not subjective day. The aim of the present study was to ascertain when and where within the SCN the photic sensitivity of Per1, Per2 and c-fos develops during early postnatal ontogenesis. The specific aim was to find out when the circadian clock starts to gate photic sensitivity. The effect of a light pulse administered during either the subjective day or the first or second part of the subjective night on gene expression within the rat SCN was determined at postnatal days (P) 1, 3, 5 and 10. Per1, Per2 and c-fos mRNA levels were assessed 30 min, 1 and 2 h after the start of each light pulse by in situ hybridization histochemistry. Expression of Per1 and c-fos was light responsive from P1, and the responses began to be gated by the circadian clock at P3 and P10, respectively. Expression of Per2 was only slightly light responsive at P3, and the response was not fully gated until P5. These data demonstrate that the light sensitivity of the circadian clock develops gradually during postnatal ontogenesis before the circadian clock starts to control the response. The photoinduction of the clock gene Per2 develops later than that of Per1.

Cytoplasmic irradiation induces mitochondrial-dependent 53BP1 protein relocalization in irradiated and bystander cells.

The accepted paradigm for radiation effects is that direct DNA damage via energy deposition is required to trigger the downstream biological consequences. The radiation-induced bystander effect is the ability of directly irradiated cells to interact with their nonirradiated neighbors, which can then show responses similar to those of the targeted cells. p53 binding protein 1 (53BP1) forms foci at DNA double-strand break sites and is an important sensor of DNA damage. This study used an ionizing radiation microbeam approach that allowed us to irradiate specifically the nucleus or cytoplasm of a cell and quantify response in irradiated and bystander cells by studying ionizing radiation-induced foci (IRIF) formation of 53BP1 protein. Our results show that targeting only the cytoplasm of a cell is capable of eliciting 53BP1 foci in both hit and bystander cells, independently of the dose or the number of cells targeted. Therefore, direct DNA damage is not required to trigger 53BP1 IRIF. The use of common reactive oxygen species and reactive nitrogen species (RNS) inhibitors prevent the formation of 53BP1 foci in hit and bystander cells. Treatment with filipin to disrupt membrane-dependent signaling does not prevent the cytoplasmic irradiation-induced 53BP1 foci in the irradiated cells, but it does prevent signaling to bystander cells. Active mitochondrial function is required for these responses because pseudo-rho(0) cells, which lack mitochondrial DNA, could not produce a bystander signal, although they could respond to a signal from normal rho+ cells.

Differential redistribution of native AMPA receptor complexes following LTD induction in acute hippocampal slices.

AMPAR trafficking is crucial for the expression of certain forms of synaptic plasticity. Here, using surface biotinylation of hippocampal slices and subsequent synaptosome isolation we assessed AMPAR surface expression in synaptosomes following NMDA-evoked long-term depression (NMDA-LTD). Surface levels of GluR1, GluR2 and GluR3 in synaptosomes were markedly reduced 90 min after NMDA-LTD induction. Consistent with endocytosis and degradation, whole-cell surface and total expression levels of GluR2 and GluR3 were also reduced. In contrast, whole-cell surface levels of GluR1 were unaltered at 90 min suggesting that AMPARs with different subunit composition are redistributed to different non-synaptic compartments following LTD induction in acute hippocampal slices.

Anti-tax interacting protein-1 (TIP-1) monoclonal antibody targets human cancers.

Radiation-inducible neo-antigens are proteins expressed on cancer cell surface after exposure to ionizing radiation (IR). These neo-antigens provide opportunities to specifically target cancers while sparing normal tissues. Tax interacting protein-1 (TIP-1) is induced by irradiation and is translocated to the surface of cancer cells. We have developed a monoclonal antibody, 2C6F3, against TIP-1.Epitope mapping revealed that 2C6F3 binds to the QPVTAVVQRV epitope of the TIP-1 protein. 2C6F3 binds to the surface of lung cancer (A549, LLC) and glioma (D54, GL261) cell lines. 2C6F3 binds specifically to TIP-1 and ELISA analysis showed that unconjugated 2C6F3 efficiently blocked binding of radiolabeled 2C6F3 to purified TIP-1 protein. To study in vivo tumor binding, we injected near infrared (NIR) fluorochrome-conjugated 2C6F3 via tail vein in mice bearing subcutaneous LLC and GL261 heterotopic tumors. The NIR images indicated that 2C6F3 bound specifically to irradiated LLC and GL261 tumors, with little or no binding in un-irradiated tumors.We also determined the specificity of 2C6F3 to bind tumors in vivo using SPECT/CT imaging. 2C6F3 was conjugated with diethylene triamine penta acetic acid (DTPA) chelator and radiolabeled with 111Indium (111In). SPECT/CT imaging revealed that 111In-2C6F3 bound more to the irradiated LLC tumors compared to un-irradiated tumors. Furthermore, injection of DTPA-2C6F3 labeled with the therapeutic radioisotope, 90Y, (90Y-DTPA-2C6F3) significantly delayed LLC tumor growth. 2C6F3 mediated antibody dependent cell-mediated cytotoxicity (ADCC) and antibody dependent cell-mediated phagocytosis (ADCP) in vitro.In conclusion, the monoclonal antibody 2C6F3 binds specifically to TIP-1 on cancer and radio-immunoconjugated 2C6F3 improves tumor control.

Tissue-engineered mucosa is a suitable model to quantify the acute biological effects of ionizing radiation.

The aim of this study was to evaluate the suitability of tissue-engineered mucosa (TEM) as a model for studying the acute effects of ionizing radiation (IR) on the oral mucosa. TEM and native non-keratinizing oral mucosa (NNOM) were exposed to a single dose of 16.5Gy and harvested at 1, 6, 24, 48, and 72h post-irradiation. DNA damage induced by IR was determined using p53 binding protein 1 (53BP1), and DNA repair was determined using Rad51. Various components of the epithelial layer, basement membrane, and underlying connective tissue were analyzed using immunohistochemistry. The expression of cytokines interleukin-1ß (IL-1ß) and transforming growth factor beta 1 (TGF-ß1) was analyzed using an enzyme-linked immunosorbent assay. The expression of DNA damage protein 53BP1 and repair protein Rad51 were increased post-irradiation. The expression of keratin 19, vimentin, collage type IV, desmoglein 3, and integrins α6 and ß4 was altered post-irradiation. Proliferation significantly decreased at 24, 48, and 72h post-irradiation in both NNOM and TEM. IR increased the secretion of IL-1ß, whereas TGF-ß1 secretion was not altered. All observed IR-induced alterations in TEM were also observed in NNOM. Based on the similar response of TEM and NNOM to IR we consider our TEM construct a suitable model to quantify the acute biological effects of IR.

Effect of radiation on the Notch signaling pathway in osteoblasts.

Notch signaling has been shown to be important in osteoblast differentiation. Therapeutic radiation has been shown to alter the skeletal system, yet little information is available on the changes in Notch signaling in irradiated osteoblasts. The purpose of this study was to analyze the effect of radiation therapy with 2 and 4 Gy on Notch signaling in osteoblasts. In order to assess the radiation damage on osteoblast differentiation, total RNA and protein were collected three days after exposure to radiation. The effects of radiation on Notch signaling at the early and terminal stages of osteoblastic MC3T3-E1 cell differentiation was analyzed by qRT-PCR and western blot analysis. Our study applied a previously established method to induce MC3T3-E1 cell differentiation into osteoblasts and osteoblast precursors. Our results showed that the expression of Notch receptors (Notch1-4), ligands (Jagged1, Jagged2 and Delta1), target of Notch signaling (Hes1) and markers (ALP, M-CSF, RANKL and OPG) were altered following 2 and 4 Gy of irradiation. The present research did not indicate a strong relationship between Notch1 regulation and suppression of osteoblast differentiation. We found Hes1 may play a role in the radiation effect on osteoblast differentiation. Our results indicate that radiated osteoblast precursors and osteoblasts promoted osteoclast differentiation and proliferation.

Light-induced COP9 signalosome expression in the Indian false vampire bat Megaderma lyra.

The COP9 signalosome (CSN) is a multi-subunit protein complex conserved in plants and animals. CSN subunits have been identified as light-mediated master regulators of eukaryotic circadian clocks from fungi to animals. The Indian false vampire bat Megaderma lyra is completely adapted to an anthropic biotope and behavioral studies have reported that M. lyra exhibits light-sampling behavior to assess environmental light. LC-MS-MS results for a 36 kDa protein were analyzed using the Sequest search engine, and COP9 signalosome subunit 5 (CSN5) was pinpointed as having the highest score with 6 matching peptides. To confirm the presence of CSN5, up-regulated cDNA was amplified, sequenced, and identified as CSN5. Furthermore, semi-quantitative RT-PCR analysis demonstrated that the level of induction of CSN5 was regulated by environmental light. We estimated the level of expression across a light-dark cycle and observed a higher level of expression at the end of the light phase. Similarly, when the animal was shifted from continuous dark to light, CSN5 expression was induced. Correspondingly, we detected the similar pattern of translated protein with JAB1 antibody. Knowledge about the circadian rhythm and its molecular mechanism in Chiroptera is very limited and this study suggests that CSN5 might be involved in the M. lyra light-signaling process.

Microwaves from Mobile Phones Inhibit 53BP1 Focus Formation in Human Stem Cells More Strongly Than in Differentiated Cells: Possible Mechanistic Link to Cancer Risk.

BACKGROUND: It is widely accepted that DNA double-strand breaks (DSBs) and their misrepair in stem cells are critical events in the multistage origination of various leukemias and tumors, including gliomas. OBJECTIVES: We studied whether microwaves from mobile telephones of the Global System for Mobile Communication (GSM) and the Universal Global Telecommunications System (UMTS) induce DSBs or affect DSB repair in stem cells. METHODS: We analyzed tumor suppressor TP53 binding protein 1 (53BP1) foci that are typically formed at the sites of DSB location (referred to as DNA repair foci) by laser confocal microscopy. RESULTS: Microwaves from mobile phones inhibited formation of 53BP1 foci in human primary fibroblasts and mesenchymal stem cells. These data parallel our previous findings for human lymphocytes. Importantly, the same GSM carrier frequency (915 MHz) and UMTS frequency band (1947.4 MHz) were effective for all cell types. Exposure at 905 MHz did not inhibit 53BP1 foci in differentiated cells, either fibroblasts or lymphocytes, whereas some effects were seen in stem cells at 905 MHz. Contrary to fibroblasts, stem cells did not adapt to chronic exposure during 2 weeks. CONCLUSIONS: The strongest microwave effects were always observed in stem cells. This result may suggest both significant misbalance in DSB repair and severe stress response. Our findings that stem cells are most sensitive to microwave exposure and react to more frequencies than do differentiated cells may be important for cancer risk assessment and indicate that stem cells are the most relevant cellular model for validating safe mobile communication signals.

An alternative mechanism for radioprotection by dimethyl sulfoxide; possible facilitation of DNA double-strand break repair.

The radioprotective effects of dimethyl sulfoxide (DMSO) have been known for many years, and the suppression of hydroxyl (OH) radicals induced by ionizing radiation has been thought to be the main cause of this effect. However, the DMSO concentration used was very high, and might be toxic, in earlier studies. In the present study, we administered a lower, non-toxic concentration (0.5%, i.e., 64 mM) of DMSO before irradiation and examined its radioprotective effects. Colony formation assay and micronucleus assay showed significant radioprotective effects in CHO, but not in xrs5, which is defective in the repair function of DNA double-strand breaks. The levels of phosphorylated H2AX and the formation of 53BP1 foci 15 minutes after irradiation, which might reflect initial DNA double-strand breaks, in DMSO-treated CHO cells were similar to those in non-treated cells, suggesting that the radioprotective effects were not attributable to the suppression of general indirect action in the lower concentration of DMSO. On the other hand, 2 hours after irradiation, the average number of 53BP1 foci, which might reflect residual DNA double-strand breaks, was significantly decreased in DMSO-treated CHO cells compared to non-treated cells. The results indicated that low concentration of DMSO exerts radioprotective effects through the facilitation of DNA double-strand break repair rather than through the suppression of indirect action.

DNA double-strand break signalling: X-ray energy dependence of residual co-localised foci of gamma-H2AX and 53BP1.

PURPOSE: The application of ionising radiation for medical purposes requires the investigation of induced and persistent DNA damages, especially for soft X-rays that are assumed to be more effective than higher energy photons. Therefore, we examined the energy dependent time and dose response of residual DNA damage foci for soft X-rays in comparison to 200 kV photons. MATERIALS AND METHODS: DNA damage present in cell line 184A1 within 48 h after irradiations with 10 kV, 25 kV and 200 kV photons was analysed by immunochemical detection of co-localised gamma-H2AX (phosphorylated histone H2AX) and 53BP1 (tumour protein 53 binding protein) foci. RESULTS: The dose dependencies of the colocated foci revealed significant energy dependent differences with increasing amounts of residual foci at decreasing X-ray energy independent on postirradiation time. Dose-dependent RBE (relative biological effectiveness) values ranging from 4 to 7 were determined for 10 kV relative to 200 kV X-rays based on the 24 hour dose responses. For 25 kV photons, ratios considerably higher than one were obtained only for doses above 2 Gy. CONCLUSIONS: The expected energy dependence with increasing DNA damage at decreasing photon energy was confirmed for the residual co-localised foci measured at different time points after irradiation.

Dose response and kinetics of foci disappearance following exposure to high- and low-LET ionizing radiation.

PURPOSE: The effect of different radiation qualities on (i) 53BP1 (p53 Binding Protein 1) and p-ATM (phosphorylated ataxia telangiectasia mutated) foci induction, and (ii) on the kinetics of foci disappearance was analysed. MATERIAL AND METHODS: Normal human skin fibroblasts were exposed to 240 kV broad-field X-rays or targeted with individually counted helium ((3)He) particles or protons ((1)H) from a Charged Particle Microbeam. Anti-p-ATM and anti-53BP1 antibodies were used for foci visualisation via immunocytochemistry. RESULTS: 1 Gy of X-rays yielded approximately 33 53BP1-positive foci/cell. The ratio between the number of delivered particles and yielded tracks was found to be 1:1 and 3:1 after targeted (3)He and (1)H irradiation, respectively. It was determined that approximately 50% of radiation-induced damage was repaired as measured by loss of foci during the first 2, 6, and 10 hours following X-ray, protons, and (3)He irradiation, respectively. CONCLUSIONS: There was significant radiation quality dependence for 53BP1- and p-ATM-positive foci induction observed. Foci disappearance was radiation dose-independent in the samples irradiated with X-rays. Our results confirm that kinetics of foci disappearance depends on radiation quality, even when individual ions are targeted to cells.