Cranial irradiation alters the behaviorally induced immediate-early gene arc (activity-regulated cytoskeleton-associated protein).

Therapeutic irradiation of the brain is commonly used to treat brain tumors but can induce cognitive impairments that can severely affect quality of life. The underlying mechanisms responsible for radiation-induced cognitive deficits are unknown but likely involve alterations in neuronal activity. To gain some mechanistic insight into how irradiation may affect hippocampal neurons known to be associated with cognitive function, we quantitatively assessed the molecular distribution of the behaviorally induced immediate-early gene Arc (activity-regulated cytoskeleton-associated protein) at the level of mRNA and the protein. Young adult C57BL/6J mice received whole-brain irradiation with 0 or 10 Gy, and 1 week or 2 months later, exploration of a novel environment was used to induce Arc expression. The fractions of neurons expressing Arc mRNA and Arc protein were detected using fluorescence in situ hybridization and immunocytochemistry, respectively. Our results showed that there was a significant reduction in the percentage of neurons expressing Arc protein 1 week after irradiation, whereas 2 months after irradiation, there was a reduction in the percentage of neurons expressing both Arc mRNA and Arc protein. Importantly, radiation-induced changes in Arc expression were not a result of neuronal cell loss. The changes observed at 2 months were associated with a significant increase in the number of activated microglia, supporting the idea that inflammation may contribute to neuronal dysfunction. These findings are the first to show that local brain irradiation initiates changes in hippocampal neurons that disrupt the activity patterns (Arc expression) associated with neuroplasticity and memory.

Altered expression of beta-catenin during radiation-induced colonic carcinogenesis.

Radiotherapy for malignant pelvic disease is commonly accompanied by treatment-induced proctitis, and rarely by colorectal cancer. Translocation of the beta-catenin protein, which is a key downstream effector of the Wnt signal transduction pathway, is frequently found in colorectal cancer. Nuclear beta-catenin enhances transcriptional activity of the cyclin D1 gene in cancer cells. Here, we evaluate the involvement of the Wnt pathway in radiation-induced colon carcinogenesis with rats (n = 36). Beta-catenin, APC, and cyclin D1 expression profiles were analyzed by immunohistochemistry in radiation-induced chronic colon injury including cancers and ulcerative lesions in rats (n = 12 in treated group, n = 12 in control group). In total, 3 cases of invasive adenocarcinomas were developed in the irradiated portion 50 weeks after a single dose of 36 Gy irradiation. Nuclear translocation of beta-catenin was observed in all radiation-induced colon cancers, whereas this protein was also found in the cytoplasm and/or nucleus of 9 cases of non-neoplastic irradiated colonocytes. Nuclear translocation of beta-catenin correlated with loss of APC and gain of cyclin D1 expression, suggesting activation of the Wnt pathway during radiation-induced colorectal carcinogenesis. A single dose of 10 Gy was also given for acute injury (n = 12: 3 each in days 0, 3, 5, and 7, respectively). Beta-catenin expression was distributed in the cytoplasm of degenerating glands at day 3 and 5, and was observed in the cell membrane of those glands with histological normalization at day 7 after irradiation. Because translocation of beta-catenin was found in irradiated-colonic mucosa as well as colon cancer, disruption of beta-catenin expression might be one of the early events in radiation-induced colonic carcinogenesis.

Micro-scale chromophore-assisted laser inactivation of nerve growth cone proteins.

Directed growth cone movement is crucial for the correct wiring of the nervous system. This movement is governed by the concerted actions of cell surface receptors, signaling proteins, cytoskeleton-associated molecules, and molecular motors. In order to investigate the molecular basis of growth cone motility, we applied a new technique to functionally inactivate proteins: micro-scale Chromophore-Assisted Laser Inactivation [Diamond et al. (1993) Neuron 11:409-421]. Micro-CALI uses laser light of 620 nm, focused through microscope optics into a 10-microm spot. The laser energy is targeted via specific Malachite green-labeled, non-function-blocking antibodies, that generate short-lived protein-damaging hydroxyl radicals [Liao et al. (1994) Proc Natl Acad Sci USA 91:2659-2663]. Micro-CALI mediates specific loss of protein function with unachieved spatial and temporal resolution. Combined with time-lapse video microscopy, it offers the possibility to induce and observe changes in growth cone dynamics on a real time base. We present here the effects of the acute and localized inactivation of selected growth cone molecules on growth cone behavior and morphology. Based on our observations, we propose specific roles for these proteins in growth cone motility and neurite outgrowth.

Effect of radiation on the expression of E-cadherin and alpha-catenin and invasive capacity in human lung cancer cell line in vitro.

PURPOSE: To investigate the effect of radiation on E-cadherin and alpha-catenin expression in a human lung cancer cell line, and also evaluate invasive capacity in the membrane invasion culture system using the Boyden Chamber. MATERIALS AND METHODS: The immunoblot and immunofluorescence analyses were performed using the human lung cancer cell line A549 to examine altered expression of E-cadherin and alpha-catenin after irradiation. We also compared invasive capacity of untreated cells with that of irradiated cells. RESULTS: Immunoblot analysis revealed that the expression of E-cadherin increased after irradiation. In a time-course analysis, the expression was increased 6 h after irradiation with 10 Gy and reached its peak level at 24 h, being 2.3 times the control value, whereas expression at 1 and 3 h after irradiation was almost equivalent to that of the control. A slight increase in expression was observed after irradiation of 2 Gy and the expression reached peak levels after 5 Gy. After fractionated irradiation, the increase in expression of both E-cadherin and alpha-catenin was observed, and the alteration of alpha-catenin was more prominent than that after a single irradiation of the same total dose. In the immunofluorescence study for E-cadherin antibody analyzed by confocal laser scanning microscopy, increased intensity in irradiated cells produced as a nondisrupted and continuous line at cell-cell contact sites. In an invasive assay, the number of migrated cells in irradiated cells after a dose of 5 and 10 Gy was reduced significantly compared to untreated cells. CONCLUSION: The results indicate that irradiation of A549 increased the expression of E-cadherin, possibly preserving their functional property.

Comparative response to UV irradiation of cytoskeletal elements in rabbit and skate lens epithelial cells.

PURPOSE: This work reports a differential effect of ultraviolet A (UVA) irradiation on the three major cytoskeletal structures, actin and vimentin filaments and microtubules of lens cells in primary culture. The effect on cells from lens of the skate (a bottom-dwelling marine elasmobranch) was compared with that on rabbit lens, in order to assess UVA sensitivity as a function of exposure to these wavelengths in the native habitat. METHODS: Exposure intervals of irradiation time up to 6 hours were selected, at fluences from 13.5 to 54.4 J/cm2 and at 365 +/- 45 nm wavelength, to represent mild to moderate physiological levels. Cultures were fixed and processed with anti-alpha-tubulin-FITC and rhodamine phalloidin, or with anti-vimentin FITC and rhodamine phalloidin conjugates. RESULTS: With epifluorescence microscopy, it was found that microtubules were most sensitive to UVA irradiation (in depolymerizing), followed by actin, with vimentin hardly at all affected. Irradiation for 6 hours followed by incubation for 3 days in fresh medium showed no recovery of actin but good recovery of microtubule organizing centers, followed by mitosis in many (rabbit) cells. Skate cells were more sensitive and showed no recovery. CONCLUSIONS: In view of the role of cytoskeletal elements in intracellular structure, cell division and transport, their disruption supports the hypothesis that UVA may damage lens epithelial cells in vivo so as to contribute to cataract formation. In addition, the data suggest that the lenses of animals exposed to sunlight require effective cytoskeletal repair mechanisms to avoid loss of function.

[Antimitotic compounds as modifiers of the radiation lesion of cultured cells]. / Antimitoticheskie soedineniia kak modifikatory luchevogo porazheniia kul'tiviruemykh kletok.

The features of antimitotic substances as radioprotectors were studied. In vitro experiments have demonstrated that taxol revealed radioprotective features concerning the process of polymerization of irradiated microtubules. These results were the basis for the use of taxol and some other substances with high affinity for cytoskeleton proteins as potential radiomodificators in vivo. Experiments with cultivated fibroblasts revealed that colchicine significantly enhances radioactive injuries of cells while taxol and phalloidin manifest their radioprotective features.

Effect of low-power He-Ne laser on deformability of stored human erythrocytes.

This study was designed to investigate the effect of the He-Ne laser (continuous wave, lambda = 632.8 nm, 8.5 mW in power) irradiation on human erythrocyte deformability. Blood samples were obtained from hematologically normal adult donors by venipuncture. Red cells were washed and adjusted to 30% Ht with 0.9% NaCl solution (pH 7.00). Red cell solution samples were assigned to three groups. Each sample was divided into seven 3-ml working aliquots. The aliquots in Group I were irradiated for 0 (control), 1, 3, 5, 10, 15, and 30 min within 2 hr after sampling. The aliquots in Group 2 and Group 3 were stored at 5 degrees C for 24 and 36 hr, respectively, and received similar irradiations after 12 hr (in both groups), 24 hr (in Group 2), and 36 hr (in Group 3) from sampling. Red cell deformability was measured by the Nuclepore filter filtration and presented as the filter filtration rate (FFR). The deformability shown as FFR was unchanged in Group 1 (fresh cell group) from the control value, but improved significantly in Groups 2 and 3 (damaged cell groups) after the irradiation. These results suggested that the irradiation of low-powered He-Ne lasers improved cytoskeletal protein activities in damaged erythrocytes.

[Protein phosphorylation during mast cell secretion in radioprophylaxis]. / Fosforilirovanie belkov v protsesse sekretsii tuchnykh kletok v usloviiakh radioprofilaktiki.

A study was made of the role of protein phosphorylation of mast cells and their cytoskeleton upon secretion induced by biogenic amines (histamine and serotonin) and bradykinin, a possible mediator of the effect of MEA, a sulfur-containing radioprotector. The data obtained indicate that the incorporation of phosphate in some proteins of mast cells is an important stage in the process of exocytosis during radioprophylaxis. Cytoskeletal proteins were shown to be involved in mast cell secretion.

Effects of gamma-irradiation on the erythrocyte membrane: ESR, NMR and biochemical studies.

The effects of gamma-irradiation on resealed erythrocyte ghosts have been examined with different techniques. Phospholipid analysis reveals peroxidative damage on the polyunsaturated chains of phosphatidylethanolamine. Gel electrophoresis and ESR measurements indicate modifications of the cytoskeletal proteins. 31P Nuclear magnetic resonance data show bilayer modifications that can be interpreted as changes in lipid-protein interactions. The overall picture from the present results favours interaction between lipids and proteins in the inner monolayer of the membrane.