2B vs. for 1224 h post-treatment. Co-localization recovered in irradiated cells within 24 h after 41.5C heating, but was inhibited longer after 42.5C heating. The decreased co-localization in heated, irradiated cells suggests that there is a decrease in protein interaction, and Mre11 complexes in nuclei disassemble after heating. Such changes could be involved, at least in part, in heat-radiosensitization and inhibition of DSB repair. Also, the kinetics of disassembly and reassembly of Mre11 complexes appears to be dependent upon treatment temperature. Keywords:DNA double-strand breaks, DNA repair foci, Mre11 complex, ionizing radiation, hyperthermia, immunofluorescence, co-localization == Introduction == Hyperthermia is currently being evaluated as a potential adjuvant to radiation therapy of cancer, since it has long been known to sensitize mammalian cells to ionizing radiation (IR) (1,2). The molecular mechanisms for thermal radiosensitization, however, have yet to be fully delineated. Although thermal treatment of cells (4045C) is capable of causing transitions and structural alterations in proteins (3-5), the major challenge has been to identify those proteins whose transitions/alterations are critical in radiosensitization. Heat-radiosensitization is believed to be mediated by an inhibition of repair of IR-induced DNA double-strand breaks (DSBs) by heat (6-11). The DSB is a critical DNA lesion which can cause transformation or cell death if mis-repaired or unrestituted (12). Normally, DSBs are recognized and then repaired via two primary pathways: non-homologous DNA end-joining and homologous recombination. An absence or deficiency in either pathway results in enhanced radiosensitivity and genomic instability (13,14). The proteins of the Mre11 complex appear to play an essential role in homologous recombination in eukaryotic cells (15). Since a significant amount of each protein of the complex is initially translocated from the nucleus to the cytoplasm when cells are irradiated before heat treatments, we have speculated that the translocation of these proteins or the disassembly of the Mre11 complex may be responsible, in part, for inhibition of DSB repair and radiosensitization by hyperthermia (16,17). Structurally, the human Mre11 complex is highly conserved. A heterotetramer of Mre11 (85 kDa) and Rad50 (153 kDa), comprised of a dimer of each protein, binds to a single Nbs1 molecule (95 kDa) (18). The Mre11-Rad50 interaction within the complex has been reported to be stable (19). The Mre11 complex, specifically Mre11 and Rad50, binds to DNA, and processes the broken ends. Nbs1 may actually serve to initiate the DNA damage-induced checkpoint response (20). Deficiency or deletion mutations in each of Mre11, Rad50, and Nbs1 genes results in hypersensitivity of cells to IR, presumably due to problems with DSB repair (21-23). The human Mre11 complex is normally uniformly distributed in the nucleus of cells. Treatment of cells with DSB-inducing agents, such as IR, results in formation of discrete and bright nuclear foci (when fluorescently stained for any of the Mre11 complex proteins), which are indicative of efficient repair of DSBs (23-28). Nuclear repair foci fluorescently stained for any of the Mre11 complex proteins or phosphorylated histone H2AX (-H2AX) (29) have often been used as an indicator of the DNA damage response, as their presence has been AR-A 014418 correlated with sites of DSBs. To date, little is known about how heat affects the integrity of the Mre11 complex and alters the formation of foci in heated, irradiated cells. Here we show that heat shock alters CD221 the interaction between proteins of the Mre11 complex in unirradiated and irradiated human U-1 melanoma cells, and inhibits the induction of repair foci after irradiation. Cells were irradiated with various doses of X-rays (012 Gy) and then heated for 2 h at 41.5C or 42.5C. At various times after the treatment AR-A 014418 (024 h), cells were fixed/permeabilized and immunostained for Mre11, Rad50, and Nbs1. Co-localization of the proteins of the Mre11 complex and formation of Mre11/Rad50 nuclear foci in heated, irradiated cells was compared with irradiated cells that were not heated, and with heated cells that were not irradiated. Measurements of co-localization of proteins and detection/scoring of fluorescent nuclear foci were performed using confocal microscopy and intensity correlation analysis of stained proteins (28). == Materials and Methods == == Cell AR-A 014418 culture, irradiation, and heat treatments == Human U-1 melanoma cells were cultured in monolayer in McCoy’s 5A medium with L-glutamine (Mediatech, Herndon, VA, USA) and 10% iron-supplemented bovine calf serum (BCS; Hyclone, Logan, UT, USA). Cells were maintained at 37C in a humidified environment of 5%.
2B vs
