These results are not consistent with the above-mentioned speculations; however, they support our opinion that this suppression of postirradiation upregulation of HIF-1 activity is usually most important for the best therapeutic benefit. This study raises the important possibility that other strategies that lead to an increase in tumour hypoxia might have the same negative influence around the therapeutic effect of radiation as RS 8359 YC-1. and the suppression of the postirradiation upregulation of HIF-1 activity is usually important for the best therapeutic benefit. leads to a rapid degradation of HIF-1protein with a half-life of 5C8?min (Berra is stabilised and interacts with HIF-1under hypoxic conditions (Wang (Harada HeLa/5HREp-ODD-Luc cells were seeded into six-well culture dish (2 105 per well) and treated with HIF-1siRNA or scramble siRNA (Invitrogen Corp., Carlsbad, CA, USA) for 12?h. The culture medium was refreshed with 1?ml of D-MEM containing 0.1% foetal bovine serum with or without YC-1 (10?antibody (BD Bioscience, San Diego, CA, USA) and with anti-imaging device (Xenogen, Alameda, CA, USA). During the imaging, the mice were anaesthetised with 2.5% isoflurane gas in the oxygen flow (1.5?l?min?1). Images were analysed using Living Image 2.50-Igor Pro 4.09 software (Xenogen). Immunohistochemical analyses HeLa/5HREp-ODD-Luc tumour xenografts were surgically excised 90?min after an intraperitoneal injection with pimonidazole hydrochloride (Natural Pharmacia International Inc., Belmont, MA, USA; 60?mg?kg?1). For diaminobenzidine staining of pimonidazole hydrochloride and CD31, the formalin-fixed and paraffin-embedded sections were treated with anti-pimonidazole antibody and anti-CD31 antibody respectively, as described earlier (Harada and pimonidazole, the tumour xenografts were embedded in OCT compound and frozen at ?80C. The frozen sections were fixed in 2% paraformaldehyde and Rabbit Polyclonal to OR10H2 ice-cold methanol sequentially for 5?min each, blocked with blocking solution (serum-free protein block solution (Dako, Glostrup, Denmark) containing 0.1% cold water fish skin (CWFS) gelatin (Sigma-Aldrich Corp., St Louis, MO, USA)) and treated with anti-HIF-1mAb (BD Bioscience) in the blocking solution. After being washed extensively with PBS, the sections were blocked with PBS made up of 0.1% RS 8359 CWFS gelatin and treated with Alexa Fluor 546 rabbit anti-mouse IgG (Invitrogen Corp.) in the blocking solution. After further extensive washing with PBS, counter staining was conducted with DAPI (Wako Pure Chemical Industries Ltd, Osaka, Japan). The sections were next treated with FITC-conjugated anti-pimonidazole mAb (Natural Pharmacia International Inc.). For the analysis of perfusion (Hoechst 33342 distribution) and the number of functional blood vessels, tumour-bearing mice were intravenously injected with 100?reporter gene is suitable for the real-time imaging of absolute HIF-1 activity (Harada gene (HeLa/5HREp-ODD-Luc cells) and monitored the postirradiation dynamics of intratumoral HIF-1 activity using an optical imaging device (Physique 1A and B). The level of activity decreased significantly and reached a minimum at 6?h after 5?Gy of protein at the edges of DAPI-positive viable regions correlated with that of bioluminescent intensity in the irradiated HeLa/5HREp-ODD-Luc xenografts (Physique 1C and D, left graph), indicating that the HIF-1level is mainly responsible for the postirradiation HIF-1 activity in the tumour xenograft. Although the radiation-induced activation of HIF-1 and the underlying mechanisms were reported earlier (Moeller expression and HIF-1 activity at several hours postirradiation. Open RS 8359 in a separate window Physique 1 Optical imaging of intratumoral HIF-1 activity after ionising radiation. (A) HeLa/EFp-Luc or HeLa/5HREp-ODD-Luc xenografts were mAb (red fluorescence) or anti-Pimonidazole mAb (green fluorescence). Counter staining was conducted with DAPI (blue fluorescence). Bar=200?mAb (red fluorescence). A perfusion marker, Hoechst 33342 (blue fluorescence), was administrated i.v. at 1?min before each tumour excision. Bar=200?protein under radiation-induced reoxygenated conditions As HIF-1is known to be rapidly degraded under oxygen-available conditions (Jaakkola expression and HIF-1 activity at 6?h postirradiation. To examine this possibility, we performed an immunohistochemical analysis using a marker of hypoxia, pimonidazole (Kennedy protein under reoxygenated conditions. To test this possibility, we took advantage of a VHL-deficient human renal cell carcinoma cell line RCC4. RCC4 cells stably transfected with the reporter gene (RCC4/Vector/5HREp-ODD-Luc cells) showed intense bioluminescence regardless of the surrounding conditions (Physique 2A). On the other hand,.