Attenuation of chronic thermotolerance by KNK437, a benzylidene lactam compound,enhances thermal radiosensitization in mild temperature hyperthermia combined with low dose-rate irradiation

Purpose: We investigated whether the attenuation of chronic thermotolerance by KNK437, a heat shock protein inhibitor, can modify the effect of thermal radiosensitization in mild temperature hyperthermia (MTH) combined with low dose-rate irradiation (LDRI).

Materials and methods: The human lung adenocarcinoma cell line A549 was simultaneously exposed to LDRI with MTH at 41°C and KNK437 at a dose of 100 μM. Cell survival was estimated by a clonogenic assay. Cell cycle change during treatment was analyzed by flow cytometry. Expression levels of the heat shock proteins hsp72, hsp27 and heat shock factor 1 (HSF-1) were measured by Western blotting.

Results: KNK437 inhibited the expression of inducible hsp72 and hsp27, but produced no change in the mobility shift of HSF-1. The cytotoxicity of LDRI was enhanced by MTH. The survival curve for LDRI + MTH revealed no development of chronic thermotolerance up to 48 h. Simultaneous LDRI and KNK437 treatment also resulted in enhanced cell killing. The radiosensitizing effect of KNK437 was enhanced by simultaneous exposure of the cells to MTH. Flow cytometry analysis of cell cycle progression demonstrated marked G2 arrest and mild G1 arrest with LDRI alone, but mild G1 arrest with MTH alone, and mild G2-M, S-phase accumulation with KNK437 alone. The marked G2 arrest caused by LDRI was partially suppressed by the addition of MTH, and was also suppressed by KNK437 treatment.

Conclusions: Exposure of A549 cells to KNK437 caused inhibition of hsp72 and hsp27 expression. The addition of KNK437 increased not only thermosensitivity to MTH, but also radiosensitivity to LDRI. KNK437 also enhanced the MTH-induced radiosensitization under these experimental conditions.     

Interaction between low dose-rate irradiation, mild hyperthermia and low-dose caffeine in a human lung cancer cell line.

PURPOSE: To investigate cell killing by means of low dose-rate irradiation (LDRI) combined with concurrent mild hyperthermia and to determine the effect of low-dose caffeine on this combination treatment. MATERIALS AND METHODS: Human lung adenocarcinoma cells, LK87, were treated with LDRI (50 cGy/h) in combination with mild hyperthermia at 41 degrees C and low-dose caffeine (1 mM). Cell survival was estimated by clonogenic assay. Flow-cytometry was performed with PI staining using FACScan. Heat-shock protein (HSP72/73) was measured by the Western blotting method. All treatments were simultaneously performed for up to 48 h (24 Gy). RESULTS: LDRI cytotoxicities were enhanced by hyperthermia at 41 degrees C. D0 calculated from the dose-response curve for LDRI combined with 41 degrees C was 3.46 Gy whereas it was 6.55 Gy for LDRI alone. The survival curve for LDRI +41 degrees C demonstrated no chronic thermotolerance up to 48 h. For LDRI + simultaneous low-dose caffeine, cell killing was also enhanced, where D0 was 3.38 Gy at 37 degrees C. Radiosensitization caused by caffeine was enhanced by combination with simultaneous mild hyperthermia at 41 degrees C, where D0=1.78 Gy. Cell cycle analysis demonstrated remarkable G2 and mild G1 arrest for LDRI alone, but only G1 arrest was observed for LDRI combined with 41 degrees C and for LDRI combined with caffeine. Strong and early G1 arrest was observed in the treatment with LDRI + caffeine at 41 degrees C. The amount of HSP72/73 in the combination of LDRI with caffeine at 41 degrees C was less than that at 41 degrees C alone. CONCLUSION: LDRI cytotoxicity was enhanced by non-lethal hyperthermia. Low dose caffeine produced further cell killing in the combination of LDRI with mild hyperthermia.     

Induction of radiation resistance by a heat shock protein inhibitor,KNK437, in human glioblastoma cells

Purpose:?We examined the effects of a heat shock protein (hsp) inhibitor, N-formyl-3, 4-methylenedioxy-γ-butyrolactam (KNK437), on the radiosensitivity of human glioblastoma cells (A-172).

Materials and methods:?Effects of KNK437 on radiosensitivity and cell cycle regulation were examined using colony formation assays, flow cytometry analysis and Western blot analysis. KNK437 was added to the culture medium 1 h before X-ray irradiation at 50, 100 or 300 μM final concentration.

Results:?KNK437 induced the resistance of A-172 cells and human squamous cell carcinoma cells (SAS) to X-rays. Flow cytometry analysis showed that KNK437 alone efficiently induced A-172 cells to enter G₂/M phase. Though A-172 cells irradiated with X-rays at 6 Gy showed no clear change in the cell cycle, the irradiated cells were induced to enter G₂/M phase when they had been pre-treated with KNK437. By Western blot analysis, p53, 14-3-3σ and cell division cycle 2 (cdc2) proteins that function in G₂ arrest were observed to be persistently accumulated or phosphorylated in KNK437-treated cells, regardless of X-ray irradiation.

Conclusions:?These results show that KNK437 causes cells to be resistant to radiation, and that this might be correlated with maintenance of G₂ arrest in the cell cycle regulation.     

Induction of radiation resistance by a heat shock protein inhibitor, KNK437, in human glioblastoma cells

PURPOSE: We examined the effects of a heat shock protein (hsp) inhibitor, N-formyl-3, 4-methylenedioxy-gamma-butyrolactam (KNK437), on the radiosensitivity of human glioblastoma cells (A-172). MATERIALS AND METHODS: Effects of KNK437 on radiosensitivity and cell cycle regulation were examined using colony formation assays, flow cytometry analysis and Western blot analysis. KNK437 was added to the culture medium 1 h before X-ray irradiation at 50, 100 or 300 microM final concentration. RESULTS: KNK437 induced the resistance of A-172 cells and human squamous cell carcinoma cells (SAS) to X-rays. Flow cytometry analysis showed that KNK437 alone efficiently induced A-172 cells to enter G2/M phase. Though A-172 cells irradiated with X-rays at 6 Gy showed no clear change in the cell cycle, the irradiated cells were induced to enter G2/M phase when they had been pre-treated with KNK437. By Western blot analysis, p53, 14-3-3sigma and cell division cycle 2 (cdc2) proteins that function in G2 arrest were observed to be persistently accumulated or phosphorylated in KNK437-treated cells, regardless of X-ray irradiation. CONCLUSIONS: These results show that KNK437 causes cells to be resistant to radiation, and that this might be correlated with maintenance of G2 arrest in the cell cycle regulation.     

The usefulness of a continuous administration of tirapazamine combined with reduced dose-rate irradiation using {gamma}-rays or reactor thermal neutrons

We clarified the usefulness of the continuous administration of tirapazamine (TPZ) in combination with reduced dose-rate irradiation (RDRI) using gamma-rays or reactor thermal neutrons. Squamous cell carcinoma (SCC) VII tumour-bearing mice received a continuous administration of 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating (P) cells. Then, they received a single intraperitoneal injection or 24 h continuous subcutaneous infusion of TPZ in combination with conventional dose-rate irradiation (CDRI) or RDRI using gamma-rays or thermal neutrons. After irradiation, the tumour cells were isolated and incubated with a cytokinesis blocker, and the micronucleus (MN) frequency in cells without BrdU labelling ( = quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. The MN frequency in the total tumour cells was determined using tumours that were not pre-treated with BrdU. The sensitivity of both total and Q cells, especially of Q cells, was significantly reduced with RDRI compared with CDRI. Combination of TPZ increased the sensitivity of both populations, with a slightly more remarkable increase in Q cells. Furthermore, the continuous administration of TPZ raised the sensitivity of both total and Q cell populations, especially the former, more markedly than the single administration, whether combined with CDRI or RDRI using gamma-rays or thermal neutrons. From the viewpoint of solid tumour control as a whole, including intratumour Q-cell control, the use of TPZ, especially when administered continuously, combined with RDRI, is useful for suppressing the reduction in the sensitivity of tumour cells caused by the decrease in irradiation dose rate in vivo.     

Exposure temperature, but not donor age, is a confounding factor for in vitro translocation production by chronic irradiation.

PURPOSE: To assess the effects of incubation temperature during irradiation, and of donor age, on the in vitro induction of chromosomal translocations in human lymphocytes. MATERIAL AND METHODS: Lymphocytes from six human male donors were scored, using fluorescence in situ hybridization, for the presence of chromosomal translocations involving chromosomes 1 to 6 after in vitro, chronic exposure (delivered continuously over 48 h at 37 degrees C or at 20 degrees C) to tritium beta-rays or 60Co gamma-rays. RESULTS: No age-related difference in the alpha coefficients of the fitted induction curves was observed for gamma-ray-exposed lymphocytes obtained from four donors whose ages ranged from 24 to 79 years, or for tritium beta-ray-exposed lymphocytes from two donors aged 36 and 62 years. Duplicate samples from one donor, irradiated concurrently at 20 degrees C or 37 degrees C, gave significantly different alpha coefficients: 0.128+/-0.008 and 0.053+/-0.004, respectively (p<0.0001). The S-ratio (the ratio of induced complete to incomplete translocations) was found to be independent of radiation dose, donor age and exposure temperature. CONCLUSIONS: For biodosimetry in chronic irradiation situations, the use of alpha coefficients derived from the dose-response curves of cells chronically irradiated in vitro at body temperature is recommended. With respect to induction rates, donor age does not appear to be a confounding factor. The S-ratio is independent of radiation doses, exposure temperatures, or donor ages.     

Molecular nature of mutations induced by irradiation with repeated low doses of X-rays in spleen, liver, brain and testis of lacZ-transgenic mice

PURPOSE: To investigate the molecular characteristics of mutations induced by repeated low doses of X-rays in spleen, liver, brain and testis of mice. MATERIALS AND METHODS: Muta mice, which harbour the lacZ gene contained in the lambda genome, were irradiated with 0.15 Gy every Monday, Wednesday and Friday for 6 months starting at 10 weeks of age for a total of 78 times. Four months after the last irradiation, DNAs were isolated from the four different tissues and the mutant frequencies of lacZ were determined. Next, the nucleotide sequences of the mutant lacZ genes were determined and compared with that of the wild-type to identify the molecular changes in the mutants. The frequencies of different types of mutations were compared with those found in age-matched non-irradiated mice. They were also compared with those found in mice irradiated with a single high dose. RESULTS: The repeated low-dose irradiation resulted in slight increases in the mutant frequency in the four kinds of tissues. The spleen, liver and brain in repeatedly irradiated mice showed higher frequencies of deletion type mutations than those of non-irradiated mice. In testis, however, the level of the increase was modest and not statistically significant. Complex type mutations were observed only in irradiated tissues. The characteristics observed in somatic tissues were similar to those induced by a single high dose of irradiation. CONCLUSIONS: The results suggest that the mechanism of mutation induction in vivo is similar between low- and high-dose irradiation in spleen, liver and brain. The low induction of deletion mutations in testis with low-dose irradiation suggests that spermatogonial cells have a unique DNA repair system against low-dose radiation-induced damage.     

Validation of the CBF, CBV, and MTT values by perfusion MRI in chronic occlusive cerebrovascular disease: a comparison with 15O-PET

RATIONALE AND OBJECTIVES: To evaluate the reliability of cerebral blood flow (CBF), cerebral blood volume (CBV), and mean transit time (MTT) values obtained by deconvolution algorithm perfusion-weighted MR imaging (D-PWI), we compared these values with those obtained by first-moment algorithm perfusion-weighted MR imaging (F-PWI) and 15O-PET. SUBJECTS AND METHODS: Six healthy volunteers and eleven patients with chronic occlusive cerebrovascular disease were studied with both perfusion-weighted MR imaging and 15O-PET, and region-of-interest analyses were performed. Normalization factors for CBF and CBV values obtained by D-PWI were determined as the mean values of 15O-PET divided by those of D-PWI in healthy volunteers. Then these values were used in analyzing the data of the patients. RESULTS: The MTT value obtained by D-PWI was 6.1 +/- 0.5 seconds on the non-occluded side, 6.4 +/- 0.7 seconds on the minimally to moderately stenosed side, and 6.7 +/- 1.2 seconds on the severely stenosed to occluded side. These values were significantly correlated with those obtained by F-PWI (r = 0.83; P < .001), and with those obtained by 15O-PET (r = 0.78; P < .05). However, the CBF and CBV values obtained by D-PWI did not correlate with those obtained by 15O-PET. CONCLUSION: MTT values obtained by D-PWI were reliable parameters of cerebral hemodynamics, but the CBF and CBV values obtained by D-PWI were not always reliable.