thus , the improvement of therapeutic effects is needed to restore chemo- and radiosensitivity to anaplastic thyroid carcinomas which have a high frequency of mutant p53 ( mp53 ) . recently , p53 gene therapy has been developed [ 6 - 8 ] and applied to patients carrying mp53[9 - 11 ] . however , overexpression of wild - type p53 ( wtp53 ) risks inducing suppression of proliferation in normal cells as well as targeted cancer cells . the suppression of proliferation of blood cells possibly gives negative effects on the physical condition of patients . we have reported a new cancer therapy strategy against mp53 cancer cells , i.e. chemical chaperon therapy . this therapy is based on glycerol - mediated conformational change of mp53 molecules . the restoration of the normal function in mp53 leads to enhanced radiosensitivity and induction of apoptosis . this novel tool for enhancing the radiosensitivity of cancer cells bearing mp53 is worth investing as p53-targeted cancer therapy , since p53 mutations are frequently found in human cancer cells . in the present study , we report the apoptotic effect of glycerol combined with radio - treatment in anaplastic thyroid carcinoma cells carrying mp53 . to evaluate the effectiveness of glycerol in radiation induced lethality for anaplastic thyroid carcinoma 8305c cells , we examined colony formation assay . 1 , when glycerol was added to culture medium before x - ray irradiation at a final concentration of 0.6 m , 8305c cells became about 3 times more x - ray - sensitive at the d10 dose . in contrast , there was no enhanced radiation sensitivity observed in 8305c cells after the addition of 0.3 m glycerol alone ( data not shown ) . these results suggest that glycerol has an ability to enhance the radiosensitivity of 8305c cells . figure . 2a shows the time - course of the frequency of x - ray induced apoptotic cells . though the frequency did not increase after x - ray irradiation without the glycerol treatment , the frequency was augmented in a time - dependent manner in the presence of glycerol . typical apoptotic bodies detected by hoechst 33342 staining analysis are shown in fig . 2c and 2d . the destruction of nuclei was observed 48 h after x - ray irradiation , especially with glycerol ( fig . we next examined the induction of dna fragmentation after x - ray irradiation using agarose gel electrophoresis of dna extracted from 8305c cells ( fig . clear dna ladders in the nucleosomal size were observed 2448 h after x - ray irradiation combined with glycerol ( fig . however , a faint dna ladder was observed 48 h after x - ray irradiation alone ( fig . these results indicate that glycerol sensitized radiation - induced apoptosis in anaplastic thyroid carcinoma in 8305c cells . dna ( p53 consensus sequence , p53con ) binding activity of whole cell extracts from x - ray ( 6 gy ) irradiated cells was analyzed with gel mobility - shift assay ( fig . 4 ) . whole cell extracts from non - irradiated cells showed no dna binding activity . similarly , the extracts from x - ray - irradiated cells showed no dna binding activity 6 h after the irradiation . however , the extracts from glycerol - treated cells showed dna binding activity and the binding activity was also observed in the extracts from the cells irradiated with x - rays 6 h after the irradiation . the time course of apoptotic body detected with hoechst33342 staining after x - ray irradiation ( 6 gy ) . a , 8305c cells were treated with ( closed circles ) or without ( opened circles ) 0.6 m glycerol . b , control ; c , 48 h after x - ray irradiation alone ; d , 48 h after x - ray irradiation combined with 0.6 m glycerol ; bars , 25 m . dna fragmentation detected with agarose gel electrophoresis of dna extracted from x - ray ( 6 gy ) irradiated cells . a and b represent with and without glycerol ( 0.6 m ) , respectively . dna ( p53con ) binding activity of whole cell extracts from x - ray ( 6 gy ) irradiated cells analyzed with gel mobility - shift assay . non - irradiated or glycerol - treated cells were collected with x - ray - irradiated cells 6 h after the irradiation . recently , the mutation of p53 has been reported to play an important role in anaplastic change in differentiated thyroid carcinomas . supporting this , the p53 gene plays a pivotal role in the pathway that controls cell growth , proliferation and apoptosis [ 21 - 24 ] . the lack of the p53-regulated pathway is a common feature in a large number of tumors , suggesting that it may be central to the pathogenesis of human cancers . we have recently reported that the effectiveness by radiotherapy or hyperthermic therapy depends on p53-status in human squamous cell carcinoma ( sas cells ) . that is , sas cells transfected with mp53 are more radiation or heat resistant than neo control cells . the present observed x - ray sensitivity enhanced by glycerol may result from the enhancement of p53-independent x - ray - induced apoptosis . glycerol possibly enhanced x - ray - sensitivity in 8305c cells through restoration of mp53 to wtp53 functions . the present data from gel mobility - shift assay suggest that the restoration is brought through conformational change of mp53 to wtp53 . the mp53 molecules from cells treated with glycerol and x - rays might affect the transactivation of p53-down stream genes , especially apoptosis - related genes such as bax . in relation to this , we have reported that glycerol has an ability to enhance bax expression in mp53 human glioblastoma cells through phosphorylation of p53 at serine 15 by pi3-k family . the conformational change of mp53 might be induced by glycerol alone because the dna binding activity of mp53 was restored after glycerol treatment alone ( fig . 4 ) . however , in this case , the transcriptional activity of mp53 is not likely to be restored . possibly , the mp53 molecules from cells treated with glycerol alone is not modified by x - ray - induced phosphorylation or acetylation . therefore , it seems that the cells treated with glycerol alone did not show apoptosis , as shown in fig . 2 and 3 . we have previously reported that the dna binding activity of mp53 was almost not restored after glycerol treatment alone in p53 mutated at codon 248 . therefore , the effect of glycerol on mp53 may depend on mutation site of p53 and amino acid sequence affecting its three - dimensional conformation . this result suggests that the investigation of mutation site of p53 is an important process for the cancer therapies based on p53 status . new strategy based on the functional restoration of mp53 the compounds can induce apoptosis in mp53 human tumor cells through restoration of the transcriptional transactivation function to mp53 . compared with these compounds , glycerol seems to be the most hopeful molecule for cancer therapy , because it has been widely used as a clinical reagent already . the present study suggests that chemical chaperones , such as a glycerol , are useful for radiotherapy of p53-mutant thyroid carcinomas . the cells used in this study were a human anaplastic thyroid carcinoma - derived cell line , 8305c bearing a point mutation at codon 273 in the p53 gene . cells were maintained in continuous culture at 37c in a 5 % co2 humidified atmosphere . penicillin ( 100 u / ml ) and streptomycin ( 100 g / ml ) were added to the media . glycerol was added into the culture medium at a final concentration of 0.6 m at 48 h before irradiation by x - ray source ( mbr-1520r , hitachi , tokyo , japan ) . thereafter the cells were incubated at 37c in the presence of glycerol until sampling of apoptosis assay . for survival assay , the medium with glycerol was changed to glycerol - free medium after 10 h incubation and thereafter cells were incubated for seven to ten days at 37c in glycerol free - medium . to measure the radiosensitivity of the cells , cell survival after x - ray irradiation thereafter , colonies were rinsed with pbs , fixed with methanol , stained with 2% giemsa solution ( merck , woodbridge , nj , usa ) . we performed the hoechst 33342 staining and the agarose gel electorophoresis for analyses of apoptosis , using the 8305c cells . induction of apoptosis was analyzed by detection of apoptotic bodies and dna fragmentation . to detect apoptotic bodies , cells were fixed with 1% glutaraldehyde ( nacalai tesque , kyoto , japan ) in pbs at 4c , washed with pbs , stained with 0.2 mm hoechst 33342 ( sigma chemical co. ) , and then observed under a fluorescence microscope . one hundred cells were judged under one field . for detection of dna fragmentation in the nucleosomal size range , cells were suspended in lysis buffer containing 10 mm tris - hcl , ph 7.4 , 10 mm nacl , 10 mm edta and 1% sodium n - lauroylsarcosinate , incubated with 100 g / ml protease k at 37c overnight , and then centrifuged at 18,500  g for 30 min . the resulting supernatants were incubated with 100 g / ml rnase a at 37c for 1 h. the dna in the solution was precipitated with ethanol at -20c , dissolved in 10 mm tris - hcl , ph 8.0 , and 1 mm edta , and electrophoresed for 30 min at 100 v through 3% nusieve 3:1 gels containing 40 mm tris - acetate , ph 7.8 , 2 mm edta and 0.5 g / ml ethidium bromide . whole cell extracts were prepared from x - ray - irradiated or non - irradiated cells ( about 2  10 cells ) according to the method described elsewhere . the binding activity of p53 to p53 consensus sequence ( p53con ) was measured by a gel mobility - shift assay   using a synthetic double - stranded dna fragment encoding the p53con ( 5'-ggacatgcccgggcatgtcc-3 ' , japan bioservice , niiza , saitama , japan ) based on a specific sequence located upstream to the bax gene as a probe . the cells used in this study were a human anaplastic thyroid carcinoma - derived cell line , 8305c bearing a point mutation at codon 273 in the p53 gene . cells were maintained in continuous culture at 37c in a 5 % co2 humidified atmosphere . penicillin ( 100 u / ml ) and streptomycin ( 100 g / ml ) were added to the media . glycerol was added into the culture medium at a final concentration of 0.6 m at 48 h before irradiation by x - ray source ( mbr-1520r , hitachi , tokyo , japan ) . thereafter the cells were incubated at 37c in the presence of glycerol until sampling of apoptosis assay . for survival assay , the medium with glycerol was changed to glycerol - free medium after 10 h incubation and thereafter cells were incubated for seven to ten days at 37c in glycerol free - medium . to measure the radiosensitivity of the cells , cell survival after x - ray irradiation was measured by plating cells into 25 cm flasks containing medium . thereafter , colonies were rinsed with pbs , fixed with methanol , stained with 2% giemsa solution ( merck , woodbridge , nj , usa ) . we performed the hoechst 33342 staining and the agarose gel electorophoresis for analyses of apoptosis , using the 8305c cells . induction of apoptosis was analyzed by detection of apoptotic bodies and dna fragmentation . to detect apoptotic bodies , cells were fixed with 1% glutaraldehyde ( nacalai tesque , kyoto , japan ) in pbs at 4c , washed with pbs , stained with 0.2 mm hoechst 33342 ( sigma chemical co. ) , and then observed under a fluorescence microscope . one hundred cells were judged under one field . for detection of dna fragmentation in the nucleosomal size range , cells were suspended in lysis buffer containing 10 mm tris - hcl , ph 7.4 , 10 mm nacl , 10 mm edta and 1% sodium n - lauroylsarcosinate , incubated with 100 g / ml protease k at 37c overnight , and then centrifuged at 18,500  g for 30 min . the resulting supernatants were incubated with 100 g / ml rnase a at 37c for 1 h. the dna in the solution was precipitated with ethanol at -20c , dissolved in 10 mm tris - hcl , ph 8.0 , and 1 mm edta , and electrophoresed for 30 min at 100 v through 3% nusieve 3:1 gels containing 40 mm tris - acetate , ph 7.8 , 2 mm edta and 0.5 g / ml ethidium bromide . whole cell extracts were prepared from x - ray - irradiated or non - irradiated cells ( about 2  10 cells ) according to the method described elsewhere . the binding activity of p53 to p53 consensus sequence ( p53con ) was measured by a gel mobility - shift assay   using a synthetic double - stranded dna fragment encoding the p53con ( 5'-ggacatgcccgggcatgtcc-3 ' , japan bioservice , niiza , saitama , japan ) based on a specific sequence located upstream to the bax gene as a probe . ko participated in the design of the study , performed the all analyses and wrote the manuscript . to conceived of the study , and participated in its design and coordination . this work was supported by grants - in - aid from the ministry of education , science , sports , technology and culture of japan .
introductionanaplastic thyroid carcinoma , which is one of the most aggressive , malignant tumors in humans , results in an extremely poor prognosis despite chemotherapy and radiotherapy . 
 the present study was designed to evaluate therapeutic effects of radiation by glycerol on p53-mutant anaplastic thyroid carcinoma cells ( 8305c cells ) . to examine 
 the effectiveness of glycerol in radiation induced lethality for anaplastic thyroid carcinoma 8305c cells , we performed colony formation assay and apoptosis analysis.resultsapoptosis was analyzed with hoechst 33342 staining and dna ladder formation assay . 
 8305c cells became radiosensitive when glycerol was added to culture medium before x - ray irradiation . 
 apoptosis was induced by x - rays in the presence of glycerol . 
 however , there was little apoptosis induced by x - ray irradiation or glycerol alone . 
 the binding activity of whole cell extracts to bax promoter region was induced by x - rays in the presence of glycerol but not by x - rays alone.conclusionthese findings suggest that glycerol is effective against radiotherapy of p53-mutant thyroid carcinomas .