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Background: Boron neutron capture therapy (BNCT) is a selective radiotherapy, being effective for the treatment of even advanced malignancies in head and neck regions as well as brain tumors and skin melanomas. To clarify the role of p53 gene, the effect of BNCT on oral squamous cell carcinoma (SCC) cells showing either wild- (SAS/neo) or mutant-type (SAS/mp53) p53 was examined. Methods: Cells were exposed to neutron beams in the presence of boronophenylalanine (BPA) at Kyoto University Research Reactor. Treated cells were monitored for modulations in colony formation, proliferation, cell cycle, and expression of cell cycle-associated proteins. Results: When SAS/neo and SAS/mp53 cells were subjected to BNCT, more suppressive effects on colony formation and cell viability were observed in SAS/neo compared with SAS/mp53 cells. Cell cycle arrest at the G1 checkpoint was observed in SAS/neo, but not in SAS/mp53. Apoptotic cells increased from 6 h after BNCT in SAS/neo and 48 h in SAS/mp53 cells. The expression of p21 was induced in SAS/neo only, but G2 arrest-associated proteins including Wee1, cdc2, and cyclin B1 were altered in both cell lines. Conclusion: These results indicate that oral SCC cells with mutant-type are more resistant to BNCT than those with wild-type p53, and that the lack of G1 arrest and related apoptosis may contribute to the resistance. At a physical dose affecting the cell cycle, BNCT inhibits oral SCC cells in p53-dependent and -independent manners. Ionizing radiation (IR) directly damages DNA by causing Background Oral squamous cell carcinoma (SCC) patients are gener- single- and double-stranded breaks. p53 is a central medi- ally treated with surgery in combination with radiation ator of the response to DNA damage and cell stress, there- therapy and/or chemotherapy [1,2]. fore, it is expected to play a role in determining the Page 1 of 8 (page number not for citation purposes) Radiation Oncology 2009, 4:63 http://www.ro-journal.com/content/4/1/63 sensitivity of tumors to apoptotic stimuli such as radiation Boron compound and BNCT for cultured cells or cytotoxic drugs [3-6]. B-enriched (>98%) BPA was obtained from Boron Bio- logicals, Inc., (Raleigh, NC) and converted to a fructose Boron neutron capture therapy (BNCT) is a binary modal- complex following the method by Coderre et al. . The ity: Boron-10 ( B)-enriched compounds such as concentration of the aqueous suspension of BPA was 250 boronophenylalanine (BPA) and borocaptate sodium are mg/ml (21.28 mg B/ml). administered at first, followed by irradiation with thermal neutrons. B to captures thermal neutrons leads to the For BNCT, cells were grown in flasks with a culture area of 10 7 2 10 nuclear reaction B (n, α) Li. Both released particles, an 25 cm and treated with BPA at a B concentration of 50 4 7 α ( He) particle and lithium ( Li) nucleus have high linear ppm for 2 h. They were exposed to neutron beams in the energy transfer (LET) properties and short path lengths in presence of BPA at Kyoto University Research Reactor. water of 5-10 μm. If the boronated compounds selectively Neutron fluence was measured by the radioactivation of accumulate in the tumor, BNCT can be used to selectively gold foils on the front and back of the dishes, as described destroy tumor cells [7,8]. It has been shown that BNCT is in previous studies [23,24]. The average fluence of ther- 12 2 n/cm , and the average flux effective for the treatment of advanced malignancies in mal neutrons was 2.1 × 10 9 2 head and neck regions as well as brain tumors and skin was 2.3 × 10 n/cm /s at 5 MW. Thermoluminescent melanomas [9-12]. dosimeters were used for gamma-ray dosimetry, and the total gamma ray dose was 0.00665 Gy. Thermal neutron The level of localized DNA damage caused by IR is fluence was converted to a dose, as described previously believed to increase with elevating LET values of radiation. . Cell inactivation induced by IR with different LET's has been analyzed, and many studies have shown that high Colony formation assay LET radiation including carbon-ion beams is more effec- Colony formation was performed as described previously tive than low LET X-rays and gamma rays regarding the . Briefly, cells were dissociated with 0.05% trypsin yield of apoptosis and reproductive death [13-16]. Car- and 0.02% EDTA, suspended in medium, and plated onto bon-ion beams have been reported to increase apoptosis 60-mm dishes at a cell density yielding approximately 500 in oral SCC and lung cancer cells regardless of the p53 sta- colonies per dish. The cells were cultured for 7 days, fixed tus [17,18]. in methanol, and stained with 1% crystal violet. Colonies composed of more than 30 cells were counted. The surviv- Approximately 50% of oral SCCs show a mutational ing cell fraction was determined by dividing the colony change of p53 [19,20]. Before the novel high LET radia- number of the treated culture by that of the non-irradiated tion therapy BNCT is used more frequently for oral SCC, control culture. its effect on the cell cycle and the cytotoxic effect on oral SCC cells irrespective of the p53 status should be clarified. 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay In the present study, we examined the effects of BNCT on the proliferation, cell cycle, and cell cycle-related proteins MTT assay was performed following the method by Mos- of oral SCC cells showing wild- or mutant-type p53 with mann . Cells were seeded in 96-well plates at a density the same background and indicated the role of p53 in the of 1 × 10 cells/well. At various intervals after BNCT, 10 μl of 5 mg/ml MTT solution was added to each well with 100 suppressive effect of BNCT. μl of medium, and cells were incubated at 37°C for 4 h. After the addition of 100 μl of 0.04 N HCl in isopropanol, Methods Cells the plates were mixed thoroughly to dissolve the dark blue The oral SCC cell line SAS showed the phenotype of wild- crystals. The plates were read on a Benchmark Plus micro- type p53 on IR-induced signal transduction. SAS cells plate spectrophotometer (Bio-Rad Laboratories, Hercules, were transfected with the plasmid pC53-248 containing CA) with a reference wavelength of 630 nm and a test an mp53 gene (codon 248, from Arg to Trp) to produce a wavelength of 570 nm. Background absorbance at 630 dominant negative mp53 protein, or with the control nm was subtracted from the 570 nm reading. The values plasmid pCMV-Neo-Bam, which contains a neo-resist- for BNCT-treated cells were calculated as a ratio in relation ance marker. The stable transfectants SAS/mp53 and SAS/ to the untreated control cells. Data are presented as the neo were used . These oral SCC cell lines were cul- means ± SD of six determinations. tured in Dulbecco's modified Eagle's medium supple- mented with 10% fetal bovine serum, 2 mM L-glutamine, Flow cytometric analysis 100 μg/ml penicillin, and 100 mg/ml streptomycin at Cells were dissociated and centrifuged, and the pellets 37°C in a humidified atmosphere with 5% CO . were fixed in ice-cold 70% ethanol at -20°C overnight. Thereafter, the cells were washed twice with ice-cold PBS Page 2 of 8 (page number not for citation purposes) Radiation Oncology 2009, 4:63 http://www.ro-journal.com/content/4/1/63 and treated with 1 mg/ml RNase at 37°C for 30 min. After survival fractions of SAS/neo and SAS/mp53 cells were 8 staining of cellular DNA with 50 μg/ml propidium iodide and 36%, respectively (Figure 1). in PBS, cells were analyzed with a fluorescence-activated cell sorter (FACSort; Becton Dickinson, Mountain View, Suppression of the proliferation of oral SCC cells by BNCT CA). The percentage of cells at different phases of the cell To determine the effect of BNCT on the proliferation of cycle was determined by employing Mod Fit LT software cells, SAS/neo and SAS/mp53 cells were treated with (Verity Software House, Topsham, ME). Based on an anal- BNCT at a dose of 6 Gy. After incubation for 6, 12, 24, and ysis of DNA histograms, the percentages of cells in sub- 48 h, cell viability was measured by employing the MTT G1, G0/G1, S, and G2/M phases were evaluated. assay. When the BNCT-treated cultures were compared with those of untreated controls, the percentage of viable Hoechst staining cells was decreased in both cell lines. The rates of viable Cells were dissociated and fixed in PBS containing 1% glu- SAS/neo and SAS/mp53 at 48 h after BNCT were 72 and taraldehyde for 2 h. After washing in PBS, cells were 86% of untreated controls, respectively (Figure 2), show- stained with 200 μM Hoechst 33342, mounted on slides, ing a significant difference (P < 0.01). and visualized using a Nikon Microphot-FXA fluorescence microscope. The number of positive cells was counted in Induction of cell cycle arrest by BNCT 3 samples, and the mean ± SD was determined. SAS/neo cells were treated with BNCT at a dose of 6 Gy and then subjected to flow cytometric analysis. Initially, Immunoblot analysis the rate of SAS/neo cells in the G0/G1 phase was 30%, Cells were lysed in a buffer containing 20 mM Tris-HCl and it increased to 39% at 6 h after BNCT. At 12 h, it (pH 7.4), 0.1% sodium dodecyl sulfate, 1% TritonX-100, decreased to 6%, and cells in the G2/M phase were 1% sodium deoxycholate, and protease inhibitor cocktail. increased to 34%. Sub-G1 peaks, indicating apoptotic After sonication, cells were centrifuged at 15,000 × g for cells, appeared from 6 h after BNCT (Figure 3). In SAS/ 10 min at 4°C, and the supernatant was harvested. Pro- mp53 cells, however, there was no increase of G0/G1 tein (20 μg) was separated through polyacrylamide gel phase cells at 6 h after BNCT; rather, they decreased electrophoresis and transferred to a polyvinylidene fluo- slightly (Figure 3). At 12 h after BNCT, the proportion of ride membrane by electroblotting. The membrane was cells in the G2/M phase was increased to 40%, indicating probed with antibodies, and antibody-binding was arrest at the G2/M checkpoint. A small sub-G1 population detected using an enhanced chemiluminescence kit appeared at 48 h after BNCT. (Amersham Life Science, Arlington Heights, IL) according to the manufacturer's instructions. The antibodies used were as follows: mouse monoclonal antibodies against p53, p53 phosphorylated at serine-15, p21, cyclin B1, and β-actin, and rabbit polyclonal antibodies against Wee 1 and cdc2 phosphorylated at tyrosine -15. Antibodies against p53 and β-actin were obtained from Oncogene (San Diego, CA) and Sigma (St.Louis, MO), respectively. Those for Wee1 and cyclin B1 were from Upstate (Lake Placid, MA). Other antibodies were from Cell Signaling Technology (Beverly, MA). The β-actin expression was assessed to ensure protein loading. Statistical analysis The mean number of apoptotic cells was analyzed using the unpaired Student's t-test. A P- value < 0.05 was consid- ered to be significant. Results Suppression of the colony formation of oral SCC cells by BNCT Supp BNCT Figure 1 ression of the colony formation of oral SCC cells by SAS/neo and SAS/mp53 cells were treated with BNCT, and Suppression of the colony formation of oral SCC cells the survival ratios were calculated based on colony forma- by BNCT. SAS/neo and SAS/mp53 cells were treated with tion. In both cell lines, the survival ratios decreased in a BNCT, and survival fractions were assessed based on colony formation. dose-dependent manner, but SAS/neo were suppressed more strongly than SAS/mp53 cells. At a dose of 6 Gy, the Page 3 of 8 (page number not for citation purposes) 3HUFHQWDJH Radiation Oncology 2009, 4:63 http://www.ro-journal.com/content/4/1/63 The expression and/or phosphorylation of G1 checkpoint- related proteins by BNCT In BNCT-treated SAS/neo cells, the expression of p53 increased and reached its maximum 6 h after BNCT. The elevation of phosphorylated p53 was observed at 6, 24, and 48 h after BNCT. An increased expression of p21 was observed from 6 h after BNCT (Figure 5). In SAS/mp53, the protein level of p53 was not specifically altered, but the phosphorylation decreased gradually after BNCT. The expression of p21 was also suppressed after BNCT in SAS/ mp53 cells. The expression and/or phosphorylation of G2 checkpoint- Suppression of the proliferatio Figure 2 n of oral SCC cells by BNCT related proteins by BNCT Suppression of the proliferation of oral SCC cells by In SAS/neo cells, the expression of Wee1 was elevated BNCT. SAS/neo and SAS/mp53 cells were treated with from 12 to 24 h after BNCT, and rapidly decreased at 48 h BNCT, and cell viability was measured by the MTT assay. (Figure 6). The protein level of cdc2 increased from 12 h The cell viability of untreated cells was also measured and after BNCT, and this was maintained until 48 h. An used as a control. *p < 0.01, SAS/neo vs. SAS/mp53. increase in the phosphorylation of cdc2 occurred at 12 h, indicating cell cycle arrest at the G2 checkpoint, and Measurement of apoptotic cells by nuclear staining declined to the initial level at 48 h. Cyclin B1 that forms Cell cycle analysis revealed the presence of a sub-G1 pop- the cdc2/cyclin B1 complex was induced at 12 h after ulation, indicating apoptosis by BNCT. After treatment BNCT. In SAS/mp53 cells, the expression of Wee1 with BNCT, nuclear DNA was stained with Hoechst increased at 12 and 24 h after BNCT (Figure 6). Although 33342, and cells showing nuclear fragmentation were the protein level of cdc2 was not specifically altered, cdc2 determined (Figure 4A). In SAS/neo cells treated with phosphorylation increased at 12 h after BNCT. The pro- BNCT, the proportion of apoptotic cells was elevated from tein level of cyclin B1 increased from 12 h after BNCT, and 6 h as compared with untreated control cells, and reached this was maintained until 48 h. 4.5% after incubation for 48 h (Figure 4B). The difference between SAS/neo and BNCT-treated SAS/neo was signifi- Discussion cant (p < 0.01). In the case of SAS/mp53, no apparent It is considered that the presence of p53 mutation might increase of apoptotic cells was observed early after BNCT, reduce the effectiveness of radiotherapy, but studies com- but the proportion increased to 3.5% at 48 h (Figure 4B). paring the presence or absence of p53 mutations in rela- The difference between SAS/mp53 and BNCT-treated SAS/ tion to the outcome following radiotherapy showed no mp53 was significant (p < 0.01). consistent relationship [26-29]. Tumors with the wild- type p53 protein may lack a functional p53 response as a result of mutations affecting other genes that function in the same pathways as p53 . It is difficult to clarify the 6$6PS 6$6QHR role of p53 in each oral SCC cell line, and so we used known mutated oral SCC cell lines, SAS/neo and SAS/ mp53, with the same background. Studies on the correlation between the cytotoxic effect of BNCT and the p53 status are limited [31,32], but more studies are employing high LET carbon-ion beams. Indeed, Iwadate et al. reported that high LET carbon- ion beams were more cytotoxic than low LET X-rays for 7LPHDIWHU%1&7KRXUV glioma cells, and the effects of the carbon-ion beams were not dependent on the p53 gene status. Tsuboi et al.  Induction of cell cycle arrest by Figure 3 BNCT reported that a glioblastoma cell line with p53 mutation Induction of cell cycle arrest by BNCT. A. SAS/neo and was sensitive to carbon-ion beams as a wild-type p53 cell SAS/mp53 cells were treated with BNCT and then subjected line at a high LET. In the present study, we performed col- to flow cytometric analysis. B. Based on an analysis of DNA ony formation assays, and confirmed that the effect of histograms, the percentages of cells in sub-G , G /G , S, and 1 0 1 G /M phases were evaluated. BNCT was more potent in SAS/neo than SAS/mp53 cells. We also examined the effect of BNCT using the MTT assay, Page 4 of 8 (page number not for citation purposes) Radiation Oncology 2009, 4:63 http://www.ro-journal.com/content/4/1/63 Induction of apoptotic cells Figure 4 with the fragmentation of nuclear DNA by BNCT Induction of apoptotic cells with the fragmentation of nuclear DNA by BNCT. SAS/neo and SAS/mp53 cells were treated with BNCT, incubated for 48 h at 37°C, and stained by Hoechst 33342. The proportion of apoptotic cells was deter- mined at various time points. *p < 0.01, SAS/neo vs. BNCT-treated SAS/neo; SAS/mp53 vs. BNCT-treated SAS/mp53. and identified a difference between SAS/neo and SAS/ cycle arrest at the G1 checkpoint only in SAS/neo cells. mp53 cells regarding their proliferative potential after Tsuboi et al.  did not identify a marked increase of BNCT. The expression of functional p53 must be involved cells in the G1 phase in glioblastoma U87 MG cells with in BNCT-induced growth suppression and/or cell death. wild-type p53 as well as TK1 with mutant-type p53 after carbon-ion beam irradiation. BNCT may differ from car- p53 is a key factor that regulates the cell cycle checkpoint bon-ion beams in terms of its ability to induce cell cycle [4,6]. In this study, it was suggested that p53 plays an arrest at the G1 checkpoint. important role in G1 arrest in SAS/neo cells. Flow cyto- metric analysis revealed a transient accumulation in the When DNA damage by IR is irreparable, the activation of G0/G population at 6 h after BNCT in SAS/neo cells. p53 leads to apoptosis via both transcription-dependent Thereafter, BNCT induced G2 arrest in both SAS/neo and SAS/mp53 cells. This indicates that BNCT induces cell A p Figure 5 o ltered ex int-related proteins pression and/or by BNCT phosphorylation of G1 check- Alter point-related proteins by BNCT Figure 6 ed expression and/or phosphorylation of G2 check- Altered expression and/or phosphorylation of G1 Altered expression and/or phosphorylation of G2 checkpoint-related proteins by BNCT. SAS/neo and checkpoint-related proteins by BNCT. SAS/neo and SAS/mp53 cells were treated with BNCT, and the expression SAS/mp53 cells were treated with BNCT, and the expression of p53 and p21 and phosphorylation of p53 were examined of Wee1, cdc2, and cyclin B1 and phosphorylation of cdc2 by immunoblot analysis. were examined by immunoblot analysis. Page 5 of 8 (page number not for citation purposes) Radiation Oncology 2009, 4:63 http://www.ro-journal.com/content/4/1/63 and -independent mechanisms. Aromando et al.  of cdc2 in both SAS/neo and SAS/mp53 cells around 12 h reported that BNCT-induced control of hamster cheek after BNCT. Therefore, it can be stated that Wee1, cdc2, pouch tumors would be an inhibitory effect on DNA syn- and cyclin B1 are associated with G2 arrest in a p53-inde- thesis and apoptosis does not have a significant role in pendent manner. tumor control. Masunaga et al.  examined the effect of BNCT on SAS xenografts in nude mice. After BNCT, the Carbon-ion beams reportedly induce apoptosis in oral tumor cells were dissociated and the cell suspension was SCC and lung cancer cells regardless of the p53 status at a cultured for colony formation, the detection of apoptotic high LET [17,18]. Why high LET BNCT leads to the p53- cells, and a micronucleus assay. The peak of apoptosis was dependent suppression of cell survival and induction of observed at 6 h after BNCT at low levels, irrespective of the cell cycle arrest at the G1 checkpoint is unclear. Probably, p53 status, suggesting that apoptosis occurred early on. each tumor cell would be equally exposed to carbon-ion We also observed an increase in the sub-G1 population beams. In the case of BNCT, however, the path lengths of and nuclear fragmentation early after BNCT in SAS/neo high LET α and Li particles are very short, so that the LET cells, and the level was maintained thereafter. In SAS/ would decrease markedly, even within a cell, being mp53 cells, however, the increase in apoptosis occurred dependent on the distance from the cytoplasmic boron to subsequent to G2 arrest. Thus, p53 seems to be responsi- the nuclear DNA [7,8]. This may generate a variety of ble for G1 arrest-associated apoptosis. In the present intracellular LET values, and yield appropriate energy to study, p53 led to a significant but limited increase of induce cell cycle arrest at G1, if the cells have functional apoptosis. Differently, in colony formation and MTT p53. It may also be ascribed to the characteristics of the assays, p53 has a much stronger impact on the survival cell lines used. Indeed, the survival curve of SAS/mp53 fraction and proliferation of treated cells. This indicates cells is not exponential, but a shoulder curve. The form of that apoptosis is a form of cell death induced by BNCT. So the curve suggests that the LET was not very high. If the far, different types of cell death have been documented. mutation may influence the intracellular accumulation of They include apoptosis, autophagy, mitotic catastrophe, BPA, it may heavily influence the LET of the radiation and necrosis and senescence . Especially, participation of relative biological effect. mitotic catastrophe, necrosis and senescence in BNCT- treated cancer cells should be clarified. In conclusion, oral SCC cells with mutant-type p53 were more resistant to the cell-killing effect of BNCT than those p21 binds to and inhibits the cyclin-dependent protein with wild-type p53 under the present experimental condi- kinases that drive the cell cycle, and is responsible for G1 tions. A functional p53 is required for the induction of arrest [34-36]. In SAS/neo cells, we found that the expres- apoptosis related to G1 arrest. BNCT inhibits oral SCC sion and phosphorylation of p53 was markedly enhanced cells via p53-dependent and -independent mechanisms. from 6 h after BNCT, and this level was maintained for 48 Recent clinical studies have shown that the delivery of h. We also detected a transient increase in the expression wild-type p53 to cancer cells with p53 mutations signifi- of p21 which inhibited the transition from the G1 to S cantly increases their radiation sensitivity [43,44]. Adeno- phase. In SAS/mp53 cells, however, p21 was not induced, viral-mediated gene therapy is a reliable method to and neither G1 arrest nor the induction of apoptosis was introduce the wild-type p53 gene [45,46]. Such an observed. This indicates that p21 is associated with cell approach may be applicable to oral SCCs with mutated cycle arrest at G1 down-stream of the p53 pathway. p53 to promote the efficiency of BNCT. After BNCT, cells that escaped G1 arrest accumulated at Conflict of interests G2 to prevent mitotic entry after potentially lethal DNA The authors declare that they have no competing interests. damage. Cdc2 protein kinase activity is required for the G2-to-mitosis transition in all eukaryotic cells. Cdc25 acti- Authors' contributions vates the cdc2/cyclin B1 complex by dephosphorylating YF carried out the experiments in the study and drafted the inhibitory threonine-14 and thyrosine-15 residues of cdc2 manuscript. IK provided the compound and carried out [37-39]. This step is indispensable to mitosis after IR. the experiments. SI carried out the experiments. KO par- Wee1 protein kinase allows cdc2 inactivation by phos- ticipated in the design of reactor irradiation. MS helped phorylation of cdc2 on tyrosine -15 [40,41]. Matsumura the measurement of boron concentration. YS helped reac- et al.  reported that carbon-ion irradiation was associ- tor irradiation. KO provided cell lines and participated in ated with the overexpression of Wee1 and phosphoryla- the design of the study. TO provided cell lines and partic- tion of cdc2, followed by the prolongation of G2 arrest ipated in the design of the study. YY conceived of the and subsequent induction of apoptosis. Consistent with study and participated in its design and coordination. All their results, we found that BNCT induced the expression authors read and approved the final manuscript. of Wee1 and cyclin B1 and increased the phosphorylation Page 6 of 8 (page number not for citation purposes) Radiation Oncology 2009, 4:63 http://www.ro-journal.com/content/4/1/63 20. Balz V, Scheckenbach K, Götte K, Bockmühl U, Petersen I, Bier H: Is Acknowledgements the p53 inactivation frequency in squamous cell carcinomas This work was supported in part by a Grant-in-Aid for Scientific Research of the head and neck underestimated? 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Radiation Oncology – Springer Journals
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