p53 -Dependent thermal enhancement of cellular sensitivity in human squamous cell carcinomas in relation to LET

Purpose : To investigate the dependence on p53 gene status of the thermal enhancement of cellular sensitivity against different levels of linear energy transfer (LET) from X-rays or carbon-ion (C-) beams. Materials and methods : Two kinds of human squamous cell carcinoma cell lines were used with an identical genotype except for the p53 gene. SAS/m p53 cells were established by transfection with mutated p53 (m p53) gene to SAS cells having functional wild-type p53 (wtp53). As the control, a neo vector was transfected to the SAS cells (SAS/ neo cells). Both cells were exposed to X-rays or accelerated C-beams (30-150 KeV w m -1) followed by heating at 44°;C. Cellular sensitivity was determined by colony-forming activity. Induction of apoptosis was analysed by Hoechst 33342 staining of apoptotic bodies and agarose-gel electrophoresis for the formation of DNA ladders. Results : It was found that (1) there was no significant difference in cellular sensitivity between SAS/ neo and SAS/m p53 cells to LET radiation of >30 KeV w m -1, although the radiosensitivity of SAS/ neo cells to X-rays was higher (1.2-fold) than that of SAS/m p53 cells; (2) there was an interactive thermal enhancement of radiosensitivity below an LET of 70 KeV w m -1 in SAS/ neo cells, although only additive thermal enhancement was observed in SAS/m p53 cells through all LET levels examined; (3) low-LET radiation induced apoptosis only in SAS/ neo cells; (4) high-LET radiation at an isosurvival dose-induced apoptosis of SAS/ neo cells at a higher frequency compared with that with low-LET radiation; (5) high-LET radiation-induced p53-independent apoptosis in SAS/m p53 cells; and (6) thermal enhancement of cellular sensitivity to X-rays was due to induction of p53-dependent apoptosis. Conclusions : The findings suggest that thermal enhancement of radiosensitivity may result from p53-dependent apoptosis induced by inhibition of p53-dependent cell survival system(s) through either regulation of the cell cycle or induction of DNA repair. It is also suggested that the analysis of p53 gene status of cancer cells may predict response to combined therapies with low-LET radiation and hyperthermia.     

Analysis of death pattern in cancer cells by using different kinds of LET radiation

We investigated the death pattern of cancer cells by using different kinds of linear energy transfer (LET) radiation. We used two human squamous cell carcinoma cell lines with an identical genotype except for the p53 gene. SAS/mp53 cells were established by transfection with the mp53 gene to SAS cells having functional p53 (wtp53). As the control, a neovector was transfected to the SAS cells (SAS/neo cells). Both types of cells were exposed to X-rays (1.5 KeV/micron) or accelerated C-beams (30-100 KeV/micron). The frequency of cell death (apoptosis and necrosis) was measured by acridine orange/ethidium bromide(AO/EB) double staining for fluorescence microscopy. We found that (1) low-LET radiation induced apoptosis only in SAS/neo cells; (2) high-LET radiation at an iso-survival dose induced apoptosis not but necrosis in SAS/neo cells at a higher frequency; (3) high-LET radiation induced p53-independent apoptosis even in SAS/mp53 cells. Our findings suggest that high-LET radiotherapy is expected to (1) have validity in its application to patients carrying mutated p53 cancer cells and (2) reduce injury to adjacent normal tissue for high-frequency-induced apoptosis without inflammatory response. We propose that elucidation of p53-independent apoptosis-related genes might provide new insights into radiotherapy for cancer.     

Apoptosis induced by high-LET radiations is not affected by cellular p53 gene status

To learn more about the biological effects of high-linear energy transfer (LET) radiations, we examined radiation-induced apoptosis in response to high-LET radiations in cells with wild-type, mutated and null p53 gene. Three human lung cancer cell lines were used. These lines had identical genotypes, except for the p53 gene. Cells were exposed to X-rays or high-LET radiations (13 - 200 keV microm(-1)) using different nuclei ion beams. Cellular radiation sensitivities were determined with the use of colony-forming assays. Apoptosis was detected and quantified using Hoechst 33342 staining with fluorescence microscopy. It was found that (1) there was no significant difference in cellular sensitivity to high-LET radiation (>85 keV microm(-1)), although the sensitivity of wild-type p53 cells to X-rays was higher than that of mutated p53 or p53-null cells; (2) X-ray-induced apoptosis at higher frequencies in wild-type p53 cells when compared with mutated p53 and p53-null cells; and (3) Fe beams (200 keV microm(-1)) induced apoptosis in a p53-independent manner. The results indicate that high-LET radiations induces apoptosis in human lung cancer cells in a manner that does not seem to depend on the p53 gene status of the cells.     

外源性p53对不同LET辐射诱导人黑色素瘤细胞系A375死亡途径的影响

目的 观察外源性P53蛋白对不同传能线密度(LET)射线辐照诱导肿瘤细胞凋亡和坏死的影响,并探讨其可能的机制。方法 人黑色素瘤细胞系A375(wild-type p53)经携带人野生型p53基因的腺病毒载体(AdCMV-p53)感染后分别给予X射线和碳离子束照射,采用克隆形成法测定细胞辐射敏感性,Hoechst 33258和吖啶橙-溴化乙锭双染荧光显微镜下观察细胞凋亡和坏死。结果1高LET辐照时,A375细胞和转导人野生型p53基因的A375细胞(A375/p53)的辐射敏感性没有明显差异;2虽然辐射诱导细胞凋亡比例的增加依赖于LET升高,但是无论高LET或低LET,外源性P53蛋白均可有效诱导细胞凋亡。3高LET辐照时,A375细胞的坏死细胞明显高于A375/p53细胞。结论 尽管高LET辐射对A375和A375/p53细胞的存活无明显影响,但是对细胞凋亡的诱导却部分依赖于P53蛋白的功能,P53蛋白可能在调节细胞死亡类型中发挥重要作用。这对临床应用高LET辐射联合p53基因治疗恶性黑色素瘤有一定参考意义。     

Different inducibility of radiation- or heat-induced p53-dependent apoptosis after acute or chronic irradiation in human cultured squamous cell carcinoma cells

PURPOSE: This study was undertaken to clarify the effects of acute or chronic pre-irradiation on the induction of p53-dependent apoptosis by X-rays or heat shock. MATERIALS AND METHODS: Having an identical genotype except for p53-status, the human cultured squamous cell carcinoma cells (SAS) were transfected with a mutant p53 gene (SAS/mp53) or neo alone (SAS/neo) as a control. After acute X-irradiation (1 Gy min(-1)), chronic gamma-irradiation (0.001 Gy min(-1)) or heat shock (44 degrees C), the cells were for the incidence of apoptotic bodies and DNA ladders, cellular levels of p53 and bax, and caspase-3 activity. RESULTS: It was found that (1) a challenge treatment with X-rays (5.O Gy) or heat shock (30 min) immediately after chronic pre-irradiation (1.5 Gy) but not acute pre-irradiation (1.5 Gy) resulted in lower levels of apoptosis than those observed after challenge treatment only in SAS/neo cells; (2) a challenge treatment-induced apoptosis was observed 48 h after cessation of chronic pre-irradiation in SAS/neo cells; (3) apoptosis was barely increased in SAS/mp53 cells; and (4) the levels of apoptosis-related proteins after challenge treatments were strongly correlated with the above phenomena. CONCLUSIONS: Chronic pre-irradiation at a low dose-rate suppressed induction of p53-dependent apoptosis via bax and caspase-3. These findings suggest that chronic pre-irradiation suppressed p53 function through radiation-induced signalling and/or p53 stability.     

WAF1 accumulation by carbon-ion beam and alpha-particle irradiation in human glioblastoma cultured cells

PURPOSE: There have been no reports about the effects of heavy-ion beams on the expression of the WAF1 gene, although ionizing radiation such as y-rays and X-rays is well known to induce WAF1 (p21/CIP1/sdi1) gene expression in a p53-dependent manner. In the present study, it was examined whether WAF1 accumulation was induced after carbon-ion (C-) beam or alpha-particle irradiation in four glioblastoma cell lines. MATERIALS AND METHODS: A colony assay for radiosensitivity and Western blot analysis of WAF1 were applied to two human glioblastoma cell lines, A-172 bearing wild-type p53 (wtp53) and T98G bearing mutated p53 (mp53). A-172/neo and A-172/mp53 were transfected with a control vector (containing only a neo selection marker) and a mp53 expression vector respectively. RESULTS: The amount of WAF1 increased markedly after X-ray irradiation in A-172 and A-172/neo cells but not in T98G and A-172/mp53 cells. The level of WAF1 reached a plateau at 3-10 h after X-ray irradiation at 5 Gy in A-172 and A-172/neo cells. Likewise, the levels of WAF1 in A-172 and A-172/neo cells reached a plateau at 3-10 h and 6-24 h after C-beam (3.0 Gy) and alpha-particle (4.5 Gy) irradiation respectively. The amount of WAF1 increased markedly in a dose-dependent manner 10 h after X-ray, C-beam or alpha-particle irradiation in A-172 and A-172/neo cells but not in T98G or A-172/mp53 cells. In addition, cell survival assay showed that these cell lines were most sensitive to C-beams, less sensitive to alpha-particles and least sensitive to X-rays at 10% survival. There was no difference in sensitivity among these cell lines against C-beam and alpha-particle irradiation whereas wtp53 cells (A-172 and A-172/neo) were more sensitive to X-rays than mp53 cells (A-172/mp53 and T98G). CONCLUSIONS: These results indicate that C-beams and alpha-particles induce p53-dependent WAF1 accumulation as well as is the case with X-rays, suggesting that WAF1 protein accumulation may not contribute to cell killing.     

WAF1 accumulation by carbon-ion beam and alpha-particle irradiation in human glioblastoma cultured cells

Purpose : There have been no reports about the effects of heavy-ion beams on the expression of the WAF1 gene, although ionizing radiation such as gamma-rays and X-rays is well known to induce WAF1 (p21/CIP1/sdi1) gene expression in a p53 -dependent manner. In the present study, it was examined whether WAF1 accumulation was induced after carbon-ion (C-) beam or alpha-particle irradiation in four glioblastoma cell lines. Materials and methods : A colony assay for radiosensitivity and Western blot analysis of WAF1 were applied to two human glioblastoma cell lines, A-172 bearing wild-type p53 (wt p53) and T98G bearing mutated p53 (m p53) . A-172/neo and A-172/mp53 were transfected with a control vector (containing only a neo selection marker) and a m p53 expression vector respectively. Results : The amount of WAF1 increased markedly after X-ray irradiation in A-172 and A-172/neo cells but not in T98G and A-172/mp53 cells. The level of WAF1 reached a plateau at 3-10 h after X-ray irradiation at 5 Gy in A-172 and A-172/neo cells. Likewise, the levels of WAF1 in A-172 and A-172/neo cells reached a plateau at 3-10 h and 6-24 h after C-beam (3.0Gy) and alpha-particle (4.5 Gy) irradiation respectively. The amount of WAF1 increased markedly in a dose-dependent manner 10 h after X-ray, C-beam or alpha-particle irradiation in A-172 and A172/neo cells but not in T98G or A-172/mp53 cells. In addition, cell survival assay showed that these cell lines were most sensitive to C-beams, less sensitive to alpha-particles and least sensitive to X-rays at 10% survival. There was no difference in sensitivity among these cell lines against C-beam and alpha-particle irradiation whereas wt p53 cells (A-172 and A-172/neo) were more sensitive to X-rays than m p53 cells (A-172/mp53 and T98G). Conclusions : These results indicate that C-beams and alpha-particles induce p53 -dependent WAF1 accumulation as well as is the case with X-rays, suggesting that WAF1 protein accumulation may not contribute to cell killing.     

Different inducibility of radiation- or heat-induced p53 -dependent apoptosis after acute or chronic irradiation in human cultured squamous cell carcinoma cells

Purpose : This study was undertaken to clarify the effects of acute or chronic pre-irradiation on the induction of p53 -dependent apoptosis by X-rays or heat shock. Materials and methods : Having an identical genotype except for p53 -status, the human cultured squamous cell carcinoma cells (SAS) were transfected with a mutant p53 gene (SAS/m p53) or neo alone (SAS/ neo) as a control. After acute X-irradiation (1Gy min -1) , chronic gamma-irradiation (0.001 Gy min -1) or heat shock (44°C), the cells were for the incidence of apoptotic bodies and DNA ladders, cellular levels of p53 and bax, and caspase-3 activity. Results : It was found that (1) a challenge treatment with X-rays (5.0 Gy) or heat shock (30 min) immediately after chronic pre-irradiation (1.5Gy) but not acute pre-irradiation (1.5 Gy) resulted in lower levels of apoptosis than those observed after challenge treatment only in SAS/ neo cells; (2) a challenge treatment-induced apoptosis was observed 48h after cessation of chronic pre-irradiation in SAS/ neo cells; (3) apoptosis was barely increased in SAS/m p53 cells; and (4) the levels of apoptosis-related proteins after challenge treatments were strongly correlated with the above phenomena. Conclusions : Chronic pre-irradiation at a low dose-rate suppressed induction of p53 -dependent apoptosis via bax and caspase-3. These findings suggest that chronic pre-irradiation suppressed p53 function through radiation-induced signalling and/or p53 stability.     

Effects of a heat shock protein inhibitor KNK437 on heat sensitivity and heat tolerance in human squamous cell carcinoma cell lines differing in p53 status

PURPOSE: The effects of a heat shock protein (hsp) inhibitor KNK437 (N-formyl-3,4-methylenedioxy-benzylidene-gamma-butyrolactam) were examined on the heat sensitivity and heat tolerance of human cancer cells with special reference to p53 status. MATERIALS AND METHODS: Human squamous cell carcinoma (SAS) and glioblastoma cell lines (A-172) transfected with mutant p53 (mp53) or control neo genes were used. KNK437 was added in culture medium at a final concentration of 50, 100 or 300 microM 1 h before heating (42 degrees C). Surviving fractions of cells were measured by use of a clonogenic assay. Effects of KNK437 on the accumulation of heat shock proteins and DNA binding activity of heat shock factor 1 were examined with Western blot analysis and gel mobility-shift assay, respectively. Heat-induced apoptotic bodies were detected by Hoechst 33342 staining. RESULTS: The mp53-transfected SAS (SAS/mp53) and A-172 (A-172/mp53) cells were more resistant to heat than the neomycin (neo)-transfected SAS (SAS/neo) and A-172 (A-172/neo) cells. The constitutive amount of hsp27 was larger in SAS/mp53 than in SAS/neo cells. Clear differences in the constitutive amounts of hsp40, hsp72 and hsp90 were not observed between SAS/mp53 and SAS/neo cells. KNK437 enhanced the heat sensitivity in SAS/mp53 and A-172/mp53 cells more effectively than in neo control cells. Heat tolerance was suppressed by KNK437 in SAS/mp53 and SAS/neo cells and also in A-172/mp53 and A-172/neo cells. Along with suppression of heat tolerance, KNK437 suppressed heat-induced accumulation of both hsp27 and hsp72. Heat-induced apoptotic bodies were enhanced by KNK437 in SAS/mp53 and SAS/neo cells. CONCLUSION: The results suggest a possible mechanism for the heat sensitivity of SAS cells. Heat sensitivity depends on p53 status regulating the amount of hsp27. Heat tolerance is suppressed by KNK437 through the suppression of heat-induced accumulations of hsp27 and hsp72 and the induction of p53-independent apoptosis.     

High-LET radiation induces apoptosis in lymphoblastoid cell lines derived from ataxia-telangiectasia patients

Purpose : To investigate and compare the propensity of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL), derived from ataxia-telangiectasia (A-T) patients and from unaffected healthy individuals (controls), to undergo apoptosis after exposure to high-linear energy transfer (LET) radiation. Materials and methods : Four A-T (ARO, BMA, CSA and RJO) and two control (JAC and KKB3) LCL were exposed to doses of up to 4 Gy of accelerated nitrogen ions (32-45 MeV/u, 8-12 Gy/min). For comparative purposes X-ray irradiation (1.36 Gy/min) was also performed. The induction of apoptosis was studied 0-48h after irradiation with the use of two methods: (1) monitoring of high molecular weight (HMW) DNA fragments by field inversion pulse gel electrophoresis (FIGE); and (2) morphological characterization of apoptotic cells after fluorescent staining. In parallel, cell-cycle distribution, monitored by DNA flow cytometry, as well as measurements of p53/p21(WAF1) protein levels by Western blots, were investigated in these cells. Results : High-LET radiation-induced apoptosis and G2/M-arrest in both A-T and control LCL. No significant increase in the amount of p53/p21(WAF1) proteins preceded apoptosis in control or in A-T LCL after high-LET irradiation. However, low-LET radiation did induce significant enhanced levels of p53 proteins in control but not in A-T LCL. Conclusions : LCL from both A-T homozygous and unaffected healthy individuals undergo apoptosis without accumulation of p53/p21(WAF1) proteins after exposure to high-LET radiation. In contrast, low-LET radiation induces apoptosis and significantly increases levels of p53 protein in control but not in A-T LCL.     

High-LET radiation induces apoptosis in lymphoblastoid cell lines derived from atazia-telangiectasia patients

PURPOSE: To investigate and compare the propensity of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL), derived from ataxia-telangiectasia (A-T) patients and from unaffected healthy individuals (controls), to undergo apoptosis after exposure to high-linear energy transfer (LET) radiation. MATERIALS AND METHODS: Four A-T (ARO, BMA, CSA and RJO) and two control (JAC and KKB3) LCL were exposed to doses of up to 4Gy of accelerated nitrogen ions (32-45 MeV/u, 8-12Gy/min). For comparative purposes X-ray irradiation (1.36 Gy/min) was also performed. The induction of apoptosis was studied 0-48 h after irradiation with the use of two methods: (1) monitoring of high molecular weight (HMW) DNA fragments by field inversion pulse gel electrophoresis (FIGE); and (2) morphological characterization ofapoptotic cells after fluorescent staining. In parallel, cell-cycle distribution, monitored by DNA flow cytometry, as well as measurements of p53/p21(WAF1) protein levels by Western blots, were investigated in these cells. RESULTS: High-LET radiation-induced apoptosis and G2/M-arrest in both A-T and control LCL. No significant increase in the amount of p53/p21(WAF1) proteins preceded apoptosis in control or in A-T LCL after high-LET irradiation. However, low-LET radiation did induce significant enhanced levels of p53 proteins in control but not in A-T LCL. CONCLUSIONS: LCL from both A-T homozygous and unaffected healthy individuals undergo apoptosis without accumulation of p53/p21(WAF1) proteins after exposure to high-LET radiation. In contrast, low-LET radiation induces apoptosis and significantly increases levels of p53 protein in control but not in A-T LCL.     

Effects of p53 status and wortmannin treatment on potentially lethal damage repair,with emphasis on the response of intratumor quiescent cells

PURPOSE: To examine the effects of p53 status and wortmannin treatment on potentially lethal damage repair, referring to the response of intratumor quiescent cells. METHODS: Human head and neck squamous cell carcinoma cells transfected with mutant TP53 (SAS/mp53) or with neo vector as a control (SAS/neo) were injected subcutaneously into both hind legs of Balb/cA nude mice. Mice bearing the tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously to label all proliferating (P) cells in the tumors. The mice then received gamma-rays with or without subsequent wortmannin administration. Right after or 24 h after gamma-ray irradiation alone or 24 h after wortmannin administration following irradiation, the tumors were excised, minced, and trypsinized. The tumor cell suspensions thus obtained were incubated with a cytokinesis blocker (cytochalasin-B), and the micronucleus (MN) frequency in cells without BrdU labeling [quiescent (Q) cells] was determined using immunofluorescence staining for BrdU. The MN frequency in total (P+Q) tumor cells was determined from the tumors that were not pretreated with BrdU. RESULTS: On the whole, larger values of MN frequency and surviving fraction were observed in SAS/mp53 cells than in SAS/neo cells, and Q cells showed lower MN frequencies than total cells. Without wortmannin, SAS/neo tumor cells, especially Q cells within SAS/neo tumors, showed large potentially lethal damage repair (PLDR) capacities, compared with total or Q tumor cells within SAS/mp53 tumors that showed little PLDR capacity. Wortmannin treatment inhibited the PLDR in SAS/neo tumors very effectively, but showed no apparent effect on either total or Q tumor cells within SAS/mp53 tumors. CONCLUSION: PLDR in vivo was thought to be a p53-dependent event whether in total or Q tumor cell populations.     

Induction and disappearance of G2 chromatid breaks in lymphocytes after low doses of low-LET gamma-rays and high-LET fast neutrons

PURPOSE: To determine by means of the G2 assay the number of chromatid breaks induced by low-LET gamma-rays and high-LET neutrons, and to compare the kinetics of chromatid break rejoining for radiations of different quality. MATERIALS AND METHODS: The G2 assay was performed on blood samples of four healthy donors who were irradiated with low-LET gamma-rays and high-LET neutrons. In a first set of experiments a dose-response curve for the formation of chromatid breaks was carried out for gamma-rays and neutrons with doses ranging between 0.1 and 0.5 Gy. In a second set of experiments, the kinetics of chromatid break formation and disappearance were investigated after a dose of 0.5 Gy using post-irradiation times ranging between 0.5 and 3.5 h. For the highest dose of 0.5 Gy, the number of isochromatid breaks was also scored. RESULTS: No significant differences in the number of chromatid breaks were observed between low-LET gamma-rays and high-LET neutrons for the four donors at any of the doses given. The dose-response curves for the formation of chromatid breaks are linear for both radiation qualities and RBEs = 1 were obtained. Scoring of isochromatid breaks at the highest dose of 0.5 Gy revealed that high-LET neutrons were, however, more effective at inducing isochromatid breaks (RBE = 6.2). The rejoining experiments further showed that the kinetics of disappearance of chromatid breaks following irradiation with low-LET gamma-rays or high-LET neutrons were not significantly different. Half-times of 0.92 h for gamma-rays and 0.84 h for neutrons were obtained. CONCLUSIONS: Applying the G2 assay, the results demonstrate that at low doses of irradiation, the induction as well as the disappearance of chromatid breaks is independent of the LET of the radiation qualities used (0.24 keV x microm(-1) 60Co gamma-rays and 20 keV x microm(-1) fast neutrons). As these radiation qualities produce the same initial number of double-strand breaks, the results support the signal model that proposes that chromatid breaks are the result of an exchange process which is triggered by a single double-strand break.     

Estimation of radiation tolerance to high LET heavy ions in an anhydrobiotic insect, Polypedilum vanderplanki

PURPOSE: Anhydrobiotic larvae of Polypedilum vanderplanki are known to show an extremely high tolerance against a range of stresses. We have recently reported that this insect withstands exposure to high doses of gamma-rays (linear energy transfer [LET] 0.2 keV/microm). However, its tolerance against high LET radiation remains unknown. The aim of this study is to characterize the tolerance to high-LET radiations of P. vanderplanki. MATERIALS AND METHODS: Larval survival and subsequent metamorphoses were compared between anhydrobiotic (dry) and non-anhydrobiotic (wet) samples after exposure to 1 - 7000 Gy of three types of heavy ions delivered from the azimuthally varying field (AVF) cyclotron with LET values ranging from 16.2 - 321 keV/microm. The tolerance against 4He ions was also compared among three chironomid species. RESULTS: At all LET values measured, dry larvae consistently showed greater radiation tolerance than hydrated larvae, perhaps due to the presence of high concentrations of the disaccharide trehalose in anhydrobiotic animals, and the radiation-induced damage became evident at lower doses as development progressed. Relative biological effectiveness (RBE) values based on the median inhibitory doses reached a maximum at 116 keV/microm (12C), and the maximum RBE clearly increased as development progressed. Lower D0 (dose to reduce survival from relative value 1.00 - 0.37 on the exponential part of the survival curve), and higher Dq (quasi-threshold dose) were found in individuals exposed to 4He ions, compared to gamma-rays, and in P. vanderplanki larvae compared to non-anhydrobiotic chironomids. CONCLUSION: Anhydrobiosis potentiates radiation tolerance in terms of larval survival, pupation and adult emergence of P. vanderplanki exposed to high-LET radiations as well as to low-LET radiation. P. vanderplanki larvae might have more efficient DNA damage repair after radiation than other chironomid species.     

Dose--response of initial G2-chromatid breaks induced in normal human fibroblasts by heavy ions

PURPOSE: To investigate initial chromatid breaks in prematurely condensed G2 chromosomes following exposure to heavy ions of different LET. MATERIAL AND METHODS: Exponentially growing human fibroblast cells AG1522 were irradiated with gamma-rays, energetic carbon (13 keV/ microm, 80 keV/microm), silicon (55 keV/microm) and iron (140 keV/microm, 185keV/microm, 440keV/microm) ions. Chromosomes were prematurely condensed using calyculin-A. Initial chromatid-type and isochromatid breaks in G2 cells were scored. RESULTS: The dose response curves for total chromatid breaks were linear regardless of radiation type. The relative biological effectiveness (RBE) showed a LET-dependent increase, peaking around 2.7 at 55-80keV/microm and decreasing at higher LET. The dose response curves for isochromatid-type breaks were linear for high-LET radiations, but linear-quadratic for gamma-rays and 13 keV/microm carbon ions. The RBE for the induction of isochromatid breaks obtained from linear components increased rapidly between 13keV/microm (about 7) and 80keV/microm carbon (about 71), and decreased gradually until 440 keV/microm iron ions (about 66). CONCLUSIONS: High-LET radiations are more effective at inducing isochromatid breaks, while low-LET radiations are more effective at inducing chromatid-type breaks. The densely ionizing track structures of heavy ions and the proximity of sister chromatids in G2 cells result in an increase in isochromatid breaks.     

Correlation between cell killing and residual chromatin breaks measured by PCC in six human cell lines irradiated with different radiation types

PURPOSE: To examine the relationship between cell killing and residual chromatin breaks after irradiation with qualitatively different types of radiation in six human cell lines. MATERIALS AND METHODS: Six human tumour cell lines and normal human cells were irradiated with 200 kV X-rays and two carbon-ion beams accelerated by the Heavy Ion Medical Accelerator in Chiba (HIMAC) differing in LET. At the sample position the carbon-ion beams had LET infinity values of 13.1 and 77.5 +/- 0.4 keV/microm. Cell inactivation was documented by a colony assay. Residual chromatin breaks were measured by counting the number of residual chromatin fragments at 24h, detected by the premature chromosome condensation (PCC) technique. RESULTS: The cell lines covered a broad range of radiosensitivity. D10 values ranged from 3.53 to 8.12 Gy for X-rays, 2.56 to 7.41 Gy for the lower LET carbon ions and 1.17 to 3.85 Gy for the higher LET carbon ions. The results for residual chromatin breaks indicate that the more radiosensitive cell lines showed a greater induction of residual chromatin breaks either by X-rays or carbon-ions, and that an X-ray resistant cell line also showed resistance to carbon-ions. Cellular radiosensitivity correlated with the frequency of residual chromatin breaks. CONCLUSION: The detection of residual chromatin breaks by the PCC technique could be used to predict cellular radiosensitivity among qualitatively different types of radiation.     

Complex chromosome aberrations in peripheral blood lymphocytes as a potential biomarker of exposure to high-LET alpha-particles

PURPOSE: To determine the induction and transmission, to second and third division cells, of complex chromosome aberrations in peripheral blood lymphocytes after exposure to high-LET alpha-particles in vitro. MATERIALS AND METHODS: Separated peripheral blood lymphocytes collected from four healthy donors were irradiated in vitro with either high-LET alpha-particles (121 keV/microm; 0.5 Gy) or low-LET X-rays (250kV constant potential; 3 Gy). Cells were harvested in first, second and third division post-irradiation and chromosome aberrations observed at each cell division were analysed by combining the techniques of FISH and DAPI/Hoechst 33258 harlequin staining. Whole chromosome probes were used for chromosomes 1, 2 and 5, together with a pan-centromeric probe and the resulting chromosome 'painting' patterns were classified according to the Savage and Simpson (S & S) scheme (Savage and Simpson 1994a, Savage and Tucker 1996). RESULTS: A greater proportion of complex chromosome aberrations was observed, defined as involving three or more breaks in two or more chromosomes, relative to total exchanges, after exposure to 0.5 Gy alpha-particles (mean 1 track/cell) than after the high reference dose of 3 Gy X-rays (49-56% and 20-22%, respectively). Qualitatively, alpha-particles induced chromosome aberrations of far greater complexity than those observed after X-rays. This was reflected by both the rapid reduction in the percentage of damaged cells between first and second division indicative of cell death, and the spectrum of aberration types observed in second and third division cells post-irradiation. Regardless of this complexity, 15% of the complexes induced by alpha-particles at first division were potentially transmissible and by third division, transmissible-type complexes, specifically insertions, represented the predominant complex type (65%). CONCLUSION: Transmissible-type complexes were observed, specifically insertions, in both second and third division cells after exposure to high-LET alpha-particles (0.5 Gy) in vitro. The authors predict these cells to be stable and to be capable of persisting through many cell generations. Considering that the induction of complex chromosome aberrations by low-LET radiation is strongly dependent on dose, so that they are expected to be undetectable at environmental exposures, insertions are much more likely to be a characteristic feature of high-LET radiation at all doses. From this the usefulness of insertions as biomarkers of past exposure to environmentally relevant doses of high-LET alpha-particles is supported.     

辐射增强启动子调控的p53基因联合照射对肿瘤细胞的作用

目的 研究辐射增强启动子调控的野生型-p53抑癌基因系统联合照射对人肿瘤细胞系HeLa和A549细胞的特异性杀伤作用。方法 构建辐射增强启动子pE6（TATA）-p53,Western blot检测不同射线剂量诱导下人肺腺癌A549细胞系和人宫颈癌HeLa细胞系中P53蛋白的表达水平,筛选出最适的照射剂量;AnnexinV-FITC试剂盒检测肿瘤细胞系早期凋亡率;利用克隆形成实验检测此系统对肿瘤细胞放射敏感性的影响。结果 在HeLa和A549细胞中,P53蛋白表达均受放射线诱导增高,且在6 Gy时辐射诱导活性最高;实验组质粒的细胞早期凋亡率与转染对照组质粒的细胞早期凋亡率相比有明显提高（F=11.018、10.736,P<0.05）。HeLa细胞和A549细胞的放射增敏比（SER）分别为2.56和2.36。结论 辐射增强启动子调控的p53基因系统具有显著的诱导肿瘤细胞凋亡的作用,可以提高肿瘤细胞的辐射敏感性,对肿瘤的治疗提供了新思路。     

Effect of oxygen pressure during incubation with a 10B-carrier on 10B uptake capacity of cultured p53 wild-type and mutated tumor cells: dependency on p53 status of tumor cells and types of 10B-carriers

Purpose To evaluate the effect of oxygen pressure during incubation with a ¹⁰B-carrier on ¹⁰B uptake capacity of cultured p53 wild-type and mutated tumor cells.

Materials and methods Cultured human head and neck squamous cell carcinoma cell line transfected with mutant TP53 (SAS/mp53), or with a neo vector as a control (SAS/neo) was incubated with L-para-boronophenylalanine-¹⁰B (BPA) or sodium mercaptoundecahydrododecaborate-¹⁰B (BSH) as a ¹⁰B-carrier at the ¹⁰B concentration of 60 ppm for 24?h under aerobic (20.7% of oxygen) or hypoxic (0.28% of oxygen) conditions. Immediately after incubation, cultured tumor cells received reactor thermal neutron beams, and a cell survival assay was performed. ¹⁰B concentration of cultured SAS/neo or SAS/mp53 cells incubated under aerobic or hypoxic conditions was determined with a thermal neutron guide tube.

Results Hypoxic incubation significantly decreased ¹⁰B concentration of cultured cells with a clearer tendency observed following BPA than BSH treatment in both SAS/neo and SAS/mp53 cells. Following neutron beam irradiation, SAS/mp53 cells showed significantly higher relative biological effectiveness values than SAS/neo cells because of the significantly lower radiosensitivity of SAS/mp53 to γ-rays than SAS/neo cells.

Conclusion Oxygen pressure during incubation with a ¹⁰B-carrier had a critical impact on ¹⁰B uptake of cultured tumor cells.     

Restoration by glycerol of p53-dependent apoptosis in cells bearing the mutant p53 gene.

PURPOSE: Effective heat-induced cell death in cultured cells bearing a mutant p53 (mp53) gene was sought by glycerol treatment which led to conformational change from mp53 to wild-type p53 (wtp53) in p53-null murine fibroblasts transfected with mp53. MATERIALS AND METHODS: Heat sensitivity was measured using a colony-forming assay. For heat-induced apoptosis, gel electrophoresis was applied to detect DNA fragmentation and Hoechst33342 staining for apoptotic bodies. Glycerol (0.6 M) was applied to the cultured cells 48 h before heating at 44 degrees C in a water bath. RESULTS: Wtp53 transfectants (MT158/wtp53-1) were sensitive to heat stress compared with mp53 transfectants (MT158/mp53-2 cells), and the combined treatment with glycerol enhanced cell killing only in the MT158/mp53-2 cells. After glycerol pretreatment for 48 h, the subsequent heat treatment enhanced DNA fragmentation and apoptosis in MT158/mp53-2 cells, while DNA fragmentation and apoptotic bodies were not enhanced with heat treatment alone in these cells. In contrast, DNA fragmentation or apoptotic bodies were clearly observed in MT158/wtp53-1 cells 3-24h after heat treatment. Treatment with glycerol alone did not induce apoptosis in the transfectants. CONCLUSIONS: Glycerol appears to function as a chemical chaperone that restores mp53 to wtp53 function.