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.     

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 degrees C and KNK437 at a dose of 100 microM. 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.     

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.     

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.     

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.     

Induction of radioresistance to accelerated carbon-ion beams in recipient cells by nitric oxide excreted from irradiated donor cells of human glioblastoma

PURPOSE: To investigate whether nitric oxide excreted from cells irradiated with accelerated carbon-ion beams modulates cellular radiosensitivity against irradiation in human glioblastoma A-172 and T98G cells. MATERIALS AND METHODS: Western-blot analysis of inducible nitric oxide synthase, hsp72 and p53, the concentration assay of nitrite in medium and cell survival assay after irradiation with accelerated carbon-ion beams were performed. RESULTS: The accumulation of inducible nitric oxide synthase was caused by accelerated carbon-ion beam irradiation of T98G cells but not of A-172 cells. The accumulation of hsp72 and p53 was observed in A-172 cells after exposure to the conditioned medium of the T98G cells irradiated with accelerated carbon-ion beams, and the accumulation was abolished by the addition of an inhibitor for inducible nitric oxide synthase to the medium. The radiosensitivity of A-172 cells was reduced in the conditioned medium of the T98G cells irradiated with accelerated carbon-ion beams compared with conventional fresh growth medium, and the reduction of radiosensitivity was abolished by the addition of an inducible nitric oxide synthase inhibitor to the conditioned medium. CONCLUSIONS: Nitric oxide excreted from the irradiated donor cells with accelerated carbon-ion beams could modulate the radiosensitivity of recipient cells. These findings indicate the importance of an intercellular signal transduction pathway initiated by nitric oxide in the cellular response to accelerated heavy ions.     

Induction of radioresistance to accelerated carbon-ion beams in recipient cells by nitric oxide excreted from irradiated donor cells of human glioblastoma

Purpose : To investigate whether nitric oxide excreted from cells irradiated with accelerated carbon-ion beams modulates cellular radiosensitivity against irradiation in human glioblastoma A-172 and T98G cells. Materials and methods : Western-blot analysis of inducible nitric oxide synthase, hsp72 and p53, the concentration assay of nitrite in medium and cell survival assay after irradiation with accelerated carbon-ion beams were performed. Results : The accumulation of inducible nitric oxide synthase was caused by accelerated carbon-ion beam irradiation of T98G cells but not of A-172 cells. The accumulation of hsp72 and p53 was observed in A-172 cells after exposure to the conditioned medium of the T98G cells irradiated with accelerated carbon-ion beams, and the accumulation was abolished by the addition of an inhibitor for inducible nitric oxide synthase to the medium. The radiosensitivity of A-172 cells was reduced in the conditioned medium of the T98G cells irradiated with accelerated carbonion beams compared with conventional fresh growth medium, and the reduction of radiosensitivity was abolished by the addition of an inducible nitric oxide synthase inhibitor to the conditioned medium. Conclusions : Nitric oxide excreted from the irradiated donor cells with accelerated carbon-ion beams could modulate the radiosensitivity of recipient cells. These findings indicate the importance of an intercellular signal transduction pathway initiated by nitric oxide in the cellular response to accelerated heavy ions.     

反义表皮生长因子受体对人肺癌细胞放射敏感性的影响

目的 观察反义表皮生长因子受体(EGFR)对人肺腺癌细胞系放射敏感性的影响。方法在脂质体介导下用质粒pcDNA3-反义EGFR(pcDNA3-antiEGFR)转染spc-a-1细胞,并以未转染(对照组)和空质粒pcDNA3转染(pcDNA3组)细胞作对照。用G418筛选稳定表达的细胞克隆,以RT-PCR和Western blot检测转染后EGFR的mRNA及蛋白表达抑制情况,流式细胞仪检测转染后细胞周期及单次照射8 Gy后各组细胞凋亡比例。3组细胞分别给予6 MV X射线照射0、2、4、6和8 Gy,计算克隆形成率,拟合生长曲线。结果pcDNA3-antiEGFR组与对照组、pcDNA3组比较,EGFR mRNA和蛋白表达量明显减少,G₂/M期比例分别为(29.53±1.91)％、(13.7±1.30)%和(12.40±1.34)％,单次照射8Gy后,3个组细胞的凋亡率分别为(39.24±1.57)%、(13.79±0.63)%和(15.02±0.85)%。与对照组相比较,pcDNA3-antiEGFR组细胞的D₀、D_q、SF₂值分别由2.11、2.49和0.84降至1.19、0.15和0.32,提示抑制EGFR表达,可降低spc-a-1细胞对射线所致亚致死性损伤的修复能力。结论反义EGFR可以降低人肺癌spc-a-1细胞中EGFR的表达,改变细胞周期分布,促进凋亡,降低亚致死性损伤的修复能力,增加肺癌细胞系spc-a-1的放射敏感性。     

粉防己碱对放射线的增敏作用与机理研究

目的探讨粉防己碱(Tet)在结肠癌中对X射线的增敏作用及机理。方法采用克隆形成测定法、流式细胞术、Western Blotting与分裂指数法检测Tet在体外对X射线的增敏作用与机理。使用肿瘤生长延缓实验检测Tet在体内对X射线的增敏作用。结果结肠癌HT-29细胞在受到X射线照射后，明显阻滞于G2／M期；Tet可以清除这种阻滞，并起到显著的增敏作用，其增敏比为1．63。进一步研究显示，HT-29细胞在受到x射线照射后，其chkl蛋白表达水平升高，Cyclin Bl表达水平明显降低，分裂指数也明显降低；在用Tet处理后，chkl蛋白表达水平降低，Cyclin Bl的表达水平明显增高，分裂指数也增高。体内实验结果表明，Tet明显引起受照射小鼠结肠癌C26生长延缓。Tet与X射线联合使用组的肿瘤生长延缓天数是单用X射线组的1．63倍，对照组的2．82倍。结论Tet是一种细胞周期关卡清除剂．体外和体内对X射线都有明显的增敏作用．     

二氢青蒿素对人宫颈癌细胞株照射后周期的影响及相关机制

目的 观察二氢青蒿素及X射线对肿瘤细胞周期的影响,并研究其具体作用机制。方法 选用已知p53突变的人宫颈癌HeLa细胞,并以p53功能正常的人宫颈癌SiHa细胞作为对照。采用流式细胞术分析X射线(6 Gy)、二氢青蒿素(20及100 μmol/L)对两种细胞的细胞周期的影响;应用蛋白印迹法(Western blot)检测细胞周期相关蛋白表达量的变化。结果 X射线照射明显导致HeLa细胞G₂期阻滞,照射后G₂期细胞比例由14.45%上升至73.58%,在二氢青蒿素联合照射作用后,HeLa细胞G₂期细胞比例由单纯照射组的73.58%降至48.31%;而对照组SiHa细胞G₂期变化不明显。在单纯照射组,随着细胞G₂期阻滞的增加,HeLa细胞中Wee1蛋白表达量增加,Cyclin B1蛋白表达量降低,而在二氢青蒿素联合照射作用后,细胞内Wee1蛋白表达量较单纯照射组减少,Cyclin B1蛋白表达量较单纯照射组增高,与该药能去除电离辐射导致细胞G₂期阻滞过程相一致。结论 对于p53突变的人宫颈癌HeLa细胞,二氢青蒿素能抑制辐射所引起的细胞G₂期阻滞,其机理可能与细胞周期调控蛋白Wee1、Cyclin B1表达变化有关;对p53功能正常的SiHa细胞,辐射主要引起细胞G₁期阻滞,故二氢青蒿素对其周期的影响作用不明显。     

p53 Levels, Cell Cycle Kinetics and Radiosensitivity in Two SV40 Transformed Wi38VA13 Fibroblast Strains

BACKGROUND: The tumor suppressor protein p53 which can mediate an ionizing radiation-induced G1 arrest in mammalian cells, forms complexes with SV40 large T antigen (L-T-Ag). We have analyzed the p53 levels, the capability to undergo a G1 arrest and the radiosensitivity of two SV40 transformed fibroblast strains differing in their large T antigen expression. MATERIAL AND METHODS: One of the two strains (VA13F) is the commercially available form of Wi38VA13, the other (VA13E) arose spontaneously from the original one in our laboratory. Their p53 levels were measured by means of flow cytometry (FCM) and Western blot (WB) with two p53 antibodies (Ab-3, clone PAb240; Ab-6, clone DO-1; both Oncogene Science). Cell cycle distributions were determined flow cytometrically after BrdU labeling at regular time intervals after exposure to 250 kV X-rays. Radiosensitivity was assessed in a clonogenicity assay. RESULTS: The p53 levels of the two strains corresponded to their large T antigen expression, presumably due to complex formation between the two proteins. The strain with a high p53 level did not show a G1 arrest and had a relatively high radiosensitivity, whereas the strain with a low p53 level showed a significant G1 arrest and a lower radiosensitivity. CONCLUSION: These results suggest that 1. complex formation between the large T antigen and p53 reduces the latter's functionality; 2. in these two strains the G1 arrest is one of the factors determining radiosensitivity.     

不同LET射线对SMMC-7721细胞辐射敏感性与周期的影响

目的研究不同LET射线对SMMC-7721肝癌细胞辐射敏感性、周期进程和凋亡的影响，为重离子治疗癌症的临床应用积累基础数据。方法以0、0．5、1、2、4和8Gy的^12C^6＋离子及X射线分别照射处于指数生长期的SMMC-7721细胞，用克隆形成率测定细胞辐射敏感性，通过流式细胞术测定细胞DNA含量以确定各时相细胞的比例及细胞凋亡情况。结果^12C^6＋离子所致的SMMC-7721细胞存活率明显低于x射线。随着吸收剂量的增加和修复时间的延长，^12C^6＋离子能导致更显著的细胞S期阻滞、G2/M期阻滞延迟和细胞凋亡。结论与X射线相比，^12C^6＋离子辐照能更有效地杀伤HepG2肝癌细胞，并诱导其凋亡。     

蒿甲醚对人鼻咽癌细胞CNE-1放射敏感性的影响及其机制研究

目的 研究蒿甲醚(artemether)对人鼻咽癌CNE-1细胞放射敏感性的影响及其作用机理。方法 用MTT法检测蒿甲醚对CNE-1细胞生长的抑制效应;克隆形成法检测蒿甲醚对CNE-1细胞放射敏感性的影响;流式细胞仪检测不同处理因素对CNE-1细胞的细胞周期的影响;Western blot分析蒿甲醚对电离辐射诱导的CNE-1细胞中的细胞周期调控蛋白Cyclin B1和Wee1表达水平的影响。结果 蒿甲醚可抑制CNE-1细胞的增殖,且药物浓度和作用时间相关;20 μmol/L蒿甲醚对CNE-1细胞有放射增敏作用,其放射增敏作用随作用时间的延长而延长,作用24 h 后增敏作用最强,放射增敏比(SER)为1.481。流式细胞仪分析表明,单纯⁶⁰Co γ射线照射后G₂/M期细胞比例上升,而G₀/G₁期细胞比例下降;药物和射线联合作用后,与单纯照射组相比,G₀/G₁期细胞比例上升(t=4.59,P<0.05),G₂/M期细胞比例下降(t=10.60,P<0.05)。蒿甲醚可降低照射后CNE-1细胞内Wee1蛋白表达水平,提高Cyclin B1蛋白表达水平。结论 低浓度蒿甲醚对CNE-1 细胞有放射增敏作用;蒿甲醚通过改变细胞周期调控蛋白Cyclin B1和 Wee1表达水平,诱导G₂/M 期阻滞而发挥放射增敏作用。     

粉防己碱增加人乳腺癌细胞对X射线敏感性及其机理研究

目的 研究粉防己碱(Tet)与X射线对人乳腺癌细胞的作用和机理。方法 采用克隆形成分析法，流式细胞术，Western Blotting以及分裂指数法进行实验。结果 在p53突变型MCF-7/ADR细胞中，Tet显著增加X射线的杀伤作用，其增敏比为1．5l；在X射线照射后，细胞明显阻滞于G2期，Tet可以降低这种阻滞。而在p53野生型MCF-7细胞中，Tet增加X射线的杀伤作用不明显，增敏比为1．10；在X射线照射后，细胞阻滞于G1期，部分阻滞于G2期，加入Tet，对于这种阻滞作用降低也不明显。进一步研究显示，MCF-7/ADR细胞在受到X射线照射后，其Cyclin Bl与Cdc2蛋白表达水平明显降低；Tet可以逆转X射线对Cyclin Bl与Cdc2蛋白的表达的抑制作用。分裂指数结果显示Tet可以促使阻滞于G2期的MCF-7/ADR细胞进入M期。结论 Tet是一种G2期阻滞清除剂，能显著增加X射线对人乳腺癌细胞的杀伤作用，这种作用在p53突变型细胞中更明显。     

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‐γ‐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?µM 1?h before heating (42°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.     

电离辐射对不同肿瘤细胞细胞周期的影响

目的 研究电离辐射对不同肿瘤细胞细胞周期的影响为肿瘤放疗及化疗提供科学依据。方法 处于细胞周期各时相的细胞百分数采用流式细胞术进行检测。结果 研究表明：电离辐射作用后,ＨｅｌａＳ３和Ｓ１８０细胞发生了Ｓ和Ｇ２期阻滞,而ＤＬ－４细胞则发生Ｇ１和Ｇ２期阻滞,Ｂ１６各时相细胞数无列出较高的辐射抗性。结论 电离辐射作用后,不同肿瘤细胞的辐射抗性、即辐射敏感性有较大差异。其细胞周期的变化规律亦不相同。     

miR-21反义寡核苷酸对SHG-44放射增敏作用的体外研究

目的 探讨抑制miR-21表达对SHG-44人脑胶质瘤细胞系放射敏感性的影响及其机制。方法 脂质体介导采用瞬间转染法转染反义miR-21寡核苷酸(AS-miR-21)及阴性对照寡核苷酸于胶质瘤SHG-44细胞中。设空白对照组、阴性对照转染组及AS-miR-21转染组,分别给予6 MeV X射线单次照射0、1、2、4、6和8 Gy,采用成克隆实验计算放射增敏比,并绘制细胞存活曲线。流式细胞仪检测上述3组及联合单次6 Gy照射后细胞凋亡率、细胞周期变化。结果 转染AS-miR-21组的D₀、D_q较空白对照组及阴性转染组降低,放射增敏比SER_D0、SER_Dq分别为1.32和2.10。细胞周期分析示,转染组较空白对照及阴性对照组的G₀/G₁期比例增高,S期比例降低(t =8.18、-4.52,P<0.05)。凋亡分析示,单纯照射组、AS-miR-21转染组及照射联合AS-miR-21转染组的早期凋亡率均高于对照组(t =20.14、11.11、50.07, P <0.05)。结论 AS-miR-21可增加胶质瘤SHG-44细胞系放射敏感性,其机制可能与促进细胞凋亡及周期再分布有关。     

脑胶质瘤SHG-44细胞株照射后子代辐射敏感性

目的 探讨脑胶质瘤SHG-44细胞株照射子代生长特性及辐射敏感性变化。方法 培养人脑胶质瘤SHG-44细胞株照射后的存活子代细胞,测定其群体倍增时间,并进行集落形成实验和流式细胞仪检测,分析其辐射敏感性和细胞周期变化。结果 SHG-44细胞群体倍增时间为(22.78±2.61) h,SHG-44细胞经6 MV X射线10 Gy照射后存活子代SHG-44.10细胞群体倍增时间为(30.46±2.73) h (F=7.878,P<0.05)。SHG-44细胞和SHG-44.10细胞再次2 Gy照射后的存活分数分别为70.8%、80.6%。与SHG-44细胞相比,SHG-44-10细胞在G2/M期比例减少,S期比例增多。结论 SHG-44细胞照射后存活子代细胞增殖延缓,辐射敏感性下降。     

降低B7-H3蛋白对受照肺癌细胞A549细胞周期和凋亡的影响

目的观察用小干扰RNA（siRNA）敲降B7-H3蛋白的表达对人肺癌细胞A549的细胞周期和凋亡的影响。 方法培养人肺癌A549细胞,将B7-H3蛋白siRNA瞬时转染于A549细胞（称为siB7-H3转染组）。实验分为4组,即对照组、siB7-H3转染组、照射组、照射+siB7-H3转染组。采用137Cs γ射线一次性照射,照射剂量为4 Gy;用Western blotting法和实时定量PCR分别检测B7-H3蛋白和mRNA的表达;流式细胞仪检测细胞周期和细胞凋亡的改变。 结果与对照组相比,siB7-H3转染组的B7-H3蛋白水平明显降低,mRNA表达量也明显低于对照组且差异有统计学意义（t=-4.222,P=0.013）。siB7-H3转染组细胞的G0/G1期细胞阻滞,S和G2/M期细胞数量减少;与对照组相比,照射组出现轻微的G0/G1期阻滞和明显的G2/M期细胞阻滞,照射+siB7-H3转染组的G0/G1、G2/M期均有明显的阻滞。照射后48 h,与对照组相比,照射组细胞的坏死率和凋亡率明显升高,siB7-H3转染组和照射+siB7-H3转染组的坏死率和凋亡率均无明显改变。 结论降低肺癌A549细胞中B7-H3蛋白表达水平可明显增加辐射诱导的G0/G1期阻滞,从而提示B7-H3表达水平的改变可能通过调节G0/G1细胞周期检查点而对肺癌细胞放射敏感性产生重要的调节功能。