Famitinib enhances nasopharyngeal cancer cell radiosensitivity by attenuating radiation-induced phosphorylation of platelet-derived growth factor receptor and c-kit and inhibiting microvessel formation

Purpose: Famitinib is a novel tyrosine kinase inhibitor. We investigated the effects of famitinib on the radiosensitivity of human nasopharyngeal carcinoma (NPC) cell radiosensitivity in vitro and in vivo, and explored its possible mechanisms.

Materials and methods: Human nasopharyngeal carcinoma cell line (CNE-2) were treated with famitinib and radiation, and analyzed by3-(4,5-dimethylthaizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), clonogenic survival assay, and Western blot. A xenograft model using CNE-2 cells was established to analyze the effects of famitinib and radiation on tumor volume and microvessel density (MVD).

Results: Famitinib dose-dependently inhibited CNE-2 cells growth and significantly reduced clonogenic survival (p < 0.05), with a sensitivity enhancement ratio (SER) of 1.45. The tumor inhibition rate of the combined treatment group was 91%, which was significantly higher than the radiation group (35%, p < 0.05) and famitinib group (46%, p < 0.05). Famitinib attenuated radiation-induced phosphorylation of the platelet-derived growth factor receptor (PDGFR) and stem cell factor (c-kit) at 0, 30, 60 min after radiation treatment. Furthermore, radiation combined with famitinib decreased tumor MVD (p < 0.05).

Conclusions: Famitinib significantly increased CNE-2 cell radiosensitivity in vitro and in vivo by attenuating radiation-induced PDGFR and c-kit phosphorylation and by inhibiting microvessel formation.     

鼻咽癌细胞放射敏感性的比较蛋白质组学研究

目的 探讨与鼻咽癌细胞放射敏感性相关的并可能预测鼻咽癌细胞放射敏感性的蛋白质。方法 以人鼻咽癌细胞株CNE-2诱导建立放射抗拒的鼻咽癌细胞株CNE-2(R743),通过克隆形成实验及流式细胞术检测以比较CNE-2和CNE-2(R743)细胞的放射敏感性及细胞周期的特征。提取细胞总蛋白质,进行双向凝胶电泳,Image Master 7.0图像分析软件分析图谱以识别差异蛋白质点,MALDI-TOF/TOF肽质量指纹图谱分析,蛋白质质谱数据库搜索鉴定差异蛋白。应用RT-PCR和Western blot验证2种细胞中相关差异蛋白的表达。 结果 得到7个差异表达较显著的蛋白质,与CNE-2相比较,CNE-2(R743)细胞中6个上调,1个下调。Western blot及 RT-PCR证实膜联蛋白A2、原肌球蛋白及糖调节蛋白78在CNE-2(R743)的表达均上调(t=24.22、24.20和29.19,P<0.05),与蛋白质组学结果一致。结论 不同放射敏感性鼻咽癌细胞存在差异表达的蛋白质,其中膜联蛋白A2、原肌球蛋白及糖调节蛋白78有可能成为预测鼻咽癌细胞放射敏感性的候选生物标志物。     

鼻咽癌细胞系CNE-2Z放射敏感性的异质性及其机理探讨

目的　进一步证实人鼻咽癌细胞系CNE 2Z确实存在着放射敏感性的异质性 ,并探讨其有关机理。方法　观察人鼻咽癌细胞系CNE 2Z亚克隆株H5和S1的裸鼠移植瘤放射敏感性的不同 ;用流式细胞仪、荧光显微镜、Western blot检测H5和S1凋亡能力的不同及凋亡相关蛋白表达的差异 ;用3H掺入法说明DNA合成抑制率与放射敏感性的不同 ;用RT PCR观察相关癌基因 (fas、p5 3)的表达与放射敏感性的关系。结果　鼻咽癌细胞系CNE 2Z中确实存在放射敏感性的异质性 ,H5、S1两种细胞的凋亡率都随着照射后时间的延长而增加 ,但H5比S1高且差异有统计学意义 (P <0 0 5 )。两种细胞的DNA合成率于放射前无差别 ;照射后都较放射前受到抑制 (P <0 0 5 ) ,H5受抑制程度大 (P <0 0 5 )。H5与S1细胞fas、p5 3基因的表达差异无统计学意义 (P >0 0 5 )。结论　本实验再次证实了鼻咽癌细胞系CNE 2Z确实存在放射敏感性异质性 ,并证实了其异质性的机理与细胞受射线照射后引起凋亡的能力不同有关、与DNA合成的抑制率成正相关、与癌基因fas、p5 3的表达无关。     

沉默STAT1基因增强放射抗拒鼻咽癌CNE-2R细胞放射敏感性

目的 研究基因STAT1沉默对人放射抗拒鼻咽癌细胞放射敏感性的影响.方法 慢病毒介导的STAT1基因转染放射抗拒鼻咽癌细胞CNE-2R,荧光定量RT-PCR技术检测沉默效果.MTT法检测转染前后细胞增殖活性,流式细胞技术检测细胞周期及凋亡,克隆形成实验检测转染前后细胞放射敏感性变化.结果 慢病毒转染后放射抗拒鼻咽癌细胞STAT1表达降低(F=429.87,P<0.05)、细胞生长抑制(F3=.88~4.63,P<0.05)、凋亡率增加(F=38.13,P<0.05)、放射敏感性增加(F=252.80,P<0.05)、细胞周期中G₀/G₁、S和G₂/M期未见明显差异(P>0.05).结论 沉默STAT1基因能增加放射抗拒鼻咽癌细胞 CNE-2R的放射敏感性.     

沉默GRAMD1A及抑制STAT5信号通路对肝癌细胞Huh7放射敏感性的影响

目的 探讨沉默GRAMD1A及抑制STAT5信号对肝癌细胞Huh7放射敏感性的影响,旨在为肝癌临床联合治疗提供新思路。方法 慢病素感染构建沉默GRAMD1A的Huh7细胞株,采用qPCR和Western blot进行验证,qPCR和荧光素酶报告实验检测沉默GRAMD1A后对Huh7细胞中STAT5及其下游基因表达的影响;以克隆形成率和细胞凋亡为指标检测沉默GRA株。结果 构建后的Huh7细胞经2 Gy照射后,沉默GRAMD1A联合照射组细胞克隆形成能力较阴性对照联合照射组显著降低,差异有统计学意义（t=8.494,P<0.05）;沉默GRAMD1A联合照射组细胞凋亡较阴性对照联合照射组显著增加,差异有统计学意义（t=3.560,P<0.05）。沉默GRAMD1A后Huh7细胞放射敏感性明显增加,且细胞中STAT5及其下游基因表达显著降低。SH-4-54抑制剂联合照射组较二甲基亚砜联合照射组细胞克隆存活能力显著降低,差异有统计学意义（t=8.660,P<0.05）,SH-4-54抑制STAT5通路后,Huh7细胞放射敏感性显著增加。结论 沉默GRAMD1A可能通过STAT5信号通路增强肝癌细胞Huh7的放射敏感性,表明GRAMD1A在肝癌发生发展中起重要作用,将可能为肝癌靶向治疗及联合治疗提供新靶点。     

鼻咽癌细胞辐射抗拒的比较蛋白质组学分析

目的 通过分离并鉴定具有不同辐射抗拒的人鼻咽癌细胞株CNE-2R与CNE-2的差异表达蛋白,探讨与鼻咽癌细胞辐射抗拒相关的分子机制.方法 分别提取鼻咽癌辐射抗拒细胞株CNE-2R及其亲本细胞株CNE-2的总蛋白,采用双向凝胶电泳分离,经软件分析识别差异表达蛋白点,应用基质辅助激光解析电离飞行时间串联质谱技术(MALDI-TOF-MS)鉴定差异蛋白质.结果 两种不同辐射敏感性的鼻咽癌细胞株CNE-2R和CNE-2中,共筛选出差异表达明显的蛋白质点有32个,其中11个蛋白质被鉴定成功,在CNE-2R中上调的蛋白有3个,下调的蛋白有8个.结论 不同辐射敏感性的鼻咽癌细胞的差异表达蛋白,主要涉及调节凋亡、DNA损伤与修复、细胞周期调控、RNA转录、细胞信号转导、细胞骨架组成及辐射应激反应等多个方面.     

自噬活性的降低增加人鼻咽癌CNE-2细胞的放射敏感性

目的 研究自噬与人鼻咽癌CNE-2细胞放射敏感性之间的关系。方法 采用慢病毒介导的RNA干扰技术建立稳定沉默自噬相关基因ATG5的人鼻咽癌CNE-2细胞系,实验分为未转染的CNE-2细胞组(对照组)、转染NC-shRNA的CNE-2细胞组(NC组)及转染ATG5-shRNA的CNE-2细胞组(ATG5组),应用CCK-8法、流式细胞术及克隆形成实验检测细胞增殖、凋亡及放射敏感性的变化。结果 CCK-8实验结果显示,与对照组和NC组相比,各剂量点ATG5组的细胞存活率均显著降低(F=3.755、46.086、8.609、44.160,P<0.05),绘制细胞生存曲线可见下调ATG5的表达后可以增加CNE-2细胞的放射敏感性;流式细胞术结果显示,经6 Gy X射线照射后,ATG5组细胞的凋亡率较NC组及对照组明显升高(F=394.876,P<0.05);克隆形成实验结果提示沉默ATG5基因可增加鼻咽癌CNE-2细胞的放射敏感性。结论 降低鼻咽癌CNE-2细胞的自噬活性可以增强其放射敏感性。     

Exploring the radiosensitizing potential of magnetotherapy: a pilot study in breast cancer cells

Abstract

Aim: To explore the influence of electromagnetic fields (EMFs) on the cell cycle progression of MDA-MB-231 and MCF-7 breast cancer cell lines and to evaluate the radiosensitizing effect of magnetotherapy during therapeutic co-exposure to EMFs and radiotherapy.

Material and methods: Cells were exposed to EMFs (25, 50 and 100?Hz; 8 and 10?mT). In the co-treatment, cells were first exposed to EMFs (50?Hz/10?mT) for 30?min and then to ionizing radiation (IR) (2?Gy) 4?h later. Cell cycle progression and free radical production were evaluated by flow cytometry, while radiosensitivity was explored by colony formation assay.

Results: Generalized G1-phase arrest was found in both cell lines several hours after EMF exposure. Interestingly, a marked G1-phase delay was observed at 4?h after exposure to 50?Hz/10?mT EMFs. No cell cycle perturbation was observed after repeated exposure to EMFs. IR-derived ROS production was enhanced in EMF-exposed MCF-7 cells at 24?h post-exposure. EMF-exposed cells were more radiosensitive in comparison to sham-exposed cells.

Conclusions: These results highlight the potential benefits of concomitant treatment with magnetotherapy before radiotherapy sessions to enhance the effectiveness of breast cancer therapy. Further studies are warranted to identify the subset(s) of patients who would benefit from this multimodal treatment.     

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.     

Upregulating DAB2IP expression via EGR-1 inhibition,a new approach for overcoming fractionated-irradiation-induced cross-tolerance to ionizing radiation and mitomycin C in tumor cells

Purpose: To evaluate the effect of fractionated irradiation (FI) on tumor cells’ sensitivity to ionizing radiation (IR) and antineoplastic drugs, and examine the potential of early growth response-1 (EGR-1) inhibition to sensitize tumor cells to IR.

Materials and methods: PC3 and HepG2 cells were subjected 10 times to γ-rays at 2?Gy. The surviving cells were named PC3/R and HepG2/R, respectively. The cells’ sensitivity to irradiation and chemotherapeutic drugs, including cisplatin (PT), doxorubicin (DOX), mitomycin C (MMC) and 5-fluorouracil (5-FU), were identified by colony formation assay and MMT method, respectively. Quantitative real-time polymerase chain reaction (RT-qPCR) analysis was utilized to compare the difference of gene expression between radioresistant cells and parental cells. The small interfering RNA system was implemented to inhibit endogenous EGR-1 expression in radiation-resistant cells. Western blot was employed to identify the possible mechanism by which EGR-1 regulates cells’ radiosensitivity.

Results: FI induced cross-resistant to IR and MMC in tumor cells. Along with the reduction of ovarian cancer-2/disabled homolog 2 (DOC-2/DAB2) interactive protein (DAB2IP) expression, EGR-1 gene was upregulated in FI-treated cells. On the other hand, downregulation of EGR-1 gene expression sensitized radioresistant cells to IR accompanied by DAB2IP overexpression and STAT3 inactivation. In addition, NF-κB inhibitor, BAY11-7082 enhanced resistant cells’ radiosensitivity and chemosensitivity.

Conclusions: Conventionally FI has a higher risk of forming acquired radioresistance (ARR) in vitro. EGR-1 gene-targeted drug design could be an effective strategy to overcome DAB2IP-dysregulation-induced ARR in tumor patients.     

Enhanced cellular radiosensitivity induced by cofilin-1 over-expression is associated with reduced DNA repair capacity

Abstract

Purpose: A previous report has indicated that over-expression of cofilin-1 (CFL-1), a member of the actin depolymerizing factor (ADF)/cofilin protein family, enhances cellular radiosensitivity. This study explores the involvement of various DNA damage responses and repair systems in the enhanced cellular radiosensitivity as well as assessing the role of CFL-1 phosphorylation in radiosensitivity.

Materials and methods: Human non-small lung cancer H1299 cells harboring a tet-on gene expression system were used to induce exogenous expression of wild-type CFL-1. Colony formation assays were used to determine cell survival after γ-ray exposure. DNA damage levels were determined by Comet assay. DNA repair capacity was assessed by fluorescence-based DNA repair analysis and antibody detection of various repair proteins. The effects of CFL-1 phosphorylation on radiation responses were explored using two mutant CFL-1 proteins, S3D and S3A. Finally, endogenous CFL-1 phosphorylation levels were investigated using latrunculin A (LA), cytochalasin B (CB) and Y27632.

Results: When phosphorylatable CFL-1 was expressed, radiosensitivity was enhanced after exposure to γ-rays and this was accompanied by DNA damage. Phosphorylated histone H2AX (γ-H2AX) and p53-binding protein-1 (53BP1) foci, as well as Chk1/2 phosphorylation, were apparently suppressed, although ataxia telangiectasia mutated (ATM) kinase activation was apparently unaffected. In addition, two radiation-induced double-strand break (DSB) repair systems, namely homologous recombination repair (HRR) and non-homologous end joining (NHEJ), were suppressed. Moreover, over-expression of CFL-1 S3D and CFL-1 S3A both enhanced radiosensitivity. However, enhanced radiosensitivity and reduced γ-H2AX expression were only detected in cells treated with LA which increased endogenous phospho-CFL-1, and not in cells treated with Y27632, which dephosphorylates CFL-1.

Conclusion: CFL-1 over-expression enhances radiosensitivity and this is associated with reduced DNA repair capacity. Although phosphorylated CFL-1 seems to be involved in radiosensitivity, further studies are required to address the importance of CFL-1 activity to the regulation of radiosensitivity.     

1-Methylxanthine enhances the radiosensitivity of tumor cells

Purpose:?To determine the efficacy of a caffeine derivative 1-methylxanthine (1-MTX) in increasing radiosensitivity of cancer cells and elucidate the underlying mechanisms in vitro.

Materials and methods:?RKO human colorectal cancer cells carrying wild type protein 53 kDa (p53) were incubated with 3 mM 1-MTX for 30 min, exposed to 4 Gy ionizing radiation, and further incubated with 1-MTX for three days. The clonogenic cell death was determined, and the cell cycle distribution and apoptosis were studied with flow cytometry at different times after irradiation. The DNA double strand break (DNA DSB) was examined using phosphorylated Histone2A (γ-H2AX) foci formation, and the expression/activity of checkpoint 2 kinase (Chk2), cell division cycle 25 (Cdc25) phosphatase and cyclin B1/Cdc2 kinase were also investigated using western blotting and in vitro kinase assays.

Results:?The treatment with 3 mM 1-MTX increased the radiation-induced clonogenic and apoptotic cell death. The radiation-induced phosphorylation of Chk2 and Cdc25c and the radiation-induced increase in the cyclin B1/Cdc2 kinas activity were little affected by 1-MTX. The radiation-induced G2/M arrest was only slightly shortened and the expression of radiation-induced γ-H2AX was markedly prolonged by 1-MTX.

Conclusions:?1-MTX significantly increased the radiosensitivity of RKO human colorectal cancer cells carrying wild type p53 mainly by inhibiting the repair of radiation-induced DNA DSB without causing significant alteration in radiation-induced G2/M arrest. Such a radiosensitization occurred at 1-MTX concentrations almost non-toxic to the target tumor cells.     

The ATM inhibitor KU55933 sensitizes radioresistant bladder cancer cells with DAB2IP gene defect

Abstract

Purpose: Our preliminary results showed that differentially expressed in ovarian cancer-2/disabled homolog 2 (DOC-2/DAB2) interactive protein (DAB2IP), a putative tumor suppressor gene, is down-regulated in bladder cancer (BCa) with aggressive phenotypes. In this study, we investigated how DAB2IP knockdown influenced BCa cell response to ionizing radiation (IR) and discussed possible ways to enhance cell radiosensitivity.

Methods and materials: The small interfering RNA (siRNA) system was implemented to inhibit endogenous DAB2IP expression in two human BCa cell lines, T24 and 5637. Cell sensitivity to IR alone or combined treatment was measured by a colony formation assay (CFA). Western blot was used to determine the phosphorylation levels of ataxia-telangiectasia mutated (ATM), catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) and related DNA damage repair (DDR) proteins. Immunofluorescence as well as a flow cytometry assay were employed to detect DNA double-strand break (DSB) repair and cell cycle distribution, respectively.

Results: DAB2IP-knockdown of BCa cells (i.e., siDAB2IP) exhibit increased clonogenic survival in response to IR compared with control cells (i.e., siCON) expressing an endogenous level of DAB2IP. The mechanism in siDAB2IP cells could be explained by elevated ATM expression and activation, increased S phase cell distribution as well as faster DSB repair kinetics. 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one (KU55933) significantly sensitized siDAB2IP cells to IR due to inhibition of the phosphorylation of ATM and its downstream targets following IR and slower DSB repair kinetics.

Conclusions: Loss of DAB2IP expression in BCa cells signifies their radioresistance. KU55933, which suppresses ATM phosphorylation upon irradiation, could be applied in the radiotherapy of BCa patients with a DAB2IP gene defect.     

Radiosensitization of tumor cells by modulation of ATM kinase

Purpose: To elucidate the relationship between the radiation-induced activation of ataxia telangiectasia mutated (ATM) kinase, G2 arrest and the caffeine-induced radiosensitization.

Method: RKO cells (human colorectal cancer cells) and ATM kinase over-expressing RKO/ATM cells were used. The cellular radiosensitivity was determined with clonogenic survival assay and the cell cycle progression, including G2 arrest, was studied with flow cytometry. The activity of ATM kinase, check point 2 (Chk2) kinase and cycline B1/cell division cycle 2 (Cdc2) kinase was investigated. The radiosensitivity of RKO xenografts grown in nude mice was studied.

Results: RKO/ATM cells were radioresistant as compared with RKO cells. There was a greater increase in ATM kinase activity and G2 arrest in RKO/ATM cells than in RKO cells. Caffeine also sensitized both RKO cells and RKO/ATM cells to radiation. The caffeine treatment suppressed the radiation-induced activation of ATM kinase, suppressed the activation of Chk2 kinase and inhibited the accumulation of cells in G2 phase. The activity of cycline B1/Cdc2 kinase increased earlier but decayed rapidly in the presence of caffeine. Caffeine enhanced radiation-induced growth delay of RKO xenografts.

Conclusions: Caffeine inhibited the radiation-induced activation of ATM kinase, thereby preventing the accumulation of cells in G2 phase. Consequently, radiosensitivity of cells increased in the presence of caffeine both in vitro and in vivo.     

Hypoxia-regulated p53 and its effect on radiosensitivity in cancer cells

Purpose: To determine the response of tumor suppressor p53 to hypoxia in different tumor cell lines and the involvement of p53 activity regulation in the effect of hypoxia on tumor cell sensitivity to radiation and hyperthermia.

Materials and methods: Three tumor cell lines with functional p53 were treated with chronic or cyclic hypoxia followed by radiation or hyperthermia to investigate p53 activity and cell survival. Flow cytometry was used to investigate the effect of hypoxia-induced cell cycle arrest on radiosensitivity in KHT-C (mouse fibrosarcoma) cells. Transient transfection was performed to determine the role of altered p53 activity in KHT-C and SCC VII (mouse squamous-cell carcinoma) radiosensitivity.

Results: Aerobic radiosensitivity was decreased in KHT-C and SCC VII cells after in vitro chronic or cyclic hypoxia pretreatment, but in HT1080 cells, it was slightly increased after chronic hypoxia, and was unchanged after acute hypoxia pretreatment. Decreased radiosensitivity in hypoxia-pretreated KHT-C and SCC VII cells was unlikely due to hypoxia-induced cell cycle arrest, but rather seemed to be associated with increased expression of Mdm2 (mouse double minute-2) and decreased p53. Furthermore, hypoxia pretreatment inhibited the activation of p53 by radiation. Similar results were observed in hyperthermia treated KHT-C cells. Finally, decreased radiosensitivity was observed in both KHT-C and SCC VII cells transiently transfected with Mdm2 or anti-sense p53 cDNA.

Conclusion: We demonstrated that hypoxia may decrease tumor cell radiosensitivity through the suppression of p53 activity in some tumor cell lines. These results suggested the response of p53 to hypoxia can be cell type specific and contribute to radiosensitivity of hypoxic cells.     

阻断血管内皮生长因子表达对食管癌细胞放射敏感性的影响

目的 观察转染反义VEGF cDNA质粒和反义寡核苷酸后,TE-1食管癌细胞在体外的生长状况、VEGF表达水平及其放射敏感性的变化。方法 分别用反义VEGF cDNA质粒及空载体质粒和反义VEGF寡核苷酸经Lipofectamine^TM2000介导转染TE-1细胞,然后经γ射线照射。采用RT-PCR、Western blotting、流式细胞术、MTT法和克隆形成实验分别检测4组细胞照射前后VEGF基因的表达、凋亡率和细胞周期变化、细胞增殖情况以及转染细胞放射敏感性的变化。结果 将反义VEGF cDNA和反义寡核苷酸成功转入食管癌TE-1细胞后,VEGF表达水平明显降低,细胞增殖反应、细胞周期无明显变化,亦未见明显凋亡发生。照射后4组细胞增殖情况未见明显差异;反义组细胞凋亡率略有上升, 放射敏感性增加。结论 转染反义VEGF cDNA质粒和反义寡核苷酸可抑制食管癌TE-1细胞VEGF表达,联合放射线作用,对TE-1细胞的增殖无明显影响,而对其放射敏感性有增强作用。     

Time and dose-dependent activation of p53 serine 15 phosphorylation among cell lines with different radiation sensitivity

Purpose:?Proper detection of DNA damage and signal transduction to other proteins following irradiation (IR) is essential for cellular integrity. The serine 15 (Ser15) on p53 is crucial for p53 stabilization and a requirement for transient and permanent cell cycle arrest. Here, we sought to determine the relationship between p53 serine 15 phosphorylation (p53-p-Ser15) on cellular sensitivity and if this modification is associated with DNA double-strand break (DSB) repair.

Materials and methods:?Eight lymphoblastoid cell lines including ataxia-telangiectasia (A-T), Nijmegen breakage syndrome (NBS) and radiosensitive patient derived cell lines were irradiated with 1 Gy, 2 Gy and 5 Gy. Then growth inhibition, p53 induction and phosphorylation on Ser15 as assessed by immunoblotting and DNA DSB repair as assessed by constant field gel electrophoresis were examined.

Results:?Phosphorylation of p53 at Ser15 in control cells rapidly increased, peaking at 3 – 6 hours and then sustained a low level of phosphorylation for up to 6 days following IR. For these cell lines, the amount of p53-p-Ser15 corresponded to the sensitivity of cells and the amount of DNA DSB. In A-T cells, p53-p-Ser15 was reduced in spite of increased DNA DSB. NBS cells had similar phosphorylation dynamics as the control cell line, which was not consistent with their increased sensitivity. Radiosensitive patients' cell lines differed only slightly from controls.

Conclusions:?Cells that are competent in signal transduction have p53-p-Ser15 kinetics corresponding to cellular radiosensitivity as assessed by clonogenicity and DNA DSB repair, and cells impaired in signal transduction lack this correspondence. Therefore, using p53-p-Ser15 as a general marker of radiation sensitivity has confounding factors which may impair proper radiosensitivity prediction.     

RNA干扰抑制c-jun基因表达对放射抗拒人鼻咽癌细胞系CNE-2R放射敏感性的影响

目的 利用RNA干扰技术抑制放射抗拒人鼻咽癌细胞系CNE-2R中高表达的c-jun基因,研究其对CNE-2R细胞放射敏感性的影响及其潜在机制.方法 设计合成3组特异性针对c-jun基因的siRNA,构建慢病毒vshRNA载体,感染CNE-2R细胞,采用RT-PCR和Western blot方法检测各组对目的基因的抑制效果;并通过克隆形成实验测定其放射敏感性,CCK-8实验检测细胞的存活能力,流式细胞术检测细胞凋亡率.结果 重组慢病毒c-jun-RNAi-LV2能明显下调CNE-2R细胞中c-jun的mRNA和蛋白水平的表达(F=217.20、42.07, P<0.05).6 MV X射线联合c-jun-RNAi-LV2组在0、2、4、6和8 Gy照射后细胞的吸光度值均显著低于未转染组和阴性对照组(F=42.70~200.67, P<0.05).克隆形成实验显示,c-jun-RNAi-LV2组与未转染组及阴性对照组相比,SF₂、D₀和D_q值均减小,放射增敏比(SER_D0)为1.41.细胞凋亡实验结果显示,未经照射的c-jun-RNAi-LV2组与联合2 Gy照射的凋亡率分别为(20.93±1.99)%和(38.17±0.83)%,均高于未转染组和阴性对照组的凋亡率[0 Gy:(10.97±0.70)%和(20.43±0.25)%;2 Gy:(10.80±1.25)%和(19.53±1.50)%;F=50.54、330.14, P<0.05].结论 RNA干扰技术可有效抑制放射抗拒人鼻咽癌细胞系CNE-2R中高表达的c-jun基因,使细胞放射敏感性增高,其作用可能与c-jun基因的下调使细胞增殖能力减弱,细胞凋亡增多有关.