α-Lipoic Acid Inhibits Apoptosis by Suppressing the Loss of Ku Proteins in Helicobacter pylori-Infected Human Gastric Epithelial Cells

Helicobacter pylori (H. pylori) is a Gram-negative bacterium that colonizes the gastric mucosa and triggers various stomach diseases. H. pylori induces reactive oxygen species (ROS) production and DNA damage. The heterodimeric Ku70/Ku80 protein plays an essential role in the repair of DNA double-strand breaks (DSB). Oxidative stress stimulate apoptosis and DNA damage that can be repaired by Ku70/80. However, excessive reactive oxygen species (ROS) can cause Ku protein degradation, resulting in DNA fragmentation and apoptosis. α-lipoic acid (α-LA), which is found in organ meats such as liver and heart, spinach, broccoli, and potatoes, quenches free radicals, chelates metal ions, and reduces intracellular DNA damage induced by oxidative stress. Here, we investigated whether H. pylori decreases Ku70/80 and induces apoptosis, and whether α-LA inhibits changes induced by H. pylori. We analyzed ROS, DNA damage markers (γ-H2AX, DNA fragmentation), levels of Ku70/80, Ku–DNA binding activity, Ku80 ubiquitination, apoptosis indices (Bcl-2, Bax, apoptosis-inducing factor (AIF), and caspase-3), and viability in a human gastric epithelial adenocarcinoma cell line (AGS). H. pylori increased ROS, DNA damage markers, Ku80 ubiquitination, and consequently induced apoptosis. It also decreased nuclear Ku70/80 levels and Ku–DNA-binding activity; increased Bax expression, caspase-3 cleavage, and truncated AIF; but decreased Bcl-2 expression. These H. pylori-induced alterations were inhibited by α-LA. The antioxidant N-acetylcysteine and proteasome inhibitor MG-132 suppressed H. pylori-induced cell death and decreased nuclear Ku70/80 levels. The results show that oxidative stress induced Ku70/80 degradation via the ubiquitin–proteasome system, leading to its nuclear loss and apoptosis in H. pylori-infected cells. In conclusion, α-LA inhibited apoptosis induced by H. pylori by reducing ROS levels and suppressing the loss of Ku70/80 proteins in AGS cells.     

Diminished or inversed dose-rate effect on clonogenic ability in Ku-deficient rodent cells

The biological effects of ionizing radiation, especially those of sparsely ionizing radiations like X-ray and γ-ray, are generally reduced as the dose rate is reduced. This phenomenon is known as ‘the dose-rate effect’. The dose-rate effect is considered to be due to the repair of DNA damage during irradiation but the precise mechanisms for the dose-rate effect remain to be clarified. Ku70, Ku86 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are thought to comprise the sensor for DNA double-strand break (DSB) repair through non-homologous end joining (NHEJ). In this study, we measured the clonogenic ability of Ku70-, Ku86- or DNA-PKcs-deficient rodent cells, in parallel with respective control cells, in response to high dose-rate (HDR) and low dose-rate (LDR) γ-ray radiation (~0.9 and ~1 mGy/min, respectively). Control cells and murine embryonic fibroblasts (MEF) from a severe combined immunodeficiency (scid) mouse, which is DNA-PKcs-deficient, showed higher cell survival after LDR irradiation than after HDR irradiation at the same dose. On the other hand, MEF from Ku70^−/− mice exhibited lower clonogenic cell survival after LDR irradiation than after HDR irradiation. XR-V15B and xrs-5 cells, which are Ku86-deficient, exhibited mostly identical clonogenic cell survival after LDR and HDR irradiation. Thus, the dose-rate effect in terms of clonogenic cell survival is diminished or even inversed in Ku-deficient rodent cells. These observations indicate the involvement of Ku in the dose-rate effect.     

Enhancement of radiosensitivity by roscovitine pretreatment in human non-small cell lung cancer A549 cells

Roscovitine has been reported to have anti-proliferative properties and is in process of undergoing clinical trials. In addition to its intrinsic anticancer properties, it has recently been suggested that roscovitine may also enhance the activity of traditional chemo- and radio- therapies in certain cancer cell lines. The purpose of this study was to define the activity of roscovitine in increasing radiosensitivity of human non-small cell lung cancer (NSCLC) cell line A549 cells in vitro. A549 cells were exposed to ionizing radiation (IR) of gamma-ray with or without roscovitine pretreatment. Clonogenic assay was performed and cell cycle and apoptosis were analyzed by flow cytometry. Expression of PARP, Ku70 and Ku80 proteins was detected by Western blot. The active form of caspase-3 positive cells were measured by flow cytometry. Our results showed that roscovitine caused dose-dependent apoptosis in A549 cells. Pretreatment with minimally toxic concentration of roscovitine significantly radiosensitized A549 cells by inhibiting colony formation. We then examined potential mechanisms that may contribute to the enhanced radiation response induced by roscovitine. Our results showed that the combination treatment significantly induced apoptosis in A549 cells compared to roscovitine or IR treatment alone. Meanwhile, in the co-treatment group, the percentage of cells with the active form of caspase-3 was markedly increased, while roscovitine or IR alone had little effect. Roscovitine decreased S phase cells when used alone or in sequential combination with IR. Furthermore, this combination treatment blocked DNA repair process after IR, indicated by down regulation of Ku70 and Ku80 proteins, while the singly used treatment did not. Taken together, these results suggest that roscovitine has the potential to act as a radio-sensitizer in A549 cells by promoting caspase-3 activity and increasing apoptosis, affecting cell cycle distribution and impairing DNA repair process.     

磷脂酰肌醇-3激酶在石英诱导的DNA双链断裂修复中的作用

目的 探讨磷脂酰肌醇-3激酶(phosphatidylinositol 3 kinase,PI3K)在石英致人胚肺成纤维细胞(HELF)DNA双链断裂修复中的作用.方法 用200μg/ml的石英刺激HELF和用显性失活突变体抑制P13K功能的HELF(DN-Δp85)不同时间.免疫印迹法检测磷酸化H2AX(γH2AX)的水平以及DNA依赖性蛋白激酶(DNA-dependent protein kinase,DNA-PK)的组成成分Ku70、Ku8和DNA-PKcs的蛋白水平,并用Image-Pro plus 6.0软件对条带光强度进行半定量分析.中性彗星试验检测DNA双链断裂损伤,用彗尾DNA百分含量值观察DNA双链断裂损伤程度变化,并计算DNA修复能力.结果 γH2AX的水平在石英刺激3 h明显增高,12 h达峰值,24 h下降.与HELF相比,石英诱导的DN-Δp85细胞γH2AX水平增高受到抑制.石英刺激HELF和DN-ΔP85细胞12 h组Ku70、Ku80和DNA-PKcs的蛋白水平(0.58±0.09、0.95±0.21、0.55±0.06,0.37±0.14、0.55±0.17、0.52±0.07)均高于相应的无石英刺激组(0.26±0.10、0.69±0.26、0.43±0.11,0.11±0.07、0.27±0.14、0.39±0.07),差异有统计学意义(P<0.05).与石英刺激HELF 12 h组比较,石英刺激DN-ΔP85 12 h组的Ku70、Ku80蛋白水平增高受到抑制,差异均有统计学意义(P<0.05).石英刺激的HELF12 h组和DN-Δp85 12h组的彗尾DNA百分含量分别为9.78±1.15和11.79±4.90,明显高于同细胞系的无石英刺激组(2.40±0.69,3.31±1.35),差异有统计学意义(P<0.05);与石英刺激HELF 12 h组相比,石英刺激HELF细胞24 h组彗尾DNA百分含量明显降低(4.19±0.47),差异有统计学意义(P<0.05).石英刺激DN-Δp85 24 h组的彗尾DNA百分含量为(7.58±4.32),明显高于无石英刺激DN-Δp85组和石英刺激HELF 24 h组,差异有统计学意义(P<0.05).HELF的DNA修复能力为75.74%,DN-Δp85的DNA修复能力为49.64%.结论 石英可诱导DNA双链断裂损伤,PI3K与DNA损伤修复有关,通过调节Ku70和Ku80的水平,可促进石英诱导的DNA双链断裂损伤的修复.     

Misrepair of DNA double-strand breaks after exposure to heavy-ion beams causes a peak in the LET–RBE relationship with respect to cell killing in DT40 cells

To determine the radiobiological mechanisms underlying relative biological effectiveness (RBE) and the repair efficiencies of DNA double-strand breaks (DSBs) as a function of linear energy transfer (LET), we exposed cells of the chicken B-lymphocyte cell line DT40 and its DSB repair pathway-deficient derivatives to heavy-ion beams produced at the Heavy-Ion Medical Accelerator in Chiba (HIMAC) at the National Institute of Radiological Sciences (NIRS), Chiba, Japan. The relationship between LET and cell lethality was investigated in the DNA DSB repair gene knockouts Ku70^−/−, Rad54^−/−, and Ku70^−/−Rad54^−/−, and in the wild-type cells. We found that cell-cycle stage and activity of the DNA DSB repair pathways influence LET-mediated biological effects. An expected LET–RBE relationship was observed in the cells capable of DNA repair, but no peak was found in the RBE with respect to cell survival in the Ku70^−/−Rad54^−/− cells or in Ku70^−/− cells in the G1 and early S cell-cycle phases (when no sister chromatids were present and homologous recombination could not occur). These findings suggest that the peak in RBE is caused by deficient repair of the DNA DSBs.     

PI3K／AKT抑制剂联合放射对CNE-1细胞Ku70、Ku80表达的影响

目的：分析体外研究磷脂酰肌醇3-激酶,丝氨酸苏氨酸激酶（P13K／AKT）信号转导通路抑制剂（LY294002）联合放射对鼻咽癌细胞CNE-1KuT0、Ku80mRNA表达的影响,探讨P13K／AKT抑制剂影响放射敏感性的机制。方法：体外培养人鼻咽癌细胞CNE-1,取指数生长期细胞分为四个实验组：对照组（A）、单纯放射组（B）、单纯药物组（C）、加药联合放射组（D）;RT-PCR检测各组Ku70、Ku80mRNA的表达水平。结果：RT-PCR结果显示,放射后Ku70mRNA的表达增高,而抑制剂LY294002单纯作用CNE-1细胞后Ku70mRNA的表达水平无改变,但联合放疗后,可以降低放射后的Ku70mRNA的表达水平（P〈0．05）;本实验中Ku80mRNA的表达水平在各实验组中未观察到明显改变。结论：LY294002联合放疗可抑制Ku70表达,通过减少CNE-1细胞DNA损伤后的再修复影响放射敏感性。     

Early increase of radiation-induced γH2AX foci in a human Ku70/80 knockdown cell line characterized by an enhanced radiosensitivity

A better understanding of the underlying mechanisms of DNA repair after exposure to ionizing radiation represents a research priority aimed at improving the outcome of clinical radiotherapy. Because of the close association with DNA double strand break (DSB) repair, phosphorylation of the histone H2AX protein (γH2AX), quantified by immunodetection, has recently been used as a method to study DSB induction and repair at low and clinically relevant radiation doses. However, the lack of consistency in literature points to the need to further validate the role of H2AX phosphorylation in DSB repair and the use of this technique to determine intrinsic radiosensitivity. In the present study we used human mammary epithelial MCF10A cells, characterized by a radiosensitive phenotype due to reduced levels of the Ku70 and Ku80 repair proteins, and investigated whether this repair-deficient cell line displays differences in the phosphorylation pattern of H2AX protein compared to repair-proficient MCF10A cells. This was established by measuring formation and disappearance of γH2AX foci after irradiating synchronized cell populations with (60)Co γ-rays. Our results show statistically significant differences in the number of γH2AX foci between the repair-deficient and -proficient cell line, with a higher amount of γH2AX foci present at early times post-irradiation in the Ku-deficient cell line. However, the disappearance of those differences at later post-irradiation times questions the use of this assay to determine intrinsic radiosensitivity, especially in a clinical setting.     

UHRF1的高表达降低人乳癌细胞对X射线敏感性的研究

目的：了解基因UHRF1的高表达对乳癌细胞MDA-MB-231辐射敏感性的影响.方法：利用克隆形成实验观察细胞存活;流式细胞术测定细胞周期;利用DNA片段分析和Annexin V 试剂盒测定细胞凋亡;Western blot 测定蛋白表达变化;利用经典的染色体分析,观察染色体畸变（着丝粒环和双着丝粒）.结果：与对照相比, UHRF1转染可明显降低MDA-MB-231细胞对X射线的敏感性.利用UHRF1-siRNA抑制UHRF1的表达,可显著增强细胞的辐射敏感性.UHRF1的高表达,可诱导G2/M期阻滞,抑制细胞凋亡,下调促凋亡蛋白Bax,上调DNA损伤修复蛋白Ku70和 Ku80的表达水平,而且,能抑制X射线诱导的染色体畸变.结论：UHRF1可能通过影响凋亡和DNA损伤修复成为乳癌放疗的新靶标.     

ShRNA-mediated Ku80 gene silencing inhibits cell proliferation and sensitizes to gamma-radiation and mitomycin C-induced apoptosis in esophageal squamous cell carcinoma lines

To investigate the effects of Ku80 depletion on cell growth and sensitization to gamma-radiation and MMC-induced apoptosis in esophageal squamous cell carcinoma lines. Six human carcinoma cell lines (LNcaP, K562, MDA-MB-231, MCF-7, EC9706, and K150) and normal HEK293 cell line were examined for basal levels of Ku80 protein by western blotting analysis. The suppression of Ku80 expression was performed using vector-based shRNA in EC9706 cells. Cell proliferation was determined with MTT assay and colony formation assay and tumorigenicity in a xenograft model in vitro and in vivo. Sensitivity of EC9706 cells treated with shRNA vector to gamma-radiation and MMC was determined with colony formation assay and MTT assay. The cell cycle distribution was determined by Flow cytometry. Apoptosis induced by gamma-radiation and MMC was analyzed using GENMED-TUNEL FACS kit. Ku80 showed higher basal levels in six carcinoma cell lines than in HEK293. The suppression of Ku80 expression decreased cellular proliferation, colony formation and inhibited tumorigenicity in a xenograft model. Furthermore, it sensitized apoptosis of the cancer cells induced by gamma-radiation and MMC. Ku80 plays an important role not only in tumorigenesis but also in radiation resistance and chemotherapy resistance in esophageal cancer cells. Hence Ku80 may serve as a promising therapeutic target, particularly for recurrent esophageal tumors.     

Relative biological effects of neutron mixed-beam irradiation for boron neutron capture therapy on cell survival and DNA double-strand breaks in cultured mammalian cells

Understanding the biological effects of neutron mixed-beam irradiation used for boron neutron capture therapy (BNCT) is important in order to improve the efficacy of the therapy and to reduce side effects. In the present study, cell viability and DNA double-strand breaks (DNA-DSBs) were examined in Chinese hamster ovary cells (CHO-K1) and their radiosensitive mutant cells (xrs5, Ku80-deficient), following neutron mixed-beam irradiation for BNCT. Cell viability was significantly impaired in the neutron irradiation groups compared to the reference gamma-ray irradiation group. The relative biological effectiveness for 10% cell survival was 3.3 and 1.2 for CHO-K1 and xrs5 cells, respectively. There were a similar number of 53BP1 foci, indicators of DNA-DSBs, in the neutron mixed-beam and the gamma-ray groups. In addition, the size of the foci did not differ between groups. However, neutron mixed-beam irradiation resulted in foci with different spatial distributions. The foci were more proximal to each other in the neutron mixed-beam groups than the gamma-ray irradiation groups. These findings suggest that neutron beams may induce another type of DNA damage, such as clustered DNA-DSBs, as has been indicated for other high-LET irradiation.     

Repair of potentially lethal damage in normal cells and ataxia telangiectasia cells; consideration of non-homologous end-joining

When cell lines are held in a quiescent state after irradiation, survival rates are greater than those from cells that are stimulated to grow immediately after irradiation. These differences in survival rates correspond to rates of potentially lethal damage repair. The effects of confluent holding recovery after gamma-irradiation were investigated using normal human fibroblasts (AG1522) and ataxia telangiectasia fibroblasts (GM02052). Calyculin-A-induced premature chromosome condensation and fluorescent in situ hybridization were applied to study G2/M chromosomal aberrations. Survival results indicated normal capacity for PLDR in AG1522 cells but that PLDR was extremely compromised in GM02052 cells. The chromosomal aberration frequency decreased when AG1522 cells were allowed to repair for 24-h, whereas 24-hour incubation had little effect on the aberration frequency in GM02052 cells. Since the main mechanism for dsbs repair during G0/G1 phases of the cells cycle involve the non-homologous end-joining (NHEJ) process, our study indicates that for AG1522 cells the NHEJ repair process is more likely to induce accurate chromosome repair under quiescent G0 conditions than proliferating G1 phase, while in GM02052 cells the fidelity of NHEJ is similarly defective at either cell cycle phase. Reduced fidelity of NHEJ may be responsible for PLDR defect and its hyper-radiosensitivity in A-T cells.     

Differences in sensitivity to DNA-damaging Agents between XRCC4- and Artemis-deficient human cells

Non-homologous end-joining (NHEJ) is the predominant pathway for the repair of DNA double-strand breaks (DSBs) in human cells. XRCC4 is indispensable to NHEJ and functions together with DNA ligase IV in the rejoining of broken DNA ends. Artemis is a nuclease required for trimming of some, but not all, types of broken DNA ends prior to rejoining by the DNA ligase IV/XRCC4 complex. To better understand the roles of these factors, we generated XRCC4- and Artemis-deficient cells from the human colon adenocarcinoma cell line HCT116 by gene targeting and examined their cellular responses to several DNA-damaging agents including X-rays. As anticipated, kinetic analyses of γ-H2AX foci and chromosomal aberrations after ionizing radiation (IR) demonstrated a serious incompetence of DSB repair in the XRCC4-deficient cells, and relatively moderate impairment in the Artemis-deficient cells. The XRCC4-deficient cells were highly sensitive to etoposide and 5-fluoro-2'-deoxyuridine as well as IR, and moderately sensitive to camptothecin, methyl methanesulfonate, cisplatin, mitomycin C, aphidicolin and hydroxyurea, compared to the parental HCT116 cells. The Artemis-deficient cells were not as sensitive as the XRCC4-deficient cells, except to cisplatin and mitomycin C. By contrast, the Artemis-deficient cells were significantly more resistant to hydroxyurea than the parental cells. These observations suggest that Artemis also functions in some DNA damage response pathways other than NHEJ in human cells.     

Ku80/p53通路在石英诱导的细胞周期改变中的作用

目的 探讨在石英刺激的人胚肺成纤维细胞(human embryo lung fibroblasts,HELF)中Ku80蛋白表达的变化及Ku80/p53通路在石英诱导的细胞周期改变中的作用.方法 RNAi技术抑制Ku80蛋白表达,流式细胞技术检测细胞周期,免疫印迹技术检测Ku80、p53、p21蛋白的表达及p53-ser15磷酸化水平,并用Image-Pro plus 6.0软件对条带光强度进行半定量分析.结果 Ku80蛋白表达与石英刺激呈剂量-反应和时间-反应关系;石英刺激阴性对照H-NC细胞,G1期细胞所占比例从89.28%±2.19%下降到68.93%±3.79%;抑制Ku80蛋白表达的HELF细胞(H-Ku80)的G1期细胞所占比例进一步减少,从85.16%±3.73%下降到59.92%±3131%,差异均有统计学意义(P<0.05);抑制Ku80蛋白表达后,石英引起的p53、p21蛋白及p53-ser15磷酸化水平增高受抑制.结论 Ku80对p53、p21蛋白表达及p53-ser15磷酸化水平起调节作用,Ku80/p53通路可能参与了石英诱导的细胞周期改变.

Abstract：

Objective To study the roles of Ku80/p53 pathway in silica-induced cell cycle changes in human embryo lung fibroblasts (HELF). Methods Ku80 siRNA expression vectors were transfected into HELF by lipofectamine. Flow cytometry was used to detect the distributions of cell cycle and western blot assay was used to determine the expression level of Ku80, p53 and p21 proteins or the phosphorylation levels of p53-serl5 after cells were exposed to silica. Results The expression levels of Ku80 protein increased in concentration-dependent and time-dependent manners after cells were exposed to silica. The proportion of G1 phases in H-NC cells (controls) decreased from 89.28%±2.19% to 68.93%±3.79% after exposure to silica, and the proportion of G1 phases in HELF cells (H-Ku80) decreased from 85.16%±3.73% to 59.92%±3.31% after exposure to silica (P<0.05). The expression levels of Ku80, p53 protecns or p21 proteins or phosphorylation level of p53-serl5 were obviously suppressed in H-Ku80, as compared with H-NC. Conclusion Ku80/p53 pathway plays a role in the cell cycle charges induced by silica in human embryo lung fibroblasts.     

Genetic factors in immunity and aging

Maximum life span is controlled by genes that regulate molecular mechanisms accounting for the synchrony of structural and functional changes in different cells and tissues of each member of a given species. The role of immune response genes was investigated in aging mice genetically selected for high (H) or low (L) antibody response (Biozzi mice). Results from genetic selection of over 1000 mice showed that genes expressed in the immune system affect life span and diseases. In most cases, the life span is longer in H than in L mice whereas the lymphoma incidence is remarkably higher in L than in H mice. Since DNA repair capacity is a property positively correlated with the maximum life span in several mammalian species, DNA repair was studied by use of hydroxyurea, a cell-synchronizing agent, and found to take place in irradiated human PBMC from young and, to a lesser extent, from adult subjects. Conversely, no repair was detected in irradiated PBMC from elderly subjects. DNA damage recognition and repair pathways involve several nuclear proteins, as double strand breaks are firstly recognized by proteins displaying helicase activity, such as ku 70/80, and then repair is carried out under the control of other proteins. Radiation-induced expression of activated ku(70/80) proteins, in terms of DNA-binding, was found in PBMC from young-adults but not from elderly subjects. Maintenance of DNA integrity is fundamental for normal immune functions, as suggested by the lack of V(D)J recombination in lymphocytes of knock-out mice deficient in ku 70 or ku 80 protein. However, whether the link between genetic factors and life span is mediated by the performance of the immune system remains to be demonstrated.     

Cellular response to pulsed low-dose rate irradiation in X-ray sensitive hamster mutant cell lines

The role of DNA repair mechanisms in the cellular response to low dose rate (LDR) irradiation was studied with the aim to gain insight in the process of sublethal damage (SLD) repair. Chinese hamster cell lines mutated in either DNA single strand break (ssb) repair or DNA double strand break (dsb) repair by non homologous end joining (NHEJ) and homologous recombination (HR), or showing an AT-like phenotype, were irradiated in plateau-phase either at high dose rate (HDR, 3.3 Gy/min) or at pulsed low dose rate (p-LDR, average 1 Gy/h). Cell survival after irradiation was assessed using the clonogenic assay. A change in sensitivity when the dose rate was decreased was observed for all parental cell lines and the DNA ssb repair mutant. No difference in cell survival after p-LDR versus. HDR irradiation was observed for the two NHEJ mutants, the AT-like mutant and the HR mutant. Based on these results we conclude that single strand break repair does not play a role in the dose rate effect. The AT like protein, functional NHEJ and XRCC3 are required for the dose rate effect.     

DNA-PK: the major target for wortmannin-mediated radiosensitization by the inhibition of DSB repair via NHEJ pathway

The effect of wortmannin posttreatment was studied in cells derived from different species (hamster, mouse, chicken, and human) with normal and defective DNA-dependent protein kinase (DNA-PK) activity, cells with and without the ataxia telangiectasia (ATM) gene, and cells lacking other regulatory proteins involved in the DNA double-strand break (DSB) repair pathways. Clonogenic assays were used to obtain all results. Wortmannin radiosensitization was observed in Chinese hamster cells (V79-B310H , CHO-K1), mouse mammary carcinoma cells (SR-1), transformed human fibroblast (N2KYSV), chicken B lymphocyte wild-type cells (DT40), and chicken Rad54 knockout cells (Rad54-/-). However, mouse mammary carcinoma cells (SX9) with defects in the DNA-PK and chicken DNA-PK catalytic subunit (DNA-PKcs) knockout cells (DNA-PKcs-/-/-) failed to exhibit wortmannin radiosensitization. On the other hand, SCID mouse cells (SC3VA2) exposed to wortmannin exhibited significant increases in radiosensitivity, possibly because of some residual function of DNA-PKcs. Moreover, the transformed human cells derived from AT patients (AT2KYSV) and chicken ATM knockout cells (ATM-/-) showed pronounced wortmannin radiosensitization. These studies demonstrate confirm that the mechanism underlying wortmannin radiosensitization is the inhibition of DNA-PK, but not of ATM, thereby resulting in the inhibition of DSB repair via nonhomologous endjoining (NHEJ).     

Synergistic effect of heat shock protein 90 inhibitor, 17-allylamino-17-demethoxygeldanamycin and X-rays, but not carbon-ion beams, on lethality in human oral squamous cell carcinoma cells

The purpose of this study is to clarify the effect of a heat shock protein 90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), in combination with X-rays or carbon-ion beams on cell killing in human oral squamous cell carcinoma LMF4 cells. Cell survival was measured by colony formation assay. Cell-cycle distribution was analyzed by flow cytometry. Expression of DNA repair-related proteins was investigated by western blotting. The results showed 17-AAG to have synergistic effects on cell lethality with X-rays, but not with carbon-ion beams. The 17-AAG decreased G(2)/M arrest induced by X-rays, but not by carbon-ion beams. Both X-ray and carbon-ion irradiation up-regulated expression of non-homologous end-joining-associated proteins, Ku70 and Ku80, but 17-AAG inhibited only X-ray-induced up-regulation of these proteins. These results show that 17-AAG with X-rays releases G(2)/M phase arrest; cells carrying misrepaired DNA damage then move on to the G(1) phase. We demonstrate, for the first time, that the radiosensitization effect of 17-AAG is not seen with carbon-ion beams because 17-AAG does not affect these changes.