曲古抑菌素A增加人肝癌Bel7402细胞对TRAIL敏感度的实验

目的 探讨曲古抑菌素A(Trichostatin A,TSA)联合肿瘤坏死因子(tumor necrosis factor,TNF)相关凋亡诱导配体(TNF related apoptosis inducing ligand,TRAIL)对肝癌细胞Bel7402增殖凋亡的影响及其机制.方法 采用四甲基偶氮唑盐(MTT)染色法分别检测TSA、TRAIL及低浓度TSA联合TRAIL处理Bel7402细胞的生长抑制率;4,6-二脒基-2-苯基吲哚二盐酸盐(DAPI)染色法对药物联合处理后的细胞进行凋亡形态学观察;免疫细胞化学和Western blot技术观察药物联合作用后p65蛋白在细胞中表达和定位的变化.结果 不同浓度TSA作用6、12和24 h对人肝癌Bel7402细胞的增殖没有明显抑制作用,而作用48 h后的细胞增殖抑制率明显升高,和对照组比较差异有统计学意义(P＜0.05);不同浓度TRAIL处理Bel7402细胞存活率没有明显改变;低浓度TSA(20 ng/ml)预处理能够增加Bel7402细胞对TRAIL治疗的敏感度,TSA预处理联合TRAIL(100ng/ml)作用细胞24 h后,细胞生存率为(57.1±5.4)％,和单独药物处理组及对照组比较差异有统计学意义(P＜0.05);DAPI染色显示TSA和TRAIL联合作用后Bel7402细胞有核凋亡出现.荧光显微镜观察证明单独应用TSA(200 ng/ml)或TRAIL(100 ng/ml)处理的细胞p65蛋白有部分核内移位,而两种药物联合应用导致p65蛋白表达量降低,并且发生明显的核内转移和集聚.结论 低浓度TSA能够增加肝癌Bel7402细胞对TRAIL的敏感度;其机制可能是两种药物联合应用降低p65的表达和活性,从而诱导Bel7402细胞凋亡.     

HDAC inhibitor confers radiosensitivity to prostate stem-like cells

Background:

Radiotherapy can be an effective treatment for prostate cancer, but radiorecurrent tumours do develop. Considering prostate cancer heterogeneity, we hypothesised that primitive stem-like cells may constitute the radiation-resistant fraction.

Methods:

Primary cultures were derived from patients undergoing resection for prostate cancer or benign prostatic hyperplasia. After short-term culture, three populations of cells were sorted, reflecting the prostate epithelial hierarchy, namely stem-like cells (SCs, α₂β₁integrin^hi/CD133⁺), transit-amplifying (TA, α₂β₁integrin^hi/CD133⁻) and committed basal (CB, α₂β₁integrin^lo) cells. Radiosensitivity was measured by colony-forming efficiency (CFE) and DNA damage by comet assay and DNA damage foci quantification. Immunofluorescence and flow cytometry were used to measure heterochromatin. The HDAC (histone deacetylase) inhibitor Trichostatin A was used as a radiosensitiser.

Results:

Stem-like cells had increased CFE post irradiation compared with the more differentiated cells (TA and CB). The SC population sustained fewer lethal double-strand breaks than either TA or CB cells, which correlated with SCs being less proliferative and having increased levels of heterochromatin. Finally, treatment with an HDAC inhibitor sensitised the SCs to radiation.

Interpretation:

Prostate SCs are more radioresistant than more differentiated cell populations. We suggest that the primitive cells survive radiation therapy and that pre-treatment with HDAC inhibitors may sensitise this resistant fraction.     

Relationship between clonogenic radiosensitivity, radiation-induced apoptosis and DNA damage/repair in human colon cancer cells

The intrinsic radiation sensitivity of normal and tumour tissue is a major determinant of the outcome of radiotherapy. There is currently no established test that can be used routinely to measure the radiosensitivity of the cells in an individual patient's cancer in a manner that can inform treatment planning. The purpose of this study was to evaluate, in four human colorectal adenocarcinoma cell lines, two possible end points as surrogate markers of radiation response--apoptosis and induction of DNA single-strand breaks--and to compare the results with those of a conventional clonogenic assay. Cell lines (SW707 SW480, SW48 and HT29) known to differ in radiosensitivity were exposed to single doses of X-rays ranging from 0.5 to 5 Gy and cell survival was measured using the clonogenic assay. Apoptosis was determined on the basis of morphology under fluorescent microscopy and DNA damage/repair was measured, as tail moment, using an adaptation of the alkaline comet assay. The relationship between surviving fraction at 2 Gy (SF2) and the percentage of apoptotic cells 24 h after the same dose was complex, but apoptosis accurately predicted the order of radiosensitivities as measured by SF2. Initial damage measured after 2 Gy using the alkaline comet assay gave a close correlation with SF2 (r2=0.95), whereas there was no correlation between initial DNA damage repair rate and SF2.     

曲古菌素A通过抑制MAPK/ERK通路上调食管癌EC1细胞CAR的表达

摘 要　目的：观察曲古菌素A（trichostatin A,TSA）对人食管癌细胞EC1膜表面柯萨奇病毒-腺病毒受体(Coxsachievirus and adenovirus receptor, CAR)表达水平的影响,探讨MAPK/ERK信号通路在TSA上调CAR表达中的作用。方法：0.3、0.5、1.0 μmol/L的TSA处理EC1细胞48 h,采用免疫荧光、RT-PCR、Western blotting检测CAR的表达。以1.0 μmol/L TSA作用EC1细胞1、6、12、24、48 h,Western blotting检测p-ERK、CAR表达水平的变化,分析CAR表达和p-ERK水平变化的相关性。结果：0.3、0.5、1.0 μmol/L TSA处理EC1细胞后,CAR蛋白和mRNA水平均明显增加（P<0.05),并呈剂量依赖关系。1.0 μmol/L TSA作用EC1细胞6、12、24、48 h后,CAR蛋白表达较对照组均明显增加（P<0.05);p-ERK表达水平均明显下降（P<0.05),两者变化呈显著负相关（r=-0.886,P<0.01)。结论：TSA能够上调人食管癌EC1细胞膜表面CAR的表达水平,其机制可能与其抑制MAPK/ERK通路有关。     

抑制USP9x表达增强食管癌Ec9706-R细胞的放射敏感性

背景与目的：泛素特异性蛋白酶9x（ubiquitin-specific protease 9x,USP9x）与多种肿瘤的发生、发展以及肿瘤细胞的放射抗拒相关。研究发现,USP9x的表达与食管鳞状细胞癌的浸润深度和淋巴结转移相关,但其食管癌细胞放射抗拒作用尚未见报道。探究USP9x对放射抗拒食管癌Ec9706-R细胞放射敏感性的作用及其机制。方法：首先通过实时荧光定量聚合酶链反应（real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR）和蛋白质印迹法（Western blot）检测放射线照射后Ec9706-R细胞及其亲本细胞Ec-9706中USP9x和抗髓样细胞白血病-1（myeloid cell leukemia-1,Mcl-1）mRNA表达和蛋白水平。然后将Ec9706-R细胞随机分成3组：放射（irradiation,IR）组、IR+对照siRNA组（IR+si-NC组,转染Control siRNA）和IR+USP9x siRNA组（IR+si-USP9x组,转染USP9x siRNA）,各组细胞均使用一定量的6 MV-X射线照射。噻唑蓝（methyl thiazolyl tetrazolium,MTT）比色法检测不同剂量（0、2、4、6和8 Gy）6 MV-X射线照射下各组细胞的活力。Transwell、流式细胞术、RTFQ-PCR和Western blot分别检测3组细胞在6 Gy照射下的细胞迁移、凋亡、Mcl-1 mRNA表达和蛋白水平以及增殖细胞核抗原（proliferating cell nuclear antigen,PCNA）和DNA损伤修复相关基因核苷酸切除修复交叉互补基因1（excision repair cross-complementing gene 1,ERCC1）蛋白水平。结果：放射后Ec9706-R和Ec-9706细胞中USP9x和Mcl-1 mRNA表达和蛋白水平均增加,Ec9706-R细胞尤为显著（P<0.05）。与IR组相比,IR+si-USP9x组中细胞活力、迁移细胞数目、PCNA、ERCC1和Mcl-1的表达均降低,细胞凋亡增加（P<0.05）。但是与IR组相比,IR+si-NC组中上述指标均无显著变化（P>0.05）。结论：抑制USP9x表达能够增强放射抗拒食管癌Ec9706-R细胞的放射敏感性,这可能是通过下调Mcl-1的表达发挥作用的。     

辐射诱导的旁观者效应研究进展

直接受到照射的细胞通过信号转导引起临近细胞的损伤,称为旁观者效应.其具体机制非常复杂,目前仍不完全清楚.本文从旁观者效应对细胞DNA损伤的类型,旁观者效应中的分子信号转导及体内存在的旁观者效应等不同方面进行综述.     

辐射诱导的旁观者效应研究进展

直接受到照射的细胞通过信号转导引起临近细胞的损伤,称为旁观者效应。其具体机制非常复杂,目前仍不完全清楚。本文从旁观者效应对细胞DNA损伤的类型,旁观者效应中的分子信号转导及体内存在的旁观者效应等不同方面进行综述。     

曲古菌素A对甲状腺癌细胞增殖及凋亡的影响

 目的 探讨组蛋白去乙酰化酶抑制剂曲古菌素A体外抑制甲状腺癌细胞增殖、诱导凋亡的作用。 方法 采用磺酰罗丹明B染色分析法、Hoechst33342/PI双染荧光显微镜检测及PI单染流式细胞仪分析等技术检测曲古菌素A对不同甲状腺癌细胞增殖、凋亡及细胞周期的影响。 结果 不同浓度的曲古菌素A处理48h,能明显抑制甲状腺癌细胞的增殖,并且随着药物浓度的增加抑制率也增大,各组间及与对照组相比差异均具有 统计学意义（F=35.67, P＜0.01）;双染后镜下观察显示曲古菌素A使甲状腺癌细胞呈明显的凋亡状态,流式细胞仪分析则表明诱导后滤泡状甲状腺癌细胞凋亡峰(Sub G1)比值升高,与对照组比较其差异有统计学意义（t=6.225, P＜0.01）。 结论 曲古菌素A能显著抑制甲状腺癌细胞体外的增殖及诱导甲状腺癌细胞的凋亡,其作用呈浓度和时间依赖性。     

The amount of DNA damage needed to activate the radiation-induced G2 checkpoint varies between single cells

Background and purpose

The radiation-induced G2 checkpoint helps facilitate DNA repair before cell division. However, recent work has revealed that human cells often escape the G2 checkpoint with unrepaired DNA breaks. The purpose was to explore whether G2 checkpoint activation occurs according to a threshold level of DNA damage.

Materials and methods

G2 checkpoint activation was assayed at 75-90 min and 24-48 h after X-ray irradiation of BJ diploid fibroblasts and U2OS osteosarcoma cells. Multiparameter flow cytometry with pacific blue barcoding, and flow cytometry-based sorting of phospho-H3 positive cells to microscope slides, were used to examine the DNA damage marker γ-H2AX in individual mitotic cells that had escaped the G2 checkpoint.

Results

For all radiation doses and times tested, the number of γ-H2AX foci varied between individual mitotic cells. At 75 min the median levels of γ-H2AX in mitotic cells increased with higher radiation doses. At 24-48 h, following a prolonged G2 checkpoint, cells were more resistant to checkpoint re-activation by a second dose of radiation.

Conclusion

Our results suggest that different amounts of DNA damage are needed to activate the G2 checkpoint in individual cells. Such single cell variation in checkpoint activation may potentially contribute to radiation-induced genomic instability.     

DNA base damage induced by ionizing radiation recognized by Escherichia coli UvrABC nuclease but not Nth or Fpg proteins

Ionizing radiation and other free radical-generating systems induce a great variety of oxidative damage to DNA bases. The major known lesions are repaired by two well-characterized DNA glycosylases of Escherichia coli, endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg), which have associated AP lyase activities. To detect and characterize potentially harmful oxidative base DNA lesions that may be repaired by alternative means, we exposed plasmid DNA to low doses γ-rays and removed the major base lesions by treatment with Nth and Fpg proteins. The closed circular DNA remaining after these treatments was used as a substrate of the UvrABC endonuclease complex from E. coli and as a template in a DNA polymerase arrest assay in vitro. The circular DNA contained lesions that were recognized and incised by the UvrABC nuclease and also lesions that blocked DNA polymerization in vitro. The blocking lesions were more abundant in DNA irradiated under nitrogen than under air and occurred mainly at tandem guanines; however, they were also frequent at tandem adenines and tandem cytosines. © 1996 Wiley-Liss, Inc.     

DNA damage and breast cancer

Cancer is intimately related to the accumulation of DNA damage, and repair failures (including mutation prone repair and hyperactive repair systems). This article relates current clinical categories for breast cancer and their common DNA damage repair defects. Information is included on the potential for accumulation of DNA damage in the breast tissue of a woman during her lifetime and the role of DNA damage in breast cancer development. We then cover endogenous and exogenous sources of DNA damage, types of DNA damage repair and basic signal transduction pathways for three gene products involved in the DNA damage response system; namely BRCA1, BRIT1 and PARP-1. These genes are often considered tumor suppressors because of their roles in DNA damage response and some are under clinical investigation as likely sources for effective new drugs to treat breast cancers. Finally we discuss some of the problems of DNA damage repair systems in cancer and the conundrum of hyper-active repair systems which can introduce mutations and confer a survival advantage to certain types of cancer cells.     

RNA干扰抑制UHRF1基因表达对食管癌细胞放射增敏作用的研究

目的 研究RNA干扰抑制UHRF₁表达对食管癌细胞系(TE-1)放射敏感性的影响及作用机制。方法 以慢病毒感染方法将UHRF₁基因的短发夹状RNA (shRNA)转入TE-1细胞,并分为未转染组、转染NC-shRNA组、转染UHRF₁-shRNA组,前二者为对照组。采用 RT-PCR和蛋白印记法检测转染前后细胞UHRF₁的mRNA和蛋白表达,成克隆法、流式细胞术及蛋白印记法检测转染UHRF₁-shRNA联合X线照射对TE-1细胞放射敏感性、周期、凋亡及DNA损伤标识蛋白γ-H₂AX的影响。     

Decreased DNA repair activity in bone marrow due to low expression of DNA damage repair proteins

The bone marrow (BM) is one of the organs that is sensitive to acute exposure of ionizing radiation (IR); however, the mechanism of its high sensitivity to IR remains to be elucidated. BM is differentiated into dendritic cells (DC) with granulocyte macrophage-colony stimulating factor (GM-CSF). Using this in vitro model, we studied whether radiosensitivity is distinctly regulated in undifferentiated and differentiated BM. We discovered that levels of DNA damage repair (DDR) proteins are extremely low in BM, and they are markedly increased upon differentiation to DC. Efficiency of both homologous recombination (HR)- and non-homologous end joining (NHEJ)-mediated repair of DNA double strand breaks (DSBs) is much lower in BM compared with that of DC. Consistent with this, immunofluorescent γH2AX is highly detected in BM after IR. These results indicate that increased radiosensitivity of BM is at least due to low expression of the DNA repair machinery.     

Growth arrest and cell death in the breast tumor cell in response to ionizing radiation and chemotherapeutic agents which induce DNA damage

Breast tumor cells are relatively refractory to apoptosis in response to modalities which induce DNA damage such as ionizing radiation and the topoisomerase II inhibitor, adriamycin. Various factors which may modulate the apoptotic response to DNA damage include the p53 status of the cell, levels and activity of the Bax and Bcl-2 families of proteins, activation of NF-kappa B, relative levels of insulin like growth factor and insulin-like growth factor binding proteins, activation of MAP kinases and PI3/Akt kinases, (the absence of) ceramide generation and the CD95 (APO1/Fas) signaling pathway. Prolonged growth arrest associated with replicative senescence may represent an alternative and reciprocal response to DNA-damage induced apoptosis that is p53 and/or p21^waf1/cip1 dependent while delayed apoptosis may occur in p53 mutant breast tumor cells which fail to maintain the growth-arrested state. Clearly, the absence of animmediate apoptotic response to DNA damage does not eliminate other avenues leading to cell death and loss of self-renewal capacity in the breast tumor cell. Nevertheless, prolonged growth arrest (even if ultimately succeeded by apoptotic or necrotic cell death) could provide an opportunity for subpopulations of breast tumor cells to recover proliferative capacity and to develop resistance to subsequent clinical intervention.     

Down-regulation of G9a triggers DNA damage response and inhibits colorectal cancer cells proliferation

G9a, a histone methyltransferase, is aberrantly expressed in some human tumor types. By comparing 182 paired colorectal cancer and peritumoral tissues, we found that G9a was highly expressed in colorectal cancer (CRC). Overexpression of G9a promoted CRC cells proliferation and colony formation, whereas knockdown of G9a inhibited CRC cells proliferation. Depletion of G9a increased the rate of chromosome aberration, induced DNA double strand breaks and CRC cells senescence. G9a inhibition synergistically increased γH2AX expression induced by topoisomerase I inhibitors and ultimately led to CRC cell death. The findings that down-regulation of G9a triggers DNA damage response and inhibits colorectal cancer cells proliferation may define G9a as potential oncotarget in CRC.     

纳米氧化锌与常规氧化锌对A549细胞的毒性与DNA损伤的比较研究

目的：比较纳米氧化锌（Nano-ZnO）和常规氧化锌（Micro-ZnO）对人肺腺癌A549细胞的毒性及DNA损伤作用。方法：采用不同浓度（25、50、100μg/ml）的纳米ZnO（30nm）和常规ZnO（≤1μm）染毒体外培养的A549细胞,实验同时设对照组（DMEM培养液）。分别于染毒后6、12、24、48 h,观察细胞形态及生长情况,采用MTT法和单细胞凝胶电泳技术（彗星实验）检测细胞毒性及对DNA的损伤作用。结果：纳米ZnO和常规ZnO对A549细胞的半数致死浓度（IC_（50））分别为53.91和190.15μg/ml;纳米ZnO在50μg/ml剂量时对A549细胞就已出现明显的生长抑制作用,并呈明显的剂量和时间依赖效应关系;而常规ZnO在100μg/ml剂量时才出现细胞生长的抑制。纳米ZnO与常规ZnO的剂量-效应曲线和时间-效应曲线的斜率比较差异均有统计学意义（P〈0.05）。与对照组相比,纳米ZnO能够导致A549细胞产生DNA损伤;常规ZnO在相同受试剂量下未观察到DNA损伤。结论：纳米ZnO与常规ZnO均对体外A549细胞产生细胞毒性,但纳米ZnO产生毒性的剂量低、出现时间早,并产生了常规ZnO未观察到的遗传毒性。     

Chemopreventive agent sulforaphane enhances radiosensitivity in human tumor cells

Sulforaphane (SFN), an isothiocyanate derived from broccoli and other cruciferous vegetables, is a positive regulator of phase II detoxification enzymes and is highly effective in protection against chemically induced cancers by inducing apoptosis and cell cycle arrest. Here, we report that SFN also enhances radiosensitivity in human tumor cells. Cell survival in HeLa human cervix carcinoma cells pretreated with SFN was significantly lower than in cells treated with radiation only. Constant‐field gel electrophoresis and a gamma‐H2AX foci assay showed marked inhibition of DSB repair in irradiated cells treated with SFN, while little inhibition was observed in cells with DMSO (control). In addition, immunofluorescence experiments revealed a significant delay in Rad51 (a key protein for homologous recombination repair) foci formation and disappearance in irradiated cells treated with SFN when compared to the cells with X‐irradiation alone. The dephosphorylation of DNA‐PKcs (a critical nonhomologous end joining protein) was also markedly delayed by SFN pretreatment in irradiated cells. These DSB repair inhibition data partially support the high apoptotic frequency of irradiated cells pretreated with SFN. Furthermore, the combined treatment of X‐rays and SFN (i.p. 300 μmol/kg) in the xenograft model with HeLa cells showed efficient inhibition of in vivo tumor growth. To the best of our knowledge, our study is the first report showing SFN‐enhanced radiosensitivity of tumor cells in vitro and in vivo, which opens the door for a multitude of clinical applications for chemoradiotherapy using SFN. © 2009 UICC     

曲古菌素A对HDAC1在K562细胞中表达的影响

目的研究表明组蛋白去乙酰化酶1(histonedeacetylase,HDAC1)在多种肿瘤中高表达,曲古菌素A(TSA)可以抑制肿瘤细胞的生长。本实验研究HDAC1在K562细胞的表达及TSA对其增殖的影响。方法100~400nmol/L的TSA分别处理K562细胞6~48h后用四甲基偶氮唑蓝(MTT法)比色检测K562细胞的生长活性;应用流式细胞仪法观察细胞凋亡。采用RT-PCR和蛋白印迹法(Westernblot)方法检测K562细胞在(24h)不同浓度(50~500nmlo/L)HDAC1mRNA和蛋白的表达以及TSA对其作用。结果TSA可显著抑制K562细胞的生长,可呈时间及剂量依赖性抑制K562细胞的增殖,抑制率为23.15%~76.63%。流式细胞仪检测Raji细胞,凋亡率为10.32%~60.16%。HDAC1的mRNAA值的半定量表达以及蛋白表达明显增强(P<0.05),但随着浓度的增加而表达下调,呈剂量依赖性下调。结论TSA对K562细胞具有明显的增生抑制及诱导凋亡作用,抑制HDAC1的表达可能是其重要作用机制之一。