Radiation-induced apoptosis in human non-small-cell lung cancer cell lines is secondary to cell-cycle progression beyond the G2-phase checkpoint

Purpose : To characterize the relationship between cell-cycle progression and radiation-induced apoptosis in NSCLC cell lines with different p53 status. Materials and methods : Cell lines with functional (H460, A549) and non-functional p53 (H661 and H520) were irradiated with 20 Gy. Multiparameter flow-cytometry was used to follow the progression of synchronized cells through the cell cycle after irradiation. Results : Delayed apoptosis was observed after cell-cycle progression beyond the G2 block, either in the late G2/M-phase of the same cell cycle being irradiated (H661, H520) or in the G1-phase of the subsequent cell cycle (H460, A549). The apoptotic fraction in H661 and H520 was 60-80% at 144 h after irradiation, higher than in A549 and H460 (5 and 35%, respectively). As an alternative to apoptosis in cells cycling beyond the G2 restriction point, hyperploid cells were generated by all cell lines. Inhibition of cell-cycle progression through the G2/M-phase efficiently reduced the induction of late apoptosis. After irradiation in S-phase, 50-60% of cells with functional p53 remained arrested at the G2 restriction point until 144 h post-irradiation, while only 20% of the H661 or H520 did so. Conclusions : These data characterize radiation-induced apoptosis in NSCLC cell lines as a removal pathway of clonogenically inactivated cells secondary to cell-cycle progression beyond G2/M, and is unlikely to be a critical factor for cellular radiation sensitivity.     

DNA-dependent protein kinase: effect on DSB repair,G2/M checkpoint and mode of cell death in NSCLC cell lines

Abstract

Purpose: To evaluate the effect of NU7026, a specific inhibitor of DNA-PKcs, on DNA-double strand break (DSB) repair in a cell cycle specific manner, on the G2/M checkpoint, mitotic progression, apoptosis and clonogenic survival in non-small-cell lung carcinoma (NSCLC) cell lines with different p53 status.

Material and methods: Cell cycle progression, and hyperploidy were evaluated using flow cytometry. Polynucleation as a measure for mitotic catastrophe (MC) was evaluated by fluorescence microscopy. DSB induction and repair were measured by constant-gel electrophoresis and γH2AX assay. The efficiency of DSB rejoining during the cell cycle was assessed by distinguishing G1 and G2/M phase cells on the basis of the DNA content in flow cytometry. The overall effect on cell death was determined by apoptosis and the surviving fraction after irradiation with 2?Gy (SF2) assessed by clonogenic survival.

Results: DSB signaling upon treatment with NU7026, as measured by γH2AX signaling, was differently affected in G1 and G2/M cells. The background level of γH2AX was significantly higher in G2/M compared to G1 cells, whereas NU7026 had no effect on the background level. The steepness of the initial dose effect relation at 1?h after irradiation was less pronounced in G2/M compared to G1 cells. NU7026 had no significant effect on the initial dose-effect relation of γH2AX signaling. In comparison, NU7026 significantly slowed down the repair kinetics and increased the residual γH2AX signal at 24?h after irradiation in the G1 phase of all cell lines, but was less effective in G2/M cells. NU7026 significantly increased the fraction of G2/M phase cells upon irradiation. Moreover, NU7026 significantly increased mitotic catastrophe and hyperploidy, as a measure for mitotic failure after low irradiation doses of about 4?Gy, but decreased both at higher doses of 20?Gy. In addition, radiation induced apoptosis increased in A549, H520 and H460 but decreased in H661 upon NU7026 treatment, with a significant reduction of SF2 in all NSCLC cell lines.

Conclusion: Overall, NU7026 significantly influences the cell cycle progression through the G2- and M-phases and thereby determines the fate of cells. The impairment of DNA-PK upon treatment with NU7026 affects the efficiency of the NHEJ system in a cell cycle dependent manner, which may be of relevance for a clinical application of DNA-PK inhibitors in tumor therapy.     

Preferential radiosensitization in p53-mutated human tumour cell lines by pentoxifylline-mediated disruption of the G2/M checkpoint control

Purpose : There is evidence that the duration of the G2/M delay following irradiation is correlated with cell survival. We studied the radiosensitizing potential of pentoxifylline (PTX) and the PTX-mediated modulation of cell-cycle progression dependent on the p53 status of various human tumour cell lines. Materials and methods : The cellular radiosensitivity of human MCF-7 (wild-type p53) and HT-29 (p53-defective) tumour cells, which were exposed to PTX (2 mM) immediately after γ-irradiation was determined by colony forming assay. The influence on cell cycle progression after irradiation (6 Gy) was assessed by flow cytometric analysis using p53 wild-type MCF-7 and HPR600 cells, and p53-defective HT-29 and WiDr cells. Results : Clonogenic survival assays up to 8 Gy demonstrated that p53-defective HT-29 cells (sensitizer enhancement ratio [SER]=1.54) were sensitized by PTX (2 mM) to a significantly higher degree than p53 wild-type MCF-7 (SER=1.14) cells. Exposure of irradiated (6 Gy) cells to PTX (2 mM) resulted in abrogation of the radiation-induced G2/M arrest in the p53-defective HT-29 and WiDr cells, whereas the p53 wild-type-expressing MCF-7 and HPR600 cells showed less significant impairment of the G2/M checkpoint. In HT-29 cells, the rate of transition into mitosis was even higher than in the sham-treated control cells. G2/M abrogation was accompanied by an increase of apoptosis only in HPR600 cells. Conclusions : Since PTX was less effective in cells expressing intact p53, the application of PTX suggests a promising strategy of pharmacological disruption of the G2/M checkpoint control by which preferentially radiation-resistant tumours with defective p53 function might be rendered more sensitive to ionizing radiation.     

Preferential radiosensitization in p53-mutated human tumour cell lines by pentoxifylline-mediated disruption of the G2/M checkpoint control

PURPOSE: There is evidence that the duration of the G2/M delay following irradiation is correlated with cell survival. We studied the radiosensitizing potential of pentoxifylline (PTX) and the PTX-mediated modulation of cell-cycle progression dependent on the p53 status of various human tumour cell lines. MATERIALS AND METHODS: The cellular radiosensitivity of human MCF-7 (wild-type p53) and HT-29 (p53-defective) tumour cells, which were exposed to PTX (2 mM) immediately after gamma-irradiation was determined by colony forming assay. The influence on cell cycle progression after irradiation (6 Gy) was assessed by flow cytometric analysis using p53 wild-type MCF-7 and HPR600 cells, and p53-defective HT-29 and WiDr cells. RESULTS: Clonogenic survival assays up to 8 Gy demonstrated that p53-defective HT-29 cells (sensitizer enhancement ratio [SER]=1.54) were sensitized by PTX (2 mM) to a significantly higher degree than p53 wild-type MCF-7 (SER=1.14) cells. Exposure of irradiated (6 Gy) cells to PTX (2 mM) resulted in abrogation of the radiation-induced G2/M arrest in the p53-defective HT-29 and WiDr cells, whereas the p53 wild-type-expressing MCF-7 and HPR600 cells showed less significant impairment of the G2/M checkpoint. In HT-29 cells, the rate of transition into mitosis was even higher than in the sham-treated control cells. G2/M abrogation was accompanied by an increase of apoptosis only in HPR600 cells. CONCLUSIONS: Since PTX was less effective in cells expressing intact p53, the application of PTX suggests a promising strategy of pharmacological disruption of the G2/M checkpoint control by which preferentially radiation-resistant tumours with defective p53 function might be rendered more sensitive to ionizing radiation.     

不同非小细胞肺癌细胞对碳离子放射敏感性的影响

目的 分析不同非小细胞肺癌（NSCLC）细胞对碳离子照射放射敏感性的影响,并探讨其相关的分子机制。方法 采用¹²C⁶⁺对肺腺癌A549细胞、鳞癌H520细胞和大细胞癌PGCL3细胞进行照射,吸收剂量分别为0、2、4和6 Gy。通过克隆实验检测3种细胞系的存活率,流式细胞术检测细胞的周期分布,利用Hoechst 33258染色检测细胞的凋亡率,实时RT-PCR方法检测细胞内DNA-PKcs基因的表达。结果 2、4、6 Gy照射后,A549、H520和PGCL3细胞的存活率明显下降,其中A549细胞辐射敏感性最低,其次是PGCL3细胞,H520细胞辐射敏感性最高。受照射后细胞均出现G₂/M期阻滞与凋亡,且随剂量的升高呈现上升的趋势。受照射H520与PGCL3细胞的阻滞率（t=4.813、20.738、25.654和t=2.790、2.977,P<0.05）和凋亡率（t=8.579、14.289、15.244和t=3.785、5.098、8.105,P<0.05）明显高于A549细胞。受照射细胞DNA-PKcs表达明显上调,A549细胞最高,其次是PGCL3细胞,H520细胞最低;并且随着剂量的升高,DNA-PKcs的表达呈现下降趋势。2、4 Gy照射后H520和PGCL3细胞DNA-PKcs的表达明显低于A549细胞（t=7.782、3.689和t=3.889、2.814,P<0.05）。结论 不同NSCLC细胞对¹²C⁶⁺离子的放射敏感性不同,H520鳞癌细胞最高,A549腺癌细胞最低。肺腺癌A549细胞放射敏感性较低可能与高表达的DNA-PKcs参与NHEJ途径修复DNA双链断裂有关。     

Lovastatin causes sensitization of HeLa cells to ionizing radiation-induced apoptosis by the abrogation of G2 blockage

PURPOSE: To investigate the effect of inhibition of Ras/Rho-regulated signalling by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) on radiation-induced cell killing and apoptosis. MATERIALS AND METHODS: Different human cell lines were pretreated or not with lovastatin before exposure to gamma-rays. Afterwards, radiation-induced cell killing, formation and repair of double-strand breaks, activation of radiation-inducible signal mechanisms (i.e. p53, p21, extracellular-signal-related kinase (ERK), NF-kappaB), changes in cell cycle progression and apoptosis were analysed. RESULTS: As shown by a colony formation assay, lovastatin sensitized HeLa cells to gamma-radiation-induced cell killing. The lovastatin effect was cell-type specific. Neither the level of gamma-ray-induced double-strand breaks nor its repair were affected by lovastatin. Sensitization was independent of p53/p21Waf1- and NF-kappaB-related mechanisms. Radiation-stimulated activation of ERKs was attenuated by lovastatin. Cell cycle analyses revealed that the level of gamma-ray-induced G2 blockage was not affected by lovastatin. However, as analysed up to 72 h after irradiation, lovastatin pretreated cells showed an accelerated abrogation of G2 blockage as compared with the control. G2 abrogation is paralleled by an increase in the frequency of apoptotic and necrotic cells. CONCLUSIONS: The data show that lovastatin can render human cells more sensitive to the cytotoxic effect of gamma-rays. This is related to abrogation of G2 blockage and a concomitant increase in apoptotic/necrotic cell death.     

Genotype-dependent radiosensitivity: clonogenic survival, apoptosis and cell-cycle redistribution

PURPOSE: We describe variations of three radiation-induced endpoints on the basis of cell genotype: Clonogenic survival, expression of apoptosis and cell-cycle redistribution. METHODS: Clonogenic survival, apoptosis and cell-cycle redistribution are measured in multiple cell lines after exposure to radiation between 2 and 16 Gy. Cell lines varied in clonogenic radiosensitivity and expression of specific genes. RESULTS: Clonal radiosensitivity is genotype-dependent, associating with four specific genes: A mutated form of Ataxia telangiectasia mutated (mutATM); with two forms of TP53, the gene that is template for tumor protein p53, wildtype TP53 (wtTP53) and mutated TP53 (mutTP53); and an unidentified gene in radioresistant glioblastoma cells. Apoptosis is also genotype-dependent showing elevated levels in cells that express mutATM and abrogated 14-3-3sigma (an isoform of the 14-3-3 gene) but less variation for different forms of TP53. Cell-cycle redistribution varied in mutATM cells. Kinetics of apoptosis are biphasic for both time and dose; cell lines did not express apoptosis at doses below 5 Gy or times before 24 hours. Kinetics of cell-cycle redistribution changed dynamically in the first 24 hours but showed little change after that time. CONCLUSIONS: Clonogenic survival, radiation-induced apoptosis and radiation-induced redistribution in the cell-cycle vary with cell genotype, but not the same genotypes. There is temporal, not quantitative, correlation between apoptosis and clonal radiosensitivity with apoptosis suppressed by lower, less toxic doses of radiation (<5 Gy) but enabled after larger, more toxic doses. Kinetic patterns for apoptosis and redistribution show a common change at approximately 24 hours.     

塞来昔布对非小细胞肺癌细胞增殖的抑制作用

目的研究塞来昔布对非小细胞肺癌（NSCLC）的抑制作用及机制. 方法应用MTT法检测塞来昔布对NSCLC细胞株的体外抑制作用,应用流式细胞学、透射电镜研究塞来昔布对NSCLC细胞株的细胞周期阻滞和诱导凋亡的作用,应用RT-PCR检测P27^KIP1、XIAP的表达,以研究细胞周期阻滞和诱导凋亡的机制. 结果 MTT比色试验表明,塞来昔布对NSCLC有明显的抑制作用,且表现为剂量依赖性和时间依赖性;而且,在相同条件下塞来昔布对NCI-H520的抑制率明显高于对A549的抑制率.流式细胞学试验证明,塞来昔布作用后G0/G1细胞比例上升,细胞阻滞于G0/G1期.流式细胞学、透射电镜证实了凋亡的存在,而且凋亡率随剂量的增加而增加.RT-PCR证实塞来昔布作用后P27^KIP1 mRNA表达增加,XIAP mRNA表达减少.结论塞来昔布在体外对NSCLC有明显的抑制增殖作用,其机制涉及细胞周期阻滞、诱导细胞凋亡两个方面.     

Epstein-Barr virus-transformed lymphoblastoid cell lines of ataxia telangiectasia patients are defective in X-ray-induced apoptosis.

PURPOSE: To investigate and compare the propensity of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) obtained from unaffected healthy individuals and ataxia telangectasia (A-T) patients to undergo apoptosis after X-ray exposure. MATERIAL AND METHODS: The LCL were exposed to 1-4 Gy X-rays at a dose-rate of 1.36 Gy/min. At various post-irradiation times (0, 24, 48 and 72 h) the induction of apoptosis was analysed by: (1) monitoring the formation of high molecular weight (HMW) DNA fragments by field inversion pulse gel electrophoresis (FIGE); and (2) morphological characterization of apoptotic cells after fluorescence staining. In parallel, cell-cycle distribution, monitored by DNA flow cytometry, was investigated in these cells. RESULTS: The LCL obtained from the A-T homozygotes were resistant to undergoing radiation-induced apoptosis during the observation time used. On the contrary, LCL from unaffected healthy controls displayed significant radiation-induced chromatin fragmentation seen at 48 h and 72 h after irradiation. In these cells, radiation-induced G -arrest (24h post-irradiation) preceded chromatin cleavage. In A-T LCL, the defective G1-arrest was not followed by apoptosis. CONCLUSIONS: In spite of a defective cell-cycle control, EBV-transformed LCL of A-T patients compared with unaffected healthy controls do not undergo X-ray-induced apoptosis, at least during their first post-irradiation cell cycle.     

Adenovirus-mediated wild-type p53 gene expression radiosensitizes non-small cell lung cancer cells but not normal lung fibroblasts

PURPOSE: We compared the ability of adenoviral-mediated wild-type p53 RPR/INGN201(Ad5/CMV/p53) to radiosensitize non-small cell lung carcinoma (NSCLC) and normal lung fibroblast cells. MATERIALS AND METHODS: NSCLC cell lines (A549 and H322) and human lung fibroblast cells (MRC-9 and CCD-16) were used in this study. Radiosensitivity was determined by clonogenic assay and tumor growth delay. Expression of p53, Bax, and p21WAF1 protein were evaluated by immunoblot. A FITC conjugate of annexin V was used for flow cytometric detection of apoptosis. RESULTS: Clonogenic and apoptotic assays indicated that Ad5/CMV/p53 enhanced the radiosensitivity of both NSCLC cell lines. On the other hand, the two normal human fibroblast cell lines appeared to be resistant to the cytotoxic effects of Ad5/CMV/p53 and were not radiosensitized compared to the NSCLC cells. According to immunoblot analysis, Bax expression was increased in the NSCLC cells treated with the combination therapy; Bax expression, however, was unchanged in normal cells. In in vivo studies, tumor growth suppression was enhanced by this combination strategy in xenograft tumors growing in nude mice compared to Ad5/CMV/p53 or radiation therapy when used alone. CONCLUSIONS: Our data indicate that therapy using Ad5/CMV/p53 and irradiation in combination is more effective than either treatment when used alone on NSCLC cells, is not limited to cells with defective endogenous p53, and does not enhance the radiosensitivity of normal cells.     

Adenovirus-mediated wild-type p53 gene expression radiosensitizes non-small cell lung cancer cells but not normal lung fibroblasts

Purpose : We compared the ability of adenoviral-mediated wildtype p53 RPR/INGN201(Ad5/CMV/p53) to radiosensitize nonsmall cell lung carcinoma (NSCLC) and normal lung fibroblast cells. Materials and methods : NSCLC cell lines (A549 and H322) and human lung fibroblast cells (MRC-9 and CCD-16) were used in this study. Radiosensitivity was determined by clonogenic assay and tumor growth delay. Expression of p53, Bax, and p21 WAF1 protein were evaluated by immunoblot. A FITC conjugate of annexin V was used for flow cytometric detection of apoptosis. Results : Clonogenic and apoptotic assays indicated that Ad5/CMV/p53 enhanced the radiosensitivity of both NSCLC cell lines. On the other hand, the two normal human fibroblast cell lines appeared to be resistant to the cytotoxic effects of Ad5/CMV/p53 and were not radiosensitized compared to the NSCLC cells. According to immunoblot analysis, Bax expression was increased in the NSCLC cells treated with the combination therapy; Bax expression, however, was unchanged in normal cells. In in vivo studies, tumor growth suppression was enhanced by this combination strategy in xenograft tumors growing in nude mice compared to Ad5/CMV/p53 or radiation therapy when used alone. Conclusions : Our data indicate that therapy using Ad5/CMV/p53 and irradiation in combination is more effective than either treatment when used alone on NSCLC cells, is not limited to cells with defective endogenous p53, and does not enhance the radiosensitivity of normal cells.     

Dcr3 inhibit p53-dependent apoptosis in γ-irradiated lung cancer cells

Purpose:?To identify genes responsible for the radiosensitivity, we investigated the role of the differential gene expression profiles by comparing radioresistant H1299 with radiosensitive H460 lung cancer cell lines.

Materials and methods:?mRNA profiles of lung cancer cell lines were assessed using microarray, and subsequent validation was performed with qRT-PCR (Quantitative real time-polymerase chain reaction). The expression levels of differentially expressed genes were determined by Western blot and the radioresistance of lung cancer cell lines was measured by clonogenic assay.

Results:?From the differentially expressed apoptosis-related genes between H1299 and H460, we found Dcr3 (Decoy receptor 3, also known as TNFRSF6B; Tumour necrosis factor receptor super family member 6B) expression was significantly (P?=?4.38 × 10^?7) higher in H1299 cells than H460 cells. Moreover, the Dcr3 mRNA expression level in the radioresistant cell lines (H1299, A549, DLD1, MB231, MB157) was increased in comparison to the radiosensitive cell lines (ME180, Caski, U87MG, MCF7, H460). Overexpression of Dcr3 increased the survival rate of radiosensitive H460, MCF7, and U87MG cells, and knockdown of Dcr3 abolished the radioresistance of A549 cells. The survival rate of p53 (Tumour protein 53)-deficient H1299 after gamma-irradiation was not affected by the suppression of Dcr3 expression. However, when we introduced p53 into H1299 cells, siDcr3 (siRNA of Dcr3) suppressed the radioresistance of H1299 cells by inducing p53-dependent Fas (Fas receptor, also known as TNFRSF6; Tumour necrosis factor receptor super family member 6)-mediated apoptosis pathway.

Conclusion:?Characterisation of gene expression profiles in two lung cancer cell lines revealed that Dcr3 expression and p53-dependent apoptosis signalling pathway regulate cellular response to ionising radiation.     

神经纤毛蛋白1对非小细胞肺癌放射敏感性的影响

目的 探讨神经纤毛蛋白1 (neuropilin 1,NRP1)在不同种类非小细胞肺癌(NSCLC)细胞系的表达情况及其与肿瘤放射敏感性的关系.方法 通过Western blot技术检测不同来源的5种人NSCLC细胞系(H358、H460、H1299、A549、SK-MES-1)NRP1的基础表达水平,MTT方法检测经不同剂量X射线照射后肺癌细胞的存活情况,初步筛选5种细胞中辐射敏感和辐射抵抗的2种细胞;采用克隆形成实验及流式细胞术分别检测细胞存活分数及凋亡率的变化,分析2种肺癌细胞的放射敏感性,采用Western blot检测2种细胞受X射线作用后 NRP1 的表达变化,通过克隆形成实验检测靶向抑制NRP1对A549细胞放射敏感性的影响.结果 5种肿瘤细胞NRP1表达量由高到低依次为:A549>SK-MES-1>H358>H460>H1299;X射线对肿瘤细胞的抑制能力呈剂量依赖性增强,其抑制率由高到低依次为:H460>H1299>H358>SK-MES-1>A549,因此选取A549细胞和 H460细胞做后续研究.克隆形成实验显示,A549在2 Gy照射下的存活分数(SF₂)是0.887,而H460细胞为0.313.A549细胞受照后的凋亡率为对照组的122.54%,明显低于H460细胞的238.88%.辐射可以诱导A549细胞NRP1的表达上调达40%,同时靶向抑制NRP1则可以增强A549细胞的放射敏感性.结论 在NSCLC细胞中A549细胞具有较强的辐射抗性,且辐射抗性的形成与其NRP1表达上调有关.     

LyGDI在非小细胞肺癌细胞A549中的辐射抗性机制研究

目的 探讨LyGDI的表达在非小细胞肺癌细胞A549中的辐射抗性机制。方法 A549和作为对照的H460细胞分别受到0、2、4和6 Gy X射线照射后,克隆形成试验分析辐射抗性差异,Western blot分析LyGDI和辐射敏感性关键基因环氧合酶COX-2的表达。实时荧光定量PCR分析miR-34a以及miR-34b/c在A549和H460细胞中的表达。A549细胞中转染50 nmol/L的miR-34c成熟序列,观察其对A549细胞辐射抗性以及LyGDI和COX-2基因表达的影响。结果 LyGDI和COX-2在辐射抗性的A549细胞中的表达水平高于H460细胞,miR-34a以及miR-34b/c在两种非小细胞肺癌中低表达。转染miR-34c可抑制LyGDI、COX-2和Bcl-2以及激活p21的表达,增强A549细胞细胞的辐射敏感性（t=3.85、5.89、5.12,P<0.05）。结论 LyGDI诱导的COX-2的上调表达以及电离辐射效应miR-34家族的下调表达,是A549细胞辐射抗性的一个主要原因。     

Cell cycle checkpoint evasion and protracted cell cycle arrest in X-irradiated small-cell lung carcinoma cells.

PURPOSE: To determine the longevity and dose-dependence of acute X-irradiation-induced cell cycle perturbations in a panel of seven small-cell lung carcinoma (SCLC) cell lines (COR-L32B, COR-L51B, COR-L88B, COR-L96C, COR-L103, COR-L266B, COR-L279), assessed for TP53 tumour suppressor gene status and showing characteristically long population doubling periods. MATERIALS AND METHODS: Cell lines were screened for abnormalities in TP53. Cell cycle arrest and nuclear fragmentation were determined by flow cytometry under culture conditions that minimized the propensity of SCLC cells to form multicellular aggregates. A faster growing SCLC cell line (NCI-H69) and two breast tumour cell lines were used as controls. RESULTS: NCI-H69 and five of the COR-SCLC cell lines showed clear evidence of TP53 abnormalities and the cycle arrest responses of the breast tumour cell lines established the effects of TP53 mutation on G1/S checkpoint loss. All SCLC lines, at 24 h after low dose irradiation, showed abrogation of the G1/S checkpoint together with a range of expression of a protracted G2/M delay. G2/M delay progressed in all panel cell lines up to 48 h post-irradiation while NCI-H69 showed significant recovery for the dose range 75-600cGy. Only NCI-H69 and one panel line showed dose-dependent progression to complete nuclear DNA fragmentation. CONCLUSIONS: The culture method permits the measurement of cell cycle effects that reflect the TP53 status of SCLC cells. G1/S checkpoint failure, long-term radiation-induced G2 arrest, highly muted apoptotic responses and delayed recovery appear to be typical responses of the recently derived COR-SCLC lines. The results imply that low levels of unrepaired DNA damage, induced at clinically relevant doses, can persist for days in SCLC cells with long cell cycle traverse times, and can remain capable of checkpoint activation with implications for S phase-targeted therapies.     

Cell cycle checkpoint evasion and protracted cell cycle arrest in X-irradiated small-cell lung carcinoma cells

Purpose: To determine the longevity and dose-dependence of acute X-irradiation-induced cell cycle perturbations in a panel of seven small-cell lung carcinoma (SCLC) cell lines (CORL32B, COR-L51B, COR-L88B, COR-L96C, COR-L103, CORL266B, COR-L279), assessed for TP53 tumour suppressor gene status and showing characteristically long population doubling periods. Materials and methods: Cell lines were screened for abnormalities in TP53. Cell cycle arrest and nuclear fragmentation were determined by flow cytometry under culture conditions that minimized the propensity of SCLC cells to form multicellular aggregates. A faster growing SCLC cell line (NCI-H69) and two breast tumour cell lines were used as controls. Results: NCI-H69 and five of the COR-SCLC cell lines showed clear evidence of TP53 abnormalities and the cycle arrest responses of the breast tumour cell lines established the effects of TP53 mutation on G1/S checkpoint loss. All SCLC lines, at 24h after low dose irradiation, showed abrogation of the G1/S checkpoint together with a range of expression of a protracted G2/M delay. G2/M delay progressed in all panel cell lines up to 48h post-irradiation while NCI-H69 showed significant recovery for the dose range 75-600cGy. Only NCI-H69 and one panel line showed dose-dependent progression to complete nuclear DNA fragmentation. Conclusions: The culture method permits the measurement of cell cycle effects that reflect the TP53 status of SCLC cells. G1/S checkpoint failure, long-term radiation-induced G2 arrest, highly muted apoptotic responses and delayed recovery appear to be typical responses of the recently derived COR-SCLC lines. The results imply that low levels of unrepaired DNA damage, induced at clinically relevant doses, can persist for days in SCLC cells with long cell cycle traverse times, and can remain capable of checkpoint activation with implications for S phase-targeted therapies.     

(32)P-磷酸铬体外诱导人非小细胞肺癌A549细胞凋亡

目的观察^32P-磷酸铬(^32P胶体)体外诱导人非小细胞肺癌(NSCLC)A549细胞周期变化和发生细胞凋亡的现象，建立剂量-效应和时间-效应关系。方法将^32P胶体加入A549细胞培养体系进行内照射，初始放射性浓度分别为0，93，180，278，370和463MBq／L，采用Giemsa染色、透射电镜、末端原位标记(TUNEL)等方法进行凋亡细胞的形态学、超微病理、生化特征检测，应用流式细胞术定量检测细胞周期变化和细胞凋亡率。结果A549细胞受照射后S期 G2-M期细胞比率在96h以内呈上升趋势，之后逐渐下降。A54g细胞受照射后72h左右出现兴奋性表现，96h起各受照组均检出凋亡，各剂量组在照射后120h达到凋亡高峰。结论在较低剂量范围内，^32P胶体内照射可以体外诱导人NSCLC A54g发生72h以后的迟发相凋亡，细胞凋亡率与内照射初始放射性浓度呈正相关。     

Radiation and hypoxia-induced non-targeted effects in normoxic and hypoxic conditions in human lung cancer cells

Purpose: Many cell lines with anaerobic metabolism do not show cytotoxic abscopal effect (AE) following irradiation. Further, there is no existing data on the radiation- and hypoxia (H)-induced AE. The purpose of this study was to investigate and compare the status of radiation-induced abscopal effect (RIAE) in normoxic and hypoxic conditions.

Methods: Lung cancer cells (A549, H460) were exposed either to hypoxia or normoxia and then irradiated (2 or 10?Gy). After 24?h, unirradiated hypoxic (H-CM) or normoxic (N-CM) conditioned media (CM) and irradiated hypoxic (H-RCM) or normoxic (N-RCM) CM was collected. Hypoxia-resistant clones (HR: A549/HR, H460/HR) were generated by continuous exposure of the cells to hypoxia. Unirradiated parental cells or HR were exposed to H-CM, N-CM, H-RCM or N-RCM. In some groups, 24?h after exposure to CM, cells were directly irradiated with 2?Gy. Cell growth was monitored using real-time cell electronic sensing system. Further, levels of hypoxia and HIF1α regulated angiogenesis related growth factors, basic fibroblast growth factor (bFGF), placental growth factor (PlGF), soluble fms-like tyrosine kinase (sFlt-1) and vascular endothelial growth factor (VEGF) were assessed in CM.

Results: In the radio-resistant A549 cells, H-RCM was much more effective in inducing growth delay compared to N-RCM. In the radio-sensitive H460 cells, both N-RCM and H-RCM induced growth delay. Interestingly, effects of N-RCM were completely reversed in HR cells. Exposure of cells to direct irradiation (2?Gy) 24?h after incubation with CM resulted in 50–60% reduction in cell proliferation in A549/HR cells and a very significant induction of death (>95%) in H460/HR cells. Direct irradiation of parental or HR clones of A549 and H460 cells exposed to H-CM 24?h with 2?Gy induced significant reduction in cell proliferation (from 40% to >95%) in all the cells. Further, levels of sFlt-1 correlated with growth delay in all the cells.

Conclusions: These results for the first time demonstrate that irradiation of hypoxic cells and exposing the cells to acute hypoxia lead to significant AE.     

外源性野生型p53基因对不同p53功能状态的人肺腺癌细胞株的放射增敏作用

目的 观察外源性野生型p53基因(wtp53)对人肺腺癌细胞株的放射增敏作用,比较不同p53基因状态对照射的影响。 方法 免疫组织化学法、聚合酶链反应-单链构象多态性分析(PCR-SSCP法)筛选p53基因状态不同的两种人肺腺癌细胞系A549及GLC-82,用腺病毒介导wtp53 (Ad-p53)转染后分别给予0、2和4 Gy照射,测定集落形成率,流式细胞仪检测细胞周期分布和凋亡。结果 A549细胞的p53基因正常,而GLC-82细胞的p53基因第7外显子突变,转染后wtp53在两种细胞内均成功表达。Ad-p53对A549及GLC-82细胞抑制率分别为55%和88%,对GLC-82抑制作用较强(P<0.01)。转染Ad-p53后照射,两种细胞集落形成率较对照组明显下降(P<0.001)。流式细胞仪分析Ad-p53使G₁期细胞比例增加和凋亡指数增高,Ad-p53＋照射组最为明显(P<0.001)。结论 Ad-wtp53可以抑制人肺腺癌细胞株的生长,增加其放射敏感性,其放射增敏作用并不依赖于细胞内源性p53基因的状态。     

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。