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.     

Response of human tumor cell lines in vitro to fractionated irradiation

The surviving fraction of human tumor cell lines after 2 Gy (SF2) varies between 0.1 and 0.8. It has been postulated that differences in inherent radiosensitivity of tumor cells are a major determinant of radiation response in vivo. Assays of inherent radiosensitivity based on acute survival are being developed as predictors of tumor response which often assume that the same inherent radiosensitivity persists throughout a fractionated treatment. We have investigated the response of 2 human tumor cell lines (A549 and MCF7) with different inherent radiosensitivities to in vitro fractionated irradiation. A549 cells had an SF2 of 0.62 and a mean inactivation dose (D) of 3.07 Gy whereas MCF7 cells had an SF2 of 0.30 and a D of 1.52 Gy. Split dose repair capacity (at equal survival levels) was less for A549 than for MCF7 cells and recovery kinetics for both cell lines were substantially longer than those of rodent cell lines. Survival after 5 fractions of 2 Gy given 12 hr apart at 37 degrees C was near to that predicted from the acute survival curve, assuming complete repair and no proliferation. Acute survival of A549 cells which survived 5 fractions of 2 Gy given 12 hr apart was similar to the acute survival of unirradiated cells. When A549 cells were incubated at 22 degrees C between 5 fractions of 2 Gy given 12 hr apart, proliferation and split dose repair were substantially inhibited. These studies support the proposals to use in vitro inherent radiosensitivity assays for the prediction of in vivo response of tumors to fractionated treatment.     

脑胶质瘤不同照射方案生物效应的实验研究

目的 通过对人脑多形性胶质母细胞瘤(BT325细胞系)裸小鼠移植瘤不同分割模式照射后生物效应的实验研究,加深对人脑胶质瘤放射反应生物学特性认识,为临床设计照射计划、制定合理分次治疗方案提供实验依据.方法 对BT325细胞系裸小鼠移植瘤进行单次10、20、30、40、60 Gy照射和2 Gy每周5次每天照射、3 Gy每周3次隔天照射、3 Gy每周5次每天照射、4 Gy每周3次隔天照射,总剂量分别为125、114、126、112 Gy.用肿瘤生长曲线评价剂量效应关系并测定肿瘤体积倍增时间.取贴壁生长的指数生长期BT325细胞,用生长曲线测定细胞倍增时间,细胞克隆形成分析法绘制细胞存活曲线(照射剂量为0、1、2、4、6、8、10 Gy)计算曲线参数值,彗星分析法测定DNA单链断裂半修复时间(T1/2).结果 单次照射各剂量点均不能有效控制肿瘤.2 Gy5次/周和3 Gy3次/周治疗方案对人脑胶质瘤实体瘤也无明显治疗效果.增加分次剂量可使脑胶质瘤实体肿瘤有较明显的消退,其中4 Gy3次/周方案好于其他方案但仍未能达到治愈肿瘤的效果.表明对肿瘤干细胞的控制不理想.各项生物学参数的测定结果:BT325细胞倍增时间为30.16 h,裸小鼠移植瘤肿瘤倍增时间为43 d;细胞存活曲线LQ模型拟合的α=0.360 Gy-1、β=0.057 Gy-2,多靶单击模型拟合的D0=1.394 Gy、Dq=2.127 Gy、SF2=0.714;5 Gy照射DNA单链断裂的T1/2=9.999 min.结论 本实验从实证实验角度印证了临床上脑胶质瘤是一种放射耐受性较高的肿瘤的看法.研究结果提示增高分割剂量有可能提高肿瘤的近期疗效(使肿瘤消退率增加),但如何提高对肿瘤干细胞的控制还需进一步深入研究.各项生物学参数测定结果提示脑胶质瘤细胞内在放射敏感性较差.     

Pre-treatment number of clonogenic cells and their radiosensitivity are major determinants of local tumour control after fractionated irradiation.

OBJECTIVE: The response of tumours to fractionated radiotherapy is determined by many factors including repopulation, reoxygenation, the number of clonogenic cells, and their intrinsic radiosensitivity. However, after single radiation doses given under conditions of clamp hypoxia, the dose to control a tumour locally is dependent only on the number of clonogenic cells and their cellular radiosensitivity. Therefore, these parameters were investigated using local control after single doses given under hypoxia, to predict the outcome of fractionated irradiation. MATERIALS AND METHODS: Ten hSCC cell lines (FaDu, UT-SCC-15, UT-SCC-14, XF354, UT-SCC-5, UT-SCC-45, SAS, CAL-33, UT-SCC-8, and HSC-4) were transplanted subcutaneously into the right hind-leg of NMRI nude mice. At 7mm in diameter, tumours were irradiated either with graded single doses under clamp blood flow conditions (n=873) or with 30 graded fractions within 6 weeks (n=905) under ambient conditions. Local tumour control was determined 120 days after irradiation. Radiation response was quantified in terms of TCD(50), i.e. the dose required to control 50% of tumours locally. RESULTS: Ten tumour lines investigated showed a pronounced heterogeneity in both TCD(50(30fx/6w)) after fractionated irradiation and TCD(50(SDclamp)) after single dose irradiation. TCD(50(30fx/6w)) varied between 45Gy for UT-SCC-45 and 127Gy for SAS; TCD(50(SDclamp)) varied between 42Gy for UT-SCC-14 and 66Gy for CAL-33. Two tumours were excluded from further analysis due to immunogenicity or non-defined TCD(50). Linear regression analysis revealed a significant positive correlation between TCD(50(SDclamp)) and TCD(50(30fx/6w)) (R(2)=0.82, p=0.002). CONCLUSIONS: Significant association between TCD(50(SDclamp)) and TCD(50(30fx/6w)) suggests that the pre-treatment number of clonogenic tumour cells and their cellular radiosensitivity have a major impact on local control after fractionated radiotherapy.     

汉防己甲素对人肺腺癌细胞的放射增敏作用及其机制的实验研究

目的 探讨汉防己甲素（Tet）对人肺腺癌SPC-A1细胞的放射敏感性的影响及其作用机制。方法 MTT法检测Tet对SPC-A1细胞的增殖抑制作用,比较单纯照射组（4 Gy）、照射（4 Gy）+Tet（1 μmol／L）组、单用Tet组（1 μmol／L）及空白对照组间SPC-A1细胞增殖抑制率的差异。采用克隆形成实验来计算受照射后的细胞存活率,拟合细胞存活曲线,计算D0、Dq、SF2。流式细胞术检测照射前后SPC-A1细胞周期的分布情况。结果 Tet对SPC-A1细胞的24、48和72 h半数抑制浓度（IC50）分别为10.77、5.78、和3.89 μmol／L。照射+Tet组24、48、72 h的细胞增殖抑制率均高于单纯照射组,差异有统计学意义（P＜0.05）。克隆形成实验显示照射+Tet组的D0、Dq和SF2值分别为（1.551±0.045）Gy、（0.522±0.023）Gy和0.503±0.008,均低于单纯照射组。放射增敏比（SER）为1.48。流式细胞仪检测结果显示照射导致了SPC-A1细胞G2期阻滞（P＜0.05）。联合Tet后可以降低G2期阻滞细胞比例（P＜0.05）。结论 Tet可以有效增加人肺腺癌SPC-A1细胞的放射敏感性,其机制可能是通过降低放射导致的G2期阻滞细胞比例,从而使DNA的损伤固定,发生增殖性死亡。     

Does skin fibroblast radiosensitivity predict squamous cancer cell radiosensitivity of the same individual?

Individualization of radiation doses is presumed to result in better radiotherapy outcome. Success rate in measuring radiosensitivity is probably the most limiting factor for present radiosensitivity assays to be introduced into clinical routine. To find a simpler predictive parameter, we compared the radiosensitivity of dermal fibroblasts and head and neck squamous cell carcinoma (SCC) cell lines established from the same individuals. The radiosensitivity was tested using the clonogenic 96-well plate assay. The surviving fraction at 2.0 Gy (SF2) was determined, as well as the mean inactivation dose (AUC) of cancer cells. SF2 of SCC cell lines and skin fibroblasts were 0.25-0.44 and 0.11-0.43, respectively. AUC of SCC cells was 1.4-2.1 Gy. Dermal fibroblasts were more radiosensitive than SCC cells in 14 of 15 cases. In 1 patient (UT-SCC-8), cancer cells were found to be more radiosensitive than corresponding dermal fibroblasts. There was a clear tendency to a correlation between radiosensitivities of these 2 cell types, but statistical significance was reached only when the data of UT-SCC-8 was excluded. In our material, the intrinsic radiosensitivity of head and neck SCC cells could in most cases be predicted from the intrinsic radiosensitivity of dermal fibroblasts established from the same individual.     

Ability to undergo apoptosis does not correlate with the intrinsic radiosensitivity (SF2) of human cervix tumor cell lines

PURPOSE: To investigate the relationship between radiation-induced apoptosis and clonogenic cell kill in 9 cervical cancer cell lines. METHODS AND MATERIALS: Cells were irradiated with 0, 2, 8, and 30 Gy. The level of apoptosis was evaluated using flow cytometry (Annexin-V binding), light microscropy (morphology), gel electrophoresis (DNA ladder formation), and TUNEL assay. Cell survival was measured using a clonogenic assay. RESULTS: Of the 9 cervical carcinoma cell lines analyzed, 3 underwent radiation-induced apoptosis: CaSki, HT3, and 778. The levels of apoptosis, obtained 72 h after a dose of 30 Gy, were 49%, 28%, and 26%, respectively. All cell lines exhibited some level of background apoptosis measured by Annexin-V binding (mean = 2.6%+/-0.8; range, 0.2-6.9%) that correlated with the level of radiation-induced apoptosis (r = 0.92, p = 0.001). In 6 of the 9 lines, necrosis was the dominant form of cell death. A significant inverse relationship was found between the level of radiation-induced apoptosis and necrosis after 30 Gy (r = -0.87, p = 0.002). No relationship was found between radiation-induced apoptosis and intrinsic radiosensitivity measured, using a clonogenic assay, as surviving fraction at 2 Gy (SF2). CONCLUSION: Cervical carcinoma cells do not readily undergo radiation-induced apoptosis in vitro. There is no relationship between ability to undergo apoptosis and intrinsic radiosensitivity measured using a clonogenic assay.     

CoCl2诱导缺氧对Eca109细胞细胞周期及放射敏感性的影响

Objective:To investigate the change of the cell cycle,apoptosis and radiosensitivity effect by CoCl2 induced hypoxia in esophageal cancer line Eca109 cells in vitro.Methods:The hypoxia culture model induced by 150 microM CoCl2 was established.The cell cycle and apoptosis were measured with flow cytometry (FCM).The radiosensitivity was analysized with clonogenic assay after irradiation alone or combined with hypoxia in Eca109 cells in vitro.Results:Eca109 cells were treated with 150 microM CoCl2 for 24 h,cell cycle arrest in G0/G1 phase increase and decreasing arrest in S phase with longer of hypoxiac time (0-24 h),the other rate of cell cycle and apoptosis did not change obviously.The G2/M phase block was arrested obviously in radiation alone comparing with the hypoxia plus irradiated group,apoptosis did not occur in Eca109 cell line following irradiation.The DO value and cell surviving fraction of Eca109 cell was 2.48 Gy,2.44 Gy and 97.33%,96.33% in hypoxia and control group,respectively;the Dq value of Eca109 cell was 2.89 Gy,0.52 Gy,the cell surviving fraction after radiation with 4 Gy was 48.3%,21.7% in hypoxia and control group,respectively.The hypoxia decreased the radiosensitivity in esophageal cancer Eca109 cells with clonogenic assay.Conclusion:Hypoxia induced by CoCl2 influences radiosensitivity of Eca109 cell through regulating cellular proliferation rates.     

siRNA干扰EGFR表达对食管鳞癌和腺癌细胞放射敏感性影响

目的 探讨siRNA干扰EGFR表达对食管鳞癌和腺癌放射敏感性的影响。方法 以食管鳞癌、腺癌细胞系ECa-109、OE-19作为研究对象。脂质体转染化学合成的不同EGFR-siRNA和阴性-siRNA,通过RT-PCR及蛋白印迹法检测转染前后EGFR表达情况,CCK8法分析转染对细胞增殖活性影响。设ECa-109、OE-19细胞空白对照组(O1、O2组)、单纯照射组(R1、R2组)及EGFR-siRNA照射组(E-R1、E-R2组),单次照射0、2、4、6、8 Gy。克隆形成实验计算细胞存活分数及放射增敏比(SER_D0值比),流式细胞仪检测EGFR-siRNA联合放射对细胞周期分布和细胞凋亡率影响,单次照射6 Gy。结果 EGFR-siRNA可明显下调两种细胞EGFR表达,且转染对细胞增殖抑制率<5%(4.9%、4.5%)。克隆形成实验结果显示E-R1、E-R2组细胞SF值低于O1、O2组,SER_D0值比分别为1.40、1.01。流式细胞术结果显示E-R1组比E-R2组照后G₂+M期比例增加、S期比例下降(P=0.016、0.028),细胞凋亡率也增高(P=0.007)。结论 与食管腺癌细胞相比,干扰EGFR表达显著提高了食管鳞癌细胞的放射敏感性。     

TECIA法体外放射敏感性实验对原发性肺鳞癌放疗个体化方案选择的意义

目的：建立人原发性肺鳞癌的组织培养放疗敏感性检测法,快速确定人原发性肺鳞癌组织的辐射敏感性的个体差异,用于筛选放疗方案及指导临床肿瘤个体化放疗方案。方法：采用TECIA法检测不同个体原发性肺鳞癌组织在不同剂量、不同分次放疗方案照射后不同时间的细胞凋亡水平。比较不同剂量、不同分次放疗方案照射后不同时间的细胞凋亡水平的差异。结果：TECIA法发现,照射前人原发性肺鳞癌组织细胞凋亡指数较低,放疗后出现较高的细胞凋亡指数,与放疗剂量呈正相关。结论：肿瘤放射敏感性实验对指导患者的个体化治疗具有重要的价值。     

Radiosensitivity testing of primary cervical carcinoma: evaluation of intra- and inter-tumour heterogeneity

Biopsies from 89 patients with cervical carcinoma were studied using a clonogenic assay to obtain values for the surviving fraction at 2 Gy (SF2). Heterogeneity in intrinsic radiosensitivity was investigated by independently processing multiple biopsies from 18 tumors. No significant differences between intra-tumour SF2 values were demonstrated (p = 0.30). The results have shown that intra-tumour heterogeneity is not a limitation to radiosensitivity testing using the Courtenay-Mills assay. A wide range of values (0.13-0.97) for SF2 was obtained with a mean value of 0.47 +/- 0.18 (+/- 1 S.D., CV = 38%) for 52 squamous cell carcinomas and 0.59 +/- 0.27 for four adenocarcinomas. There were statistically significant differences between the individual tumours (p less than 0.001). From the analysis-of-variance of all the SF2 results it appears to be the surviving fractions below about 0.40 and those above about 0.7 which show significant differences in radiosensitivity between pairs of tumours (p = 0.05). Also 36% of the values of SF2 show significant differences from the mean SF2 of all tumours. The storage of tumour cell suspensions in liquid nitrogen improved the colony-forming efficiency (CFE) but it did not alter the radiosensitivity.     

Potential use of the alkaline comet assay as a predictor of bladder tumour response to radiation

Bladder tumours show a variable response to radiotherapy with only about 50% showing good local control; currently there is no test to predict outcome prior to treatment. We have used five bladder tumour cell lines (T24, UM-UC-3, TCC-SUP, RT112, HT1376) to investigate the potential of the alkaline comet assay (ACA) to predict radiosensitivity. Radiation-induced DNA damage and repair were compared to clonogenic survival. When the five cell lines were irradiated and initial DNA damage was plotted against cell survival, at all doses (0-6 Gy), a significant correlation was found (r2=0.9514). Following 4 Gy X-irradiation, all cell lines, except T24, showed a correlation between SF2 vs half-time for repair and SF2 vs residual damage at 5, 10, 20 and 30 min. The T24 cell line showed radioresistance at low doses (0-2 Gy) and radiosensitivity at higher doses (4-6 Gy) using both cell survival and ACA end points, explaining the lack of correlation observed for this cell line. These data indicate that initial DNA damage and residual damage can be used to predict for radiosensitivity. Our data suggest that predictive tests of radiosensitivity, appropriate to the clinical situation, may require the use of test doses in the clinical range.     

Inherent radiosensitivity testing of tumor biopsies obtained from patients with carcinoma of the cervix or endometrium.

The inherent radiosensitivity of tumor biopsies obtained from a series of patients with carcinoma of the uterine cervix or endometrium has been characterized. Early passage cell lines were irradiated and assayed for cell survival using a clonogenic assay system. Survival curves were generated using the alpha/beta model and the surviving fraction at 2 Gy (SF2) was estimated. A wide range of SF2 values was observed among histologically similar tumors. The mean (+/- SD) SF2 value was 0.29 +/- 0.12 (range = 0.11-0.59) for the cervical biopsies and 0.30 +/- 0.13 (range = 0.11-0.67) for the endometrial biopsies. No correlation between inherent radiosensitivity and tumor DNA index or histopathology was observed. Patient accrual continues with the expectation that these results may help to determine whether SF2 values are of clinical value in predicting the response of individual patients to treatment with radiotherapy.     

Intrinsic radiosensitivity and prediction of patient response to radiotherapy for carcinoma of the cervix.

The intrinsic radiosensitivity of cervical carcinoma has been measured using a soft agar clonogenic assay. All patients received radical radiotherapy alone with a minimum of 2 years post-treatment follow-up. Only women with stage I, II and III disease were included in the analysis. Values for cell surviving fraction at 2 Gy (SF2) were obtained for 88 tumours with an assay success rate of 73%. The 53 patients alive and well at the time of analysis had tumours with a mean SF2 that was significantly lower than the value from the 22 patients with locoregional failure (P < 0.01). Patients with radioresistant tumours (SF2 > 0.40, the median) had a significantly lower 3 year survival level than those with sensitive tumours (SF2 < or = 0.40) (P = 0.002). Also the frequency of local recurrence was higher (P = 0.001) whether these were central (P = 0.009) or peripheral (P = 0.046). Cell surviving fraction at 3.5 Gy was obtained for 46 tumours and the 3 year patient survival rate was significantly higher for those with SF3.5 values less than the median (P = 0.043). There was, however, no difference in the level of local recurrence (P = 0.24). The ability to grow in culture was not associated with significantly poorer patient survival (P = 0.56) or failure to control the primary disease (P = 0.17). While high colony forming efficiencies were associated with an increased rate of local recurrence (P = 0.029) they did not predict for overall patient survival (P = 0.32). These data suggest that, for cervical carcinoma treated with radical radiotherapy, intrinsic radiosensitivity is important in determining treatment outcome.     

In vitro radiosensitivity of skin fibroblasts can identify a group of patients with complications in various health tissues after radiotherapy]

PURPOSE: A retrospective study of the in vitro radiosensitivity of skin fibroblasts derived from two groups of patients treated by definitive radiotherapy for a variety of tumors who either displayed or did not display severe complications. PATIENTS AND METHODS: Seven radiotherapy patients were selected: three were treated for head and neck, prostate and non-Hodgkin lymphoma tumors, and did not develop any significant complications (control group); four patients were treated for bladder, thyroid, head and neck and anal canal tumors and developed serious acute and especially late reactions (hypersensitive group). Primary cell cultures of skin fibroblasts were established and their radiosensitivity studied by the clonogenic assay after exposing to single radiation doses ranging between 1 and 8 Gy. RESULTS: The survival fraction at 2 Gy (SF2) ranged from 0.27 to 0.38, with a mean of 0.33 for the control group, and from 0.10 to 0.20 with a mean of 0.17 for the hypersensitive group. The Mann-Whitney non-parametric test showed that the difference between the two means was statistically significant (p = 0.03). CONCLUSION: The data are in favor of a correlation between the radiosensitivity of patients' fibroblasts and the reactions of different normal tissues to radiotherapy. This association supports the use of the clonogenic survival, or a surrogate test, as a predictive assay. The multiplicity of normal tissues and organs implicated in this association suggests the existence of genetic factors that determine, at least in part, the radiosensitivity of target cells involved in the expression of normal tissues complications following radiotherapy.     

Ovarian cancer: radiation sensitivity in vitro

The radiosensitivity of four human ovarian cancer cell lines was investigated in vitro by a clonogenic assay and analyzed using the linear-quadratic model. Two cell lines were found to be highly radiosensitive (mean inactivation dose (D) 0.82-0.92 Gy; surviving fraction 2 Gy (SF2) less than or equal to 0.13). Two other cell lines were less sensitive to radiation (D 1.31-1.94 Gy; SF2 0.22-0.38). Although the use of external radiotherapy in ovarian cancer has been limited due to the pattern of metastatic spread of this cancer, the present data support the view that ovarian carcinomas are radiosensitive tumors. Investigations on the effects of new approaches, such as delivering radiation more specifically to intraperitoneal ovarian cancer cells, are warranted.     

Relationship between tumour cell in vitro radiosensitivity and clinical outcome after curative radiotherapy for squamous cell carcinoma of the head and neck.

BACKGROUND AND PURPOSE: Clinically, it is recognized that individual tumours respond differently to radiation treatment. Variation in tumour cell radiosensitivity is believed to be an important underlying factor. In the current study, cellular in vitro radiosensitivity was estimated as the fraction of surviving cells after a radiation dose of 2 Gy (SF2) and related to clinical outcome after curative radiotherapy. PATIENTS AND METHODS: Thirty-eight patients with squamous cell carcinoma of the head and neck were treated with curative radiotherapy alone. Pre-treatment biopsies were disaggregated to form a single-cell suspension and cells were cultured in the modified Courtenay-Mills soft agar clonogenic assay. Directly from this assay and with no respect to cell type, overall SF2 was assessed. By collecting the obtained colonies on a preparation slide using a colony-filter technique, and with immunocytochemical staining, it was possible to measure the surviving fraction of tumour cells selectively as tumour cell SF2. RESULTS: Experimentally, a broad inter-tumour variation was found for both tumour cell SF2 and overall SF2. Using weighted linear regression, it was demonstrated that tumour cell SF2 and overall SF2 were two independent measures of tumour radiosensitivity. In general, the measures of tumour radiosensitivity were independent of patient sex and age, T- and N-category, disease stage, tumour size and plating efficiency. Among the 38 patients grouped in loco-regional failures and patients with loco-regional control, respectively, sex, age, total radiation dose, overall treatment time and tumour grade were equally distributed. Advanced stage, lymph node involvement and tumour size correlated statistically significantly with poor loco-regional control. Neither tumour cell SF2, overall SF2, nor plating efficiency predicted loco-regional tumour control probability. In a multivariate analysis with respect to the risk of loco-regional tumour failure, only disease stage yielded independent prognostic significance. This significance suggests that this patient sample was representative for the patient population with head and neck cancer. CONCLUSION: In 38 patients with squamous cell carcinoma of the head and neck, the estimated tumour radiosensitivities were not statistically related to clinical outcome after curative radiotherapy alone.     

Radiation induced DNA damage and damage repair in human tumor and fibroblast cell lines assessed by histone H2AX phosphorylation

PURPOSE: To analyze the radiation-induced levels of gammaH2AX and its decay kinetics in 10 human cell lines covering a wide range of cellular radiosensitivity (SF2, 0.06-0.63). METHODS AND MATERIALS: Five tumor cell lines included Colo-800 melanoma, two glioblastoma (MO59J and MO59K), fibrosarcoma HT 1080, and breast carcinoma MCF7. Five primary skin fibroblasts lines included two normal strains, an ataxia telangiectasia strain, and two fibroblast strains from breast cancer patients with an adverse early skin reaction to radiotherapy. Cellular radiosensitivity was assessed by colony-forming test. Deoxyribonucleic acid damage and repair were analyzed according to nuclear gammaH2AX foci intensity, with digital image analysis. RESULTS: The cell lines tested showed a wide degree of variation in the background intensity of immunostained nuclear histone gammaH2AX, which was higher for the tumor cell lines compared with the fibroblast strains. It was not possible to predict clonogenic cell survival (SF2) for the 10 cell lines studied from the radiation-induced gammaH2AX intensity. In addition, the slopes of the dose-response (0-4 Gy) curves, the rates of gammaH2AX disappearance, and its residual expression (相似文献   

吉非替尼对肺癌细胞株HCC827和H358放射敏感性的影响及其机制研究

背景与目的表皮生长因子受体(epidermal growth factor receptor,EGFR)是决定放疗效应的一个重要因素,其过表达或是下游通路的激活与包括非小细胞肺癌在内的肿瘤的放疗抵抗相关,因而阻断EGFR的信号通路可能会增强放疗敏感性。本研究旨在探讨小分子EGFR酪氨酸激酶抑制剂吉非替尼能否增加肺癌细胞株HCC827和H358的放疗敏感性及其可能的机制。方法选取HCC827和H358这两个非小细胞肺癌细胞株,分为单纯X线组和X线+吉非替尼两组。单纯X线组采用单纯X线照射,X线+吉非替尼组经1μmol/L吉非替尼作用24h后再行X线照射。克隆形成实验比较两株细胞中不同分组细胞放射敏感性,免疫荧光激光共聚焦显微镜观察X线照射后各时间点细胞核中的磷酸化H2AX(γ-H2AX)及EGFR焦点在细胞中的定位情况,Western blot法检测放疗后胞质胞核蛋白中EGFR的表达。结果克隆形成实验中,H358细胞实验组与对照组在各放疗剂量点的SF2值分别为0.355和0.433;HCC827细胞实验组与对照组在各放疗剂量点的SF2值分别为0.223和0.242,差别不明显。激光共聚焦显微镜观察照射4Gy后各时间段实验组H358细胞核中g-H2AX斑点相比对照组要多,且持续时间更长。而对照组和实验组的HCC827细胞g-H2AX斑点在各时间段并无明显差异;激光共聚焦显微镜观察照射4Gy后对照组H358的EGFR蛋白在1h内入核,而经吉非替尼处理后EGFR蛋白几乎不入核;实验组及对照组HCC827细胞的EGFR表达位置均在细胞质中,胞核中很少或者没有,可以认为并无入核现象;Western blot结果显示,H358细胞在经4Gy放射处理后有入核现象,而预先经吉非替尼处理后,EGFR蛋白几乎不在核内表达而仍位于细胞浆内。对于HCC827细胞,实验组及对照组的EGFR蛋白均在细胞质中表达,胞核中很少或没有,且两组并无明显差异。结论吉非替尼可增加肺癌细胞株H358的放射敏感性,这可能与其阻止放疗后EGFR入核、影响放疗后双链断裂(double strand break,DSB)修复有关;而对HCC827细胞无影响,可能与其放疗后EGFR不入核相关。