Thielmann H W, Popanda O, Edler L, Böing A, Jung E G
Division of Interaction of Carcinogens with Biological Macromolecules, German Cancer Research Center, Heidelberg.
J Cancer Res Clin Oncol. 1995;121(6):327-37. doi: 10.1007/BF01225684.
The DNA excision repair capacity of 23 primary fibroblast lines from patients with dysplastic nevus syndrome was investigated and DNA repair synthesis ("unscheduled DNA synthesis") was determined after UV exposure. Seventeen fibroblast lines from normal donors served as controls. The dose/response experiments included up to ten dose levels and two wavelength ranges: UV-C (using a low-pressure mercury lamp emitting predominantly 254-nm light) and UV-B (artificial "sunlamp" radiation centering around 312-nm light). For each dose level, silver grains over fibroblast nuclei were counted by visual inspection. Twelve cell lines were also evaluated for both UV wavelength ranges using a new semi-automatic image analyzing system. This system included components for rapid sequential identification of both fibroblast nuclei and silver grains sited above them. Silver grains over 100 nuclei were determined for each UV dose level. Dose/response curves were established and analyzed by linear regression. As a quantitative term for assessing DNA excision repair capacity of a cell line we calculated the linear increase (G0) in the number of grains per nucleus, when the UV dose was multiplied by the factor e (i.e. 2.72). The sensitivity of grain detection and resolution of overlapping grains was approximately threefold better in visual than in automatic counting, especially when there were more than 70 grains over nuclei. The time required for visual counting, however, was tenfold that of automatic counting. The variance-weighted mean G0v.w of all fibroblast lines from patients with dysplastic nevus syndrome was found to be 79.1 (+/- 1.8- grains/nucleus, that of fibroblast lines from normal donors was 74.2 (+/- 1.7) grains/nucleus. This difference revealed a slightly better repair capability for cell lines from patients but was at the borderline of detection and, therefore, should not be overinterpreted. From the experimental accuracy achieved by determination of the variance-weighted means of the two groups, we would have been able to detect a difference of 7 and more grains [> 2 x (sigma normal+sigma patients)]. The variance-weighted mean G0v.w of all fibroblast lines from patients with dysplastic nevus syndrome was found to be 76.4 (+/- 1.4) grains/nucleus, whereas that of fibroblast lines from normal donors was only 66.6 (+/- 1.8) grains/nucleus. This difference was statistically significant and, contrary to expectation, revealed better, not worse post-UV DNA repair capability in cell lines from patients that in those from normal donors.(ABSTRACT TRUNCATED AT 400 WORDS)
研究了发育异常痣综合征患者的23条原代成纤维细胞系的DNA切除修复能力,并在紫外线照射后测定了DNA修复合成(“非定常DNA合成”)。来自正常供体的17条成纤维细胞系作为对照。剂量/反应实验包括多达十个剂量水平和两个波长范围:UV-C(使用主要发射254nm光的低压汞灯)和UV-B(以312nm光为中心的人工“太阳灯”辐射)。对于每个剂量水平,通过目视检查对成纤维细胞核上的银颗粒进行计数。还使用一种新的半自动图像分析系统对12条细胞系在两个紫外线波长范围内进行了评估。该系统包括用于快速顺序识别成纤维细胞核及其上方银颗粒的组件。对于每个紫外线剂量水平,测定100个细胞核上的银颗粒。通过线性回归建立并分析剂量/反应曲线。作为评估细胞系DNA切除修复能力的定量指标,我们计算了当紫外线剂量乘以因子e(即2.72)时,每个细胞核中颗粒数量的线性增加(G0)。在目视计数中,颗粒检测的灵敏度和重叠颗粒的分辨率比自动计数大约高两倍,尤其是当细胞核上有超过70个颗粒时。然而,目视计数所需的时间是自动计数的十倍。发育异常痣综合征患者的所有成纤维细胞系的方差加权平均G0v.w为79.1(±1.8颗粒/细胞核),正常供体的成纤维细胞系的方差加权平均G0v.w为74.2(±1.7)颗粒/细胞核。这种差异显示患者细胞系的修复能力略好,但处于检测的临界值,因此不应过度解读。根据两组方差加权平均值测定所达到的实验精度,我们本能够检测到7个及更多颗粒的差异[>2×(正常标准差+患者标准差)]。发育异常痣综合征患者的所有成纤维细胞系的方差加权平均G0v.w为76.4(±1.4)颗粒/细胞核,而正常供体的成纤维细胞系的方差加权平均G0v.w仅为66.6(±1.8)颗粒/细胞核。这种差异具有统计学意义,与预期相反,显示患者细胞系在紫外线照射后的DNA修复能力比正常供体细胞系更好,而不是更差。(摘要截短至400字)