Heinloth Alexandra N, Shackelford Rodney E, Innes Cynthia L, Bennett Lee, Li Leping, Amin Rupesh P, Sieber Stella O, Flores Kristina G, Bushel Pierre R, Paules Richard S
Growth Control and Cancer Group, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Mol Carcinog. 2003 Jun;37(2):65-82. doi: 10.1002/mc.10122.
The human genome is exposed to many different kinds of DNA-damaging agents. While most damage is detected and repaired through complex damage recognition and repair machineries, some damage has the potential to escape these mechanisms. Unrepaired DNA damage can give rise to alterations and mutations in the genome in an individual cell, which can result in malignant transformation, especially when critical genes are deregulated. In this study, we investigated gene expression changes in response to oxidative stress, gamma (gamma) radiation, and ultraviolet (UV) radiation and their potential implications in cancer development. Doses were selected for each of the three treatments, based on their ability to cause a similar G(1) checkpoint induction and slow down in early S-phase progression, as reflected by a comparable reduction in cyclin E-associated kinase activity of at least 75% in logarithmically growing human dermal diploid fibroblasts. To investigate gene expression changes, logarithmically growing dermal diploid fibroblasts were exposed to either gamma radiation (5 Gy), oxidative stress (75 microM of tert-butyl hydroperoxide (t-butyl-OOH)), or UV radiation (UVC) (7.5 J/m(2)) and RNA was harvested 6 h after treatment. Gene expression was analyzed using the NIEHS Human ToxChip 2.0 with approximately 1901 cDNA clones representing known genes and expressed sequence tags (ESTs). We were able to identify common and distinct responses in dermal diploid fibroblasts to the three different stimuli used. Within our analysis, gene expression profiles in response to gamma radiation and oxidative stress appeared to be more similar than profiles expressed after UV radiation. Interestingly, equivalent cyclin E-associated kinase activity reduction with all the three treatments was associated with greater transcriptional changes after UV radiation than after gamma radiation and oxidative stress. While samples treated with UV radiation displayed modulations of their mitogen activated protein kinase (MAPK) pathway, gamma radiation had its major influence on cell-cycle progression in S-phase and mitosis. In addition, cell cultures from different individuals displayed significant differences in their gene expression responses to DNA damage.
人类基因组会接触到多种不同的DNA损伤剂。虽然大多数损伤可通过复杂的损伤识别和修复机制被检测和修复，但有些损伤有可能逃避这些机制。未修复的DNA损伤可导致单个细胞基因组发生改变和突变，进而可能引发恶性转化，尤其是当关键基因失调时。在本研究中，我们调查了氧化应激、γ（伽马）辐射和紫外线（UV）辐射引起的基因表达变化及其在癌症发展中的潜在影响。根据三种处理各自引起相似的G1期检查点诱导和早期S期进程减缓的能力来选择剂量，这可通过对数生长的人皮肤二倍体成纤维细胞中细胞周期蛋白E相关激酶活性至少降低75%的类似减少来反映。为了研究基因表达变化，将对数生长的皮肤二倍体成纤维细胞暴露于γ辐射（5 Gy）、氧化应激（75 μM叔丁基过氧化氢（t-丁基-OOH））或紫外线（UVC）辐射（7.5 J/m²），并在处理后6小时收集RNA。使用NIEHS人类毒理芯片2.0分析基因表达，该芯片有大约1901个代表已知基因和表达序列标签（EST）的cDNA克隆。我们能够识别皮肤二倍体成纤维细胞对所使用的三种不同刺激的共同和不同反应。在我们的分析中，响应γ辐射和氧化应激的基因表达谱似乎比紫外线辐射后表达的谱更相似。有趣的是，与γ辐射和氧化应激相比，所有三种处理导致的同等细胞周期蛋白E相关激酶活性降低与紫外线辐射后更大的转录变化相关。虽然用紫外线辐射处理的样品显示其丝裂原活化蛋白激酶（MAPK）途径有调节作用，但γ辐射对S期和有丝分裂中的细胞周期进程有主要影响。此外，来自不同个体的细胞培养物对DNA损伤的基因表达反应存在显著差异。
