Li Z, Xia L, Lee L M, Khaletskiy A, Wang J, Wong J Y, Li J J
Department of Radiation Research, Beckman Research Institute, City of Hope National Medical Center, 1500 Duarte Road, Duarte, CA 91010, USA.
Radiat Res. 2001 Apr;155(4):543-53. doi: 10.1667/0033-7587(2001)155[0543:egaimh]2.0.co;2.
Understanding the molecular mechanisms involved in the response of tumors to fractionated exposures to ionizing radiation is important for improving radiotherapy and/or radiochemotherapy. In the present study, we examined the expression of stress-related genes in an MCF-7 cell population (MCF-IR20) that has been derived through treatment with fractionated irradiation (2 Gy per fraction with a total dose of 40 Gy). MCF-IR20 cells showed a 1.6-fold increase in sensitization with dose at 10% isosurvival in a clonogenic assay, and a reduced growth delay ( approximately 15 h compared to approximately 27 h), compared to the parental MCF-7 cells treated with a single dose of 5 Gy. To determine which effector genes were altered in the MCF-IR20 cells, the expression of stress-related effector genes was measured using a filter with 588 genes (Clontech) that included major elements involved in cell cycle control, DNA repair, and apoptosis. Compared to MCF-7 cells that were not exposed to fractionated radiation, 19 genes were up- regulated (2.2-5.1-fold) and 4 were down-regulated (2.7-3.4- fold) in the MCF-IR20 cells. In agreement with the array results, 6 up-regulated genes tested by RT-PCR showed elevated expression. Also, activities of the stress-related transcription factors NFKB, TP53 and AP1 showed a 1.2-4.5-fold increase after a single dose of 5 Gy in MCF-IR20 cells compared with parental MCF-7 cells. However, when the radioresistant MCF-IR20 cell were cultured for more than 12 passages after fractionated irradiation (MCF-RV), radioresistance was lost, with the radiosensitivity being the same as the parental MCF- 7 cells. Interestingly, expression levels of CCNB1, CD9 and CDKN1A in MCF-RV cells returned to levels expressed by the parental cells, whereas the expression levels of three other genes, MSH2, MSH6 and RPA remained elevated. To determine if any of the changes in gene expression could be responsible for the induced radioresistance, CCNB1 and CDKN1A, both of which were up-regulated in MCF-IR20 cells and down-regulated in MCF-RV cells, were studied further by transfection with antisense oligonucleotides. Antisense of CCNB1 significantly reduced the clonogenic survival of MCF- IR20 cells at doses of 5 and 10 Gy, from 42% to 26% and from 5.7% to 1.0%, respectively. Antisense of CDKN1A, however, had no effect on radiation survival of MCF-IR20 cells. In summary, these results suggest that stress-related effector genes are altered in cells after treatment with fractionated irradiation, and that up-regulation of CCNB1 is responsible, at least in part, for radioresistance after fractionated irradiation.
了解肿瘤对分次电离辐射反应所涉及的分子机制对于改进放射治疗和/或放化疗至关重要。在本研究中，我们检测了通过分次照射（每次2 Gy，总剂量40 Gy）处理获得的MCF - 7细胞群体（MCF - IR20）中应激相关基因的表达。在克隆形成试验中，MCF - IR20细胞在10%等存活剂量下的敏化程度随剂量增加了1.6倍，与接受单次5 Gy照射的亲本MCF - 7细胞相比，生长延迟缩短（约15小时，而亲本细胞约为27小时）。为了确定MCF - IR20细胞中哪些效应基因发生了改变，使用包含588个基因的滤膜（Clontech）检测应激相关效应基因的表达，这些基因包括参与细胞周期调控、DNA修复和凋亡的主要成分。与未接受分次辐射的MCF - 7细胞相比，MCF - IR20细胞中有19个基因上调（2.2 - 5.1倍），4个基因下调（2.7 - 3.4倍）。与芯片结果一致，通过RT - PCR检测的6个上调基因表达升高。此外，与亲本MCF - 7细胞相比，MCF - IR20细胞在单次5 Gy照射后，应激相关转录因子NFKB、TP53和AP1的活性增加了1.2 - 4.5倍。然而，当分次照射后的耐辐射MCF - IR20细胞培养超过12代（MCF - RV）后，耐辐射性丧失，放射敏感性与亲本MCF - 7细胞相同。有趣的是，MCF - RV细胞中CCNB1、CD9和CDKN1A的表达水平恢复到亲本细胞的表达水平，而另外三个基因MSH2、MSH6和RPA的表达水平仍保持升高。为了确定基因表达的任何变化是否可能导致诱导的耐辐射性，通过反义寡核苷酸转染进一步研究了在MCF - IR20细胞中上调而在MCF - RV细胞中下调的CCNB1和CDKN1A。CCNB1反义寡核苷酸在5 Gy和10 Gy剂量下显著降低了MCF - IR20细胞的克隆存活能力，分别从42%降至26%和从5.7%降至1.0%。然而，CDKN1A反义寡核苷酸对MCF - IR20细胞的辐射存活没有影响。总之，这些结果表明，分次照射处理后的细胞中应激相关效应基因发生了改变，并且CCNB1的上调至少部分地导致了分次照射后的耐辐射性。
