Molecular Mechanisms and Visualization of Radiation-induced tumor cell repopulation

目的/目的：使用任何细胞毒性药物治疗都能使肿瘤中存活的细胞比以前分裂得更快。这种现象在放射生物学家中被广泛认为是“再繁殖”。许多临床研究也表明，再繁殖是放疗失败的主要因素之一。然而，人们对其分子机制知之甚少。为了更好地阐明其机制，我们进行了基因表达谱和病理实验。材料/方法：本研究采用小鼠纤维肉瘤细胞系QRSP （p53野生型）。使用4MV直线加速器，以1.8Gy/min的剂量率对培养基中的细胞进行10 Gy照射。将1万个辐照细胞接种到培养皿中进行菌落试验和克隆。10天后，对菌落进行染色和计数。同时，建立6个无性系，保存于-80℃备用。为了观察这些克隆的辐射抗性，细胞再次用10Gy辐照。使用亲本QRSP细胞系作为对照，对显示最多菌落的克隆（以下称为“耐受性克隆”）进行超过20,000个基因表达的寡核苷酸微阵列分析（Agilent Technologies）。将耐受克隆细胞和QRSP细胞各2万个皮下注射到5只雌性小鼠（C57BL/6）中。28 d后处死小鼠，比较两组小鼠的移植率、肿瘤体积及病理情况。为了进行组织学分析，肿瘤用10%福尔马林固定，石蜡包埋，并用H&E染色进行光镜观察。两名病理学家检查了样本，并在3个随机选择的高倍视野中计数有丝分裂细胞。结果：10Gy辐照后，QRSP对照株产生25个菌落。6个无性系在第二次10Gy辐照后产生的菌落数分别为86、42、38、34、5和3个。比较了QPSP与最耐受性克隆（产生86个菌落）的基因表达。耐受性无性系共有133个基因上调（即上调2.0倍），239个基因下调（即降低<2.0倍）。在上调的基因中，IL6（增加36.0倍）、基质金属蛋白酶（MMP） 13（增加25.8倍）、MMP3（增加22.5倍）和GRO1癌基因（增加12.8倍）的表达量尤其高。在下调的基因中，p16和p57的表达水平特别低，分别下降了14.8倍和12.0倍。牺牲时，QRSP组5只小鼠中有2只（40%）出现肿瘤，耐受克隆组5只小鼠中全部（100%）出现肿瘤（p<0.05，卡方检验）。耐受性克隆的平均肿瘤体积更大：1.92+/-1.68（SD）比0.82+/-1.04 g。耐受性克隆的5个肿瘤中，1个在触诊和显微镜下表现为深部肌肉侵犯。QRSP的平均有丝分裂细胞数为4.0+/-3.9，耐受性克隆的平均有丝分裂细胞数为12.8+/-3.4 （p<0.01，学生t检验）。结论：菌落试验表明，10Gy辐照后可建立耐辐射克隆。最耐受性克隆的微阵列数据在细胞周期调节（周期蛋白依赖性激酶抑制剂p16和p57）和侵袭性（GRO1和MMPs）相关基因方面是独特的。这些体外基因特征与体内肿瘤状态密切相关。该研究表明，种群再生与辐照引起的“克隆”基因表达变化有关，尽管尚不清楚这些变化是由于耐受细胞选择还是基因突变/修饰（如甲基化）。少

Purpose/Objective : Treatment with any cytotoxic agent can trigger surviving cells in a tumor to divide faster than before. This phenomenon is widely recognized as "repopulation" among radiobiologists. Many clinical studies have also indicated that repopulation is one of the major treatment failure factors in radiotherapy. However, little is known about its molecular mechanism. To better clarify the mechanism, gene expression profiling and pathological experiments were performed. Materials/Methods : A mouse fibrosarcoma cell line, QRSP (p53 wild type), was used in this study. Cells in culture medium were irradiated with 10 Gy using a 4MV linear accelerator at a dose rate of 1.8Gy/min. Ten thousand irradiated cells were seeded onto culture dishes for colony assay and cloning. After 10 days, colonies were stained and counted. At the same time, 6 clones were established and stored at -80C for future use. To observe radio-resistance in these clones, cells were irradiated again with 10Gy an … More d colonies were counted as described above. Oligonucleotide microarray analysis (Agilent Technologies) of the expression of over 20,000 genes was performed on the clone that showed the largest number of colonies (hereafter referred to as the "tolerant clone"), using the parental QRSP cell line as a control. Twenty thousand cells of the tolerant clone and of QRSP were injected subcutaneously into 5 female mice (C57BL/6) each. The mice were sacrificed 28 days later and the transplantation ratio, tumor volume, and pathological status between the two groups were compared. For histologic analyses, tumors were fixed in 10% formalin, embedded in paraffin, and stained with H&E for light microscopy. Two pathologists examined the samples and mitotic cells were counted in 3 randomly selected high-power fields. Results : The QRSP control line produced 25 colonies after 10Gy irradiation. For the 6 clones, the number of colonies produced after the second 10Gy irradiation was 86,42,38,34,5 and 3, respectively. Gene expression was compared between QPSP and the most tolerant clone (which produced 86 colonies). A total of 133 genes were up-regulated (i.e., >2.0-fold increase) and a total of 239 genes were down-regulated (i.e., <2.0-fold decrease) in the tolerant clone. Among the up-regulated genes, the following were expressed at a particularly high level : IL6 (36.0-fold increase), matrix metalloproteinase (MMP) 13 (25.8), MMP3 (22.5), and GRO1 oncogene (12.8). Among the down-regulated genes, p16 and p57 were expressed at a particularly low level : 14.8- and 12.0-fold decrease, respectively. On sacrifice, tumors were observed in 2 of 5 mice (40%) for QRSP, and in all 5 mice (100%) for the tolerant clone (p<0.05, chi-square test). The mean tumor volume was greater for the tolerant clone : 1.92+/-1.68(SD) versus 0.82+/-1.04 g. Of the 5 tumors from the tolerant clone, one showed deep muscle invasion on palpation and under microscope examination. The mean mitotic cell number was 4.0+/-3.9 for QRSP, and 12.8+/-3.4 for the tolerant clone (p<0.01, Student's t-test). Conclusions : Colony assay showed that radio-resistant clones were established following 10Gy irradiation. The microarray data of the most tolerant clone was distinctive in terms of genes related to cell-cycle regulation (cyclin-dependent kinase inhibitors p16 and p57) and aggressive nature (GRO1 and MMPs). These in-vitro gene signatures correlated well with in-vivo tumor status. This study implies that repopulation is related to "clonal" gene expression changes caused by irradiation, though it is unknown whether the changes are attributed to tolerant cell selection or to gene mutation/modification (such as methylation). Less