Molecular Mechanisms and Visualization of Radiation-induced tumor cell repopulation

辐射诱导肿瘤细胞增殖的分子机制和可视化

• 批准号: 16390331
• 负责人: NISHIOKA Takeshi
• 金额: $ 5.12万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16390331/
• 关键词: repopulation; cell cycle; cancer invasion; radiation therapy; DNA array

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

目的：任何细胞毒性药物治疗都可以触发肿瘤中存活的细胞比以前更快地分裂。这种现象在放射生物学家中被广泛认为是“再增殖”。许多临床研究也表明，再增殖是放射治疗失败的主要因素之一。然而，对其分子机制知之甚少。为了更好地阐明机制，进行了基因表达谱分析和病理学实验。材料/方法：本研究使用小鼠纤维肉瘤细胞系QRSP（p53野生型）。用4 MV直线加速器以1.8戈伊/min的剂量率对培养液中的细胞进行10戈伊的辐照。10天后，对集落进行染色和计数。同时，建立6个克隆并在-80 ℃下保存以备将来使用。为了观察这些克隆的辐射抗性，细胞再次用10 Gy和10 Gy的剂量照射， ...更多信息 如上所述对菌落计数。使用亲本QRSP细胞系作为对照，对显示最大菌落数的克隆（下文称为“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2万个耐受性克隆细胞和QRSP细胞皮下注射到5只雌性小鼠（C57 BL/6）中。28 d后处死小鼠，比较两组小鼠移植率、肿瘤体积及病理状态。对于组织学分析，将肿瘤固定在10%福尔马林中，包埋在石蜡中，并用H&E染色用于光学显微镜检查。两名病理学家检查样本，并在3个随机选择的高倍视野中计数有丝分裂细胞。结果：QRSP对照系经10 Gy照射后，产生25个菌落。第二次10 Gy照射后，6个克隆的菌落数分别为86、42、38、34、5和3个。在QPSP和最耐受的克隆（其产生86个集落）之间比较基因表达。总共有133个基因被上调（即，>2.0倍增加）并且总共239个基因被下调（即，<2.0倍降低）。在上调的基因中，以下基因以特别高的水平表达：IL 6（36.0倍增加）、基质金属蛋白酶（MMP）13（25.8倍）、MMP 3（22.5倍）和GRO 1癌基因（12.8倍）。在下调的基因中，p16和p57的表达水平特别低：分别降低了14.8倍和12.0倍。在处死时，对于QRSP，在5只小鼠中的2只（40%）中观察到肿瘤，并且对于耐受性克隆，在所有5只小鼠（100%）中观察到肿瘤（p<0.05，卡方检验）。耐受克隆的平均肿瘤体积更大：1.92+/-1.68（SD）vs 0.82+/-1.04 g。在来自耐受性克隆的5个肿瘤中，在触诊和显微镜检查下，1个显示深肌肉浸润。QRSP的平均有丝分裂细胞数为4.0+/-3.9，耐受克隆的平均有丝分裂细胞数为12.8+/-3.4（p<0.01，Student ′ s t检验）。结论：10 Gy照射后，克隆形成率较高，且具有一定的耐辐射性。最耐受克隆的微阵列数据在与细胞周期调节（细胞周期蛋白依赖性激酶抑制剂p16和p57）和侵袭性（GRO 1和MMPs）相关的基因方面是独特的。这些体外基因特征与体内肿瘤状态密切相关。这项研究意味着细胞再增殖与辐射引起的“克隆”基因表达变化有关，尽管目前尚不清楚这些变化是归因于耐受细胞选择还是归因于基因突变/修饰（例如甲基化）。少

项目成果

Pilot study of modified version of CHOP plus radiotherapy for early-stage aggressive non-Hodgkin's lymphoma of the head and neck

改良版 CHOP 联合放疗治疗早期侵袭性头颈部非霍奇金淋巴瘤的初步研究

• 发表时间: 2004
• 期刊: International Journal of Radiation Oncology Biology Physics 60
• 作者: Nishioka T;Tsuchiya K;Nishioka S.
• 通讯作者: Nishioka S.

Anorectal gastrointestinal stromal tumor mimicking prostatic tumor on CT and MRI

肛门直肠胃肠道间质瘤在 CT 和 MRI 上与前列腺肿瘤相似

• 发表时间: 2005
• 期刊: European Journal of Radiology Extra 53
• 作者: Hiromura T;Nishioka T;Nishioka S;Ikeda H;Tomita K
• 通讯作者: Tomita K

Long-term results of ethmoid squamous cell or undifferentiated carcinoma treated with radiotherapy with or without surgery.

筛窦鳞状细胞癌或未分化癌接受放射治疗（联合或不联合手术）的长期结果。

• 发表时间: 2005
• 期刊: Cancer J 11(2)
• 作者: Uchida D;Shirato H;Onimaru R,;Endou H;Aoyama H;Tsuchiya K;Nishioka T;Homma A;Furuta Y;Fukuda S;Miyasaka K.
• 通讯作者: Miyasaka K.

Spontaneous rupture of renal angiomyolipoma: value of multidetector CT angiography for interventional therapy

• DOI: 10.1007/s10140-005-0445-9
• 发表时间: 2005-11
• 期刊: Emergency Radiology
• 影响因子: 2.2
• 作者: T. Hiromura;T. Nishioka;Kazuo Tomita

Pilot study of modified version of CHOP plus radiotherapy for early-stage aggressive non-Hodgkin's lymphoma of the head and neck.

改良版 CHOP 加放疗治疗头颈部早期侵袭性非霍奇金淋巴瘤的初步研究。

• 发表时间: 2004
• 期刊: Int J Radiat Oncol Biol Phys. 60巻
• 作者: Nishioka T;Tsuchiya K;Nishioka S他
• 通讯作者: Nishioka S他