| Co-Investigator(Kenkyū-buntansha) |
YASUDA Motoaki Hokkaido University, Graduate school of Dental medicine, Associate Professor, 大学院・歯学研究科, 助教授 (90239765)
HOMMA Akihiro Hokkaido University, Graduate school of medicine, Instructor, 大学院・医学研究科, 助手 (30312359)
NAKADA Kunihiro Hokkaido University, Hokkaido University Hospital, Doctor, 医員 (00301010)
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| Research Abstract |
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
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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
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