Proliferation and cell death of human glioblastoma cells after carbon-ion beam exposure: Morphologic and morphometric analyses

Histological analyses of glioblastoma cells after carbon-ion exposure are still limited and ultrastructural characteristics have not been investigated in detail. Here we report the results of morphological and morphometric analyses of a human glioblastoma cell line, CGNH-89, after ionizing radiation to characterize the effect of a carbon-beam on glioblastoma cells. Using CGNH-89 cells exposed to 0-10 Gy of X-ray (140 kVp) or carbon-ions (18.3 MeV/nucleon, LET = 108 keV/mu m), we performed conventional histology and immunocytochemistry with MIB-1 antibody, transmission electron microscopy, and computer-assisted, nuclear size measurements. CGNH-89 cells with a G to A transition in codon 280 in exon 8 of the TP53 gene had nuclei with pleomorphism, marked nuclear atypia and brisk mitotic activity. After carbon-ion and X-ray exposure, living cells showed decreased cell number, nuclear condensation, increased atypical mitotic figures, and a tendency of cytoplasmic enlargement at the level of light microscopy. The deviation of the nuclear area size increased during 48 h after irradiation, while the small cell fraction increased in 336 h. In glioblastoma cells of the control, 5 Gy carbon-beam, and 10 Gy carbon-beam, and MIB-1 labeling index decreased in 24 h (12%, 11%, 7%, respectively) but increased in 48 h (10%, 20%, 21%, respectively). Ultrastructurally, cellular enlargement seemed to depend on vacuolation, swelling of mitochondria, and increase of cellular organelles, such as the cytoskeleton and secondary lysosome. We could not observe apoptotic bodies in the CGNH-89 cells under any conditions. We conclude that carbon-ion irradiation induced cell death and senescence in a glioblastoma cell line with mutant TP53. Our results indicated that the increase of large cells with enlarged and bizarre nuclei, swollen mitochondria, and secondary lysosome occurred in glioblastoma cells after carbon-beam exposure.

碳离子照射后胶质母细胞瘤细胞的组织学分析仍然有限，超微结构特征尚未得到详细研究。在此我们报告了对人胶质母细胞瘤细胞系CGNH - 89进行电离辐射后的形态学和形态计量学分析结果，以描述碳束对胶质母细胞瘤细胞的影响。利用暴露于0 - 10 Gy的X射线（140 kVp）或碳离子（18.3 MeV/核子，线性能量传递 = 108 keV/μm）的CGNH - 89细胞，我们进行了常规组织学检查、MIB - 1抗体免疫细胞化学、透射电子显微镜检查以及计算机辅助的细胞核大小测量。TP53基因第8外显子第280密码子发生G到A转换的CGNH - 89细胞具有多形性细胞核、明显的核异型性和活跃的有丝分裂活动。在碳离子和X射线照射后，在光学显微镜下可见活细胞数量减少、核浓缩、非典型有丝分裂象增加以及细胞质增大的趋势。照射后48小时内细胞核面积大小的偏差增加，而在336小时小细胞比例增加。在对照组、5 Gy碳束组和10 Gy碳束组的胶质母细胞瘤细胞中，MIB - 1标记指数在24小时下降（分别为12%、11%、7%），但在48小时上升（分别为10%、20%、21%）。在超微结构上，细胞增大似乎取决于空泡形成、线粒体肿胀以及细胞器（如细胞骨架和次级溶酶体）的增加。在任何条件下我们都未在CGNH - 89细胞中观察到凋亡小体。我们得出结论，碳离子照射在具有突变TP53的胶质母细胞瘤细胞系中诱导了细胞死亡和衰老。我们的结果表明，在碳束照射后胶质母细胞瘤细胞中出现了具有增大且奇异的细胞核、肿胀的线粒体和次级溶酶体的大细胞增加的情况。