γH2AX and cleaved PARP-1 as apoptotic markers in irradiated breast cancer BT474 cellular spheroids
γH2AX and cleaved PARP-1 as apoptotic markers in irradiated breast cancer BT474 cellular spheroids
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DOI：
10.3892/ijo_00000311
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发表时间：
2009-07-01
影响因子：
5.2
通讯作者：
Carlsson, J.
中科院分区：
文献类型：
--
作者：
Qvarnstrom, O. F.;Simonsson, M.;Carlsson, J.
化疗和放疗可诱导肿瘤及周围组织的细胞凋亡。为了寻找稳健可靠的凋亡标志物，我们在石蜡包埋的细胞球体中评估了γ H2AX和裂解PARP-1的免疫染色模式。乳腺癌BT474细胞培养成直径为700-800 pm的细胞球体。球体包括增生细胞的外层细胞层，静止细胞的深层区域和坏死的中央区域。用5 Gy辐照后，在距离球体表面0 ~ 150 μ m的时间点（0 ~ 144 h）测定细胞凋亡频率。γ - H2AX和cleaved PARP-1分别独立定量。两种标记物的凋亡频率在时间和空间上都是一致的。与裂解的PARP-1相比，γ - H2AX信号更强，背景更低。中心坏死区被裂解的PARP-1强烈染色，而未检测到γ - H2AX。各时间点细胞凋亡频率随离表面距离的增加而增加。然而，高于未辐照对照水平的凋亡频率仅在辐照后144 h的最后一个时间点检测到。我们已经证明，球体模型是一个实用的系统，用于评估染色模式和特异性的凋亡标志物。此外，径向梯度为研究同一系统内一系列生理条件下的细胞凋亡提供了机会。我们进一步证明，γ - H2AX和cleaved PARP-1是适用于球体增殖区凋亡的标记物。然而，伽马H2AX更强烈和清晰的染色模式表明，该标记物更适合用于球体和类似石蜡包埋材料的细胞凋亡定量。
Chemo- and radiotherapy induce apoptosis in tumours and surrounding tissues. In a search for robust and reliable apoptosis markers, we have evaluated immunostaining patterns of gamma H2AX and cleaved PARP-1 in paraffin-embedded cellular spheroids. Breast cancer BT474 cells were grown as cell spheroids to diameters of 700-800 pm. The spheroids contained an outer cell layer with proliferative cells, a deeper region with quiescent cells and a central area with necrosis. They were irradiated with 5 Gy and the frequency of apoptotic cells was determined at several time points (0-144 h) and distances (0-150 mu m) from the spheroids surface. gamma H2AX and cleaved PARP-1 were quantified independently. Apoptotic frequencies for the two markers agreed both temporally and spatially in the proliferative regions of the spheroids. The gamma H2AX signal was stronger and had lower background compared to cleaved PARP-1. The central necrotic region was intensely stained with cleaved PARP-1, whereas no gamma H2AX could be detected. The apoptotic frequency increased with distance from surface for all time points. However, apoptotic frequencies, above unirradiated control levels, could only be detected for the last time point, 144 h after irradiation. We have shown that the spheroid model is a practical system for evaluation of staining patterns and specificities of apoptosis markers. Also, the radial gradient provides the opportunity to study apoptosis under a range of physiological conditions within the same system. We have further shown that gamma H2AX and cleaved PARP-1 are applicable markers for apoptosis in the proliferative regions of the spheroids. However, the more intense and clear staining patterns of gamma H2AX suggests that this marker is preferable for quantification of apoptosis in spheroids and similar paraffin-embedded materials.