Apoptosis in human adenocarcinoma HT29 cells induced by exposure to hypoxia.
Apoptosis in human adenocarcinoma HT29 cells induced by exposure to hypoxia.
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DOI：
10.1093/jnci/87.2.117
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发表时间：
1995-01
期刊：
影响因子：
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通讯作者：
K. Yao;M. M. Clayton-M.;P. O'dwyer
中科院分区：
文献类型：
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作者：
K. Yao;M. M. Clayton-M.;P. O'dwyer
最近的证据表明，程序性细胞死亡或细胞凋亡可能是肿瘤化疗药物致死性的重要机制。肿瘤细胞凋亡可由多种物理应激诱导，包括辐射、紫外线、热休克和寒冷。在初步研究中，我们发现细胞暴露于缺氧环境中会迅速诱导几种直接早期基因的表达，包括c-jun、jun-D和c-fos，以及一种双功能氧化还原蛋白/核酸内切酶Ref-1。我们的目的是确定缺氧诱导的过表达核酸内切酶是否与DNA完整性改变和细胞凋亡的表现有关。方法采用电泳和免疫荧光技术检测培养细胞对凋亡的诱导作用，并将其与缺氧诱导基因表达联系起来。结果：我们发现，人腺癌HT29细胞暴露于缺氧环境8小时（仅导致14%的生存能力丧失），在缺氧8小时后和再氧之前开始诱导核小体间DNA断裂。缺氧处理细胞的免疫荧光显示，缺氧4小时后可检测到凋亡细胞，再氧化12小时达到峰值（31.3%的细胞），再氧化36小时后凋亡细胞全部从群体中清除。缺氧诱导的细胞凋亡与c-myc信使RNA （mRNA）的显著诱导有关。我们之前已经证明缺氧与ref-1 mRNA诱导有关。虽然c-myc的表达随着时间的推移而下降，与凋亡细胞消失的过程相似，但ref-1的表达仍然升高。与c-myc的下降相一致，我们观察到bcl-2的表达在再氧后24小时开始延迟增加。结论和意义这些结果提示Ref-1诱导与缺氧暴露后细胞凋亡的发生之间可能存在关系。
BACKGROUND Recent evidence indicates that programmed cell death or apoptosis may be an important mechanism for the lethality of cancer chemotherapy drugs in tumor cells. Apoptosis may be induced in tumor cells by a number of physical stresses, including radiation, UV light, heat shock, and cold. In preliminary studies, we have found that exposure of cells to hypoxia rapidly induces the expression of several immediate early genes, including c-jun, jun-D, and c-fos, and of a bifunctional redox protein/endonuclease, Ref-1. PURPOSE Our purpose was to determine if hypoxia-induced overexpression of the endonuclease was associated with altered DNA integrity and manifestations of apoptosis. METHODS We examined cultured cells for the induction of apoptosis by electrophoresis and immunofluorescence techniques and related these findings to the induction of gene expression by hypoxia. RESULTS We found that an 8-hour exposure of human adenocarcinoma HT29 cells to hypoxia (which results in only 14% loss of viability) induced internucleosomal DNA fragmentation beginning after 8 hours of hypoxia and before reoxygenation. Immunofluorescence of hypoxia-treated cells showed that apoptotic cells were detectable initially after 4 hours of hypoxia and reached a peak (31.3% of cells) at 12 hours after reoxygenation and that by 36 hours after reoxygenation all apoptotic cells had been eliminated from the population. Hypoxia-induced apoptosis was associated with a marked induction of c-myc messenger RNA (mRNA). We have previously shown that hypoxia is associated with ref-1 mRNA induction. While expression of c-myc declined with a time course similar to that of the disappearance of apoptotic cells, that of ref-1 remained elevated. Coincident with the decline of c-myc, we observed a late increase in the expression of bcl-2 beginning 24 hours after reoxygenation. CONCLUSIONS AND IMPLICATIONS These results suggest a possible relationship between Ref-1 induction and the occurrence of apoptosis following a hypoxic exposure.