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电离辐射在乳腺肿瘤细胞中诱导的p53依赖性加速衰老

p53-Dependent accelerated senescence induced by ionizing radiation in breast tumour cells.

作者信息

Jones K R, Elmore L W, Jackson-Cook C, Demasters G, Povirk L F, Holt S E, Gewirtz D A

机构信息

Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond 23298, USA.

出版信息

Int J Radiat Biol. 2005 Jun;81(6):445-58. doi: 10.1080/09553000500168549.

Abstract

Ionizing radiation has been reported to promote accelerated or premature senescence in both normal and tumour cell lines. The current studies were designed to characterize the accelerated senescence response to radiation in the breast tumour cell in terms of its dependence on functional p53 and its relationship to telomerase activity, telomere lengths, expression of human telomerase reverse transcriptase (hTERT, the catalytic subunit of telomerase) and human telomerase RNA (hTR, the RNA subunit of telomerase), as well as the induction of cytogenetic aberrations. Studies were performed in p53 wild-type MCF-7 cells, MCF-7/E6 cells with attenuated p53 function, MDA-MB231 cells with mutant p53 and MCF-7/hTERT cells with constitutive expression of hTERT. Telomerase activity was measured by the telomeric repeat amplification protocol (TRAP assay), telomere lengths by the terminal restriction fragment (TRF) assay, hTR and hTERT expression by reverse transcriptase-polymerase chain reaction (RT-PCR), senescence by beta-galactosidase staining, and apoptosis by TdT-mediated d-UTP-X nick-end labelling (TUNEL assay). Widespread and extensive expression of beta-galactosidase, a marker of cellular senescence, was evident in MCF-7 breast tumour cells following exposure to 10 Gy of ionizing radiation. Radiation did not suppress expression of either hTERT or hTR, alter telomerase activity or induce telomere shortening. Senescence arrest was also observed in irradiated MCF-7/hTERT cells, which have elongated telomeres due to the ectopic expression of the catalytic component of telomerase. In contrast to MCF-7 cells, irradiated MDA-MB231 breast tumour cells and MCF-7/E6 cells failed to senesce and instead demonstrated a delayed apoptotic cell death. Irradiation produced chromosome end associated abnormalities, including end-to-end fusions (an indicator of telomere dysfunction) in MCF-7 cells, MCF-7/hTERT cells, as well as in MCF-7/E6 cells. When cells were maintained in culture following irradiation, proliferative recovery was evident exclusively after senescence while the cell lines which responded to radiation by apoptosis continued to decline in cell number. Accelerated senescence in response to ionizing radiation is p53 dependent and associated with telomer dysfunction but is unrelated to changes in telomerase activity or telomere lengths, expression of hTERT and hTR. In the absence of functional p53, cells are unable to arrest for an extended period, resulting in apoptotic cell death while accelerated senescence in cells expressing p53 is succeeded by proliferative recovery.

摘要

据报道,电离辐射可促使正常细胞系和肿瘤细胞系加速衰老或过早衰老。当前的研究旨在从对功能性p53的依赖性、与端粒酶活性、端粒长度、人端粒酶逆转录酶(hTERT,端粒酶的催化亚基)和人端粒酶RNA(hTR,端粒酶的RNA亚基)表达的关系以及细胞遗传学异常的诱导等方面,对乳腺肿瘤细胞对辐射的加速衰老反应进行表征。研究在p53野生型MCF-7细胞、p53功能减弱的MCF-7/E6细胞、p53突变的MDA-MB231细胞以及组成性表达hTERT的MCF-7/hTERT细胞中进行。通过端粒重复序列扩增法(TRAP分析)测量端粒酶活性,通过末端限制片段(TRF)分析测量端粒长度,通过逆转录聚合酶链反应(RT-PCR)测量hTR和hTERT表达,通过β-半乳糖苷酶染色检测衰老,通过TdT介导的dUTP-X缺口末端标记法(TUNEL分析)检测凋亡。在暴露于10 Gy电离辐射后,乳腺肿瘤细胞MCF-7中细胞衰老标志物β-半乳糖苷酶广泛且大量表达。辐射并未抑制hTERT或hTR的表达,未改变端粒酶活性,也未诱导端粒缩短。在经辐射的MCF-7/hTERT细胞中也观察到衰老停滞,这些细胞由于端粒酶催化成分的异位表达而具有延长的端粒。与MCF-7细胞不同,经辐射的乳腺肿瘤细胞MDA-MB231和MCF-7/E6细胞未能衰老,而是表现出延迟的凋亡性细胞死亡。辐射产生了染色体末端相关异常,包括MCF-7细胞、MCF-7/hTERT细胞以及MCF-7/E6细胞中的端端融合(端粒功能障碍的一个指标)。辐射后当细胞在培养中维持时,仅在衰老后出现明显的增殖恢复,而通过凋亡对辐射作出反应的细胞系细胞数量持续下降。对电离辐射的加速衰老反应是p53依赖性的,与端粒功能障碍相关,但与端粒酶活性、端粒长度、hTERT和hTR的表达变化无关。在缺乏功能性p53的情况下,细胞无法长时间停滞,导致凋亡性细胞死亡,而表达p53的细胞中的加速衰老之后是增殖恢复。

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