Jaiswal Aruna S, Multani Asha S, Pathak Sen, Narayan Satya
UF Shands Cancer Center and Anatomy and Cell Biology, College of Medicine, Academic Research Building, Room R4-216, PO Box 100232, University of Florida, Gainesville, FL 32610, USA.
Mol Cancer. 2004 Jan 16;3:3. doi: 10.1186/1476-4598-3-3.
Cellular senescence is a state in which mammalian cells enter into an irreversible growth arrest and altered biological functions. The senescence response in mammalian cells can be elicited by DNA-damaging agents. In the present study we report that the DNA-damaging agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) is able to induce senescence in the HCT-116 colon cancer cell line.
Cells treated with lower concentrations of MNNG (0-25 microM) for 50 h showed a dose-dependent increase in G2/M phase arrest and apoptosis; however, cells treated with higher concentrations of MNNG (50-100 microM) showed a senescence-like G0/G1 phase arrest which was confirmed by increased expression of beta-galactosidase, a senescence induced marker. The G2/M phase arrest and apoptosis were found to be associated with increased levels of p53 protein, but the senescence-like G0/G1 phase arrest was dissociated with p53 protein levels, since the p53 protein levels decreased in senescence-like arrested cells. We further, determined whether the decreased level of p53 was a transcriptional or a translational phenomenon. The results revealed that the decreased level of p53 protein in senescence-like arrested cells was a transcriptional phenomenon since p53 mRNA levels simultaneously decreased after treatment with higher concentrations of MNNG. We also examined the effect of MNNG treatment on other cell cycle-related proteins such as p21, p27, cyclin B1, Cdc2, c-Myc and max. The expression levels of these proteins were increased in cells treated with lower concentrations of MNNG, which supported the G2/M phase arrest. However, cells treated with higher concentrations of MNNG showed decreased levels of these proteins, and hence, may not play a role in cell cycle arrest. We then examined a possible association of the expression of APC protein and telomeric DNA signals with cellular senescence in MNNG-treated cells. We found that protein and mRNA levels of APC were drastically reduced in cells treated with higher concentrations of MNNG. The loss of APC expression might lead to chromosomal instability as well as microtubular disorganization through its dissociation with tubulin. In fact, the protein level of alpha-tubulin was also drastically decreased in senescence-like arrested cells treated with higher concentrations of MNNG. The levels of telomeric DNA also decreased in cells treated with higher concentrations of MNNG.
These results suggest that in response to DNA alkylation damage the senescence-like arrest of HCT-116 cells was associated with decreased levels of APC protein, microtubular organization, and telomeric DNA.
细胞衰老指的是哺乳动物细胞进入不可逆生长停滞并伴有生物学功能改变的一种状态。DNA损伤剂可引发哺乳动物细胞的衰老反应。在本研究中，我们报告DNA损伤剂N-甲基-N'-硝基-N-亚硝基胍（MNNG）能够在HCT-116结肠癌细胞系中诱导衰老。
用较低浓度（0 - 25微摩尔）的MNNG处理细胞50小时，细胞在G2/M期停滞和凋亡方面呈现剂量依赖性增加；然而，用较高浓度（50 - 100微摩尔）的MNNG处理的细胞表现出类似衰老的G0/G1期停滞，这通过衰老诱导标志物β-半乳糖苷酶表达增加得到证实。发现G2/M期停滞和凋亡与p53蛋白水平升高有关，但类似衰老的G0/G1期停滞与p53蛋白水平无关，因为在类似衰老停滞的细胞中p53蛋白水平降低。我们进一步确定p53水平降低是转录现象还是翻译现象。结果表明，在类似衰老停滞的细胞中p53蛋白水平降低是转录现象，因为用较高浓度的MNNG处理后p53 mRNA水平同时降低。我们还检测了MNNG处理对其他细胞周期相关蛋白如p21、p27、细胞周期蛋白B1、Cdc2、c-Myc和max的影响。在用较低浓度MNNG处理的细胞中，这些蛋白的表达水平升高，这支持了G2/M期停滞。然而，用较高浓度MNNG处理的细胞这些蛋白水平降低，因此可能在细胞周期停滞中不起作用。然后我们检测了MNNG处理细胞中APC蛋白表达和端粒DNA信号与细胞衰老之间可能的关联。我们发现用较高浓度MNNG处理的细胞中APC的蛋白和mRNA水平大幅降低。APC表达缺失可能通过其与微管蛋白解离导致染色体不稳定以及微管紊乱。事实上，在用较高浓度MNNG处理的类似衰老停滞的细胞中α-微管蛋白的蛋白水平也大幅降低。用较高浓度MNNG处理的细胞中端粒DNA水平也降低。
这些结果表明，响应DNA烷基化损伤，HCT-116细胞的类似衰老停滞与APC蛋白水平降低、微管组织和端粒DNA有关。
