Alpha-tocopheryl succinate induces cytostasis and apoptosis in osteosarcoma cells: the role of E2F1

Alpha-tocopheryl succinate (alpha-TOS), a redox-inactive analog of vitamin E, induces cell cycle arrest, differentiation, and triggers apoptosis. We examined the ability of alpha-TOS to induce cytostasis and/or apoptosis in two human osteosarcoma cell lines, which carry wild-type pRb but differ in the p53 status. In the wt-p53 cells, alpha-TOS induced apoptosis, which was associated with p53 activation and enhanced E2F1 expression. Mutant p53 cells failed to undergo apoptosis when challenged with alpha-TOS. The cell growth arrest after alpha-TOS treatment was associated with a reduced expression of E2F1. Knocking down E2F1 rendered the alpha-TOS-sensitive cells rather resistant to the apoptotic stimulus inducing a marked and prolonged cell growth arrest. We conclude that alpha-TOS induces cell growth arrest or apoptosis involving E2F1.     

E2F1 mediates DNA damage and apoptosis through HCF‐1 and the MLL family of histone methyltransferases

E2F1 is a key positive regulator of human cell proliferation and its activity is altered in essentially all human cancers. Deregulation of E2F1 leads to oncogenic DNA damage and anti‐oncogenic apoptosis. The molecular mechanisms by which E2F1 mediates these two processes are poorly understood but are important for understanding cancer progression. During the G1‐to‐S phase transition, E2F1 associates through a short DHQY sequence with the cell‐cycle regulator HCF‐1 together with the mixed‐lineage leukaemia (MLL) family of histone H3 lysine 4 (H3K4) methyltransferases. We show here that the DHQY HCF‐1‐binding sequence permits E2F1 to stimulate both DNA damage and apoptosis, and that HCF‐1 and the MLL family of H3K4 methyltransferases have important functions in these processes. Thus, HCF‐1 has a broader role in E2F1 function than appreciated earlier. Indeed, sequence changes in the E2F1 HCF‐1‐binding site can modulate both up and down the ability of E2F1 to induce apoptosis indicating that HCF‐1 association with E2F1 is a regulator of E2F1‐induced apoptosis.     

E2F1 confers anticancer drug resistance by targeting ABC transporter family members and Bcl-2 via the p73/DNp73-miR-205 circuitry

Resistance to anti-neoplastic agents is the major cause of therapy failure, leading to disease recurrence and metastasis. E2F1 is a strong inducer of apoptosis in response to DNA damage through its capacity to activate p53/p73 death pathways. Recent evidence, however, showed that E2F1, which is aberrantly expressed in advanced malignant melanomas together with antagonistic p73 family members, drives cancer progression. Investigating mechanisms responsible for dysregulated E2F1 losing its apoptotic function, we searched for genomic signatures in primary and late clinical tumor stages to allow the prediction of downstream effectors associated with apoptosis resistance and survival of aggressive melanoma cells. We identified miR-205 as specific target of p73 and found that upon genotoxic stress, its expression is sufficiently abrogated by endogenous DNp73. Significantly, metastatic cells can be rescued from drug resistance by selective knockdown of DNp73 or overexpression of miR-205 in p73-depleted cells, leading to increased apoptosis and the reduction of tumor growth in vivo. Our data delineate an autoregulatory circuit, involving high levels of E2F1 and DNp73 to downregulate miR-205, which, in turn, controls E2F1 accumulation. Finally, drug resistance associated to this genetic signature is mediated by removing the inhibitory effect of miR-205 on the expression of Bcl-2 and the ATP-binding cassette transporters A2 (ABCA2) and A5 (ABCA5) related to multi-drug resistance and malignant progression. These results define the E2F1-p73/DNp73-miR-205 axis as a crucial mechanism for chemoresistance and, thus, as a target for metastasis prevention.     

E2F1 confers anticancer drug resistance by targeting ABC transporter family members and Bcl-2 via the p73/DNp73-miR-205 circuitry

Resistance to anti-neoplastic agents is the major cause of therapy failure, leading to disease recurrence and metastasis. E2F1 is a strong inducer of apoptosis in response to DNA damage through its capacity to activate p53/p73 death pathways. Recent evidence, however, showed that E2F1, which is aberrantly expressed in advanced malignant melanomas together with antagonistic p73 family members, drives cancer progression. Investigating mechanisms responsible for dysregulated E2F1 losing its apoptotic function, we searched for genomic signatures in primary and late clinical tumor stages to allow the prediction of downstream effectors associated with apoptosis resistance and survival of aggressive melanoma cells. We identified miR-205 as specific target of p73 and found that upon genotoxic stress, its expression is sufficiently abrogated by endogenous DNp73. Significantly, metastatic cells can be rescued from drug resistance by selective knockdown of DNp73 or overexpression of miR-205 in p73-depleted cells, leading to increased apoptosis and the reduction of tumor growth in vivo. Our data delineate an autoregulatory circuit, involving high levels of E2F1 and DNp73 to downregulate miR-205, which, in turn, controls E2F1 accumulation. Finally, drug resistance associated to this genetic signature is mediated by removing the inhibitory effect of miR-205 on the expression of Bcl-2 and the ATP-binding cassette transporters A2 (ABCA2) and A5 (ABCA5) related to multi-drug resistance and malignant progression. These results define the E2F1-p73/DNp73-miR-205 axis as a crucial mechanism for chemoresistance and, thus, as a target for metastasis prevention.     

G1阻滞中p21-E2F间的相互作用

前列腺素A2(PGA2)具有强的体内、外抗增殖活性,引起细胞周期阻滞,同时,可诱导cdk抑制物p21蛋白的表达,后者亦可介导多种细胞的G1阻滞.提示p21^waf1/cip1在PGA2诱导的细胞周期阻滞中具有重要作用.主要介绍了近两年来有关p21^waf1/cip1与转录因子E2F间的相互作用的研究,阐述p21^waf1/cip1在PGA2介导的细胞周期阻滞中的作用机制.     

转录因子E2F1抑制CREG表达调控体外培养的人血管平滑肌细胞分化

为探讨转录因子E2F1在血管平滑肌细胞(vascular smooth muscle cells,VSMCs)表型转化中的作用及其对E1A激活基因阻遏子(cellular repressor of E1A-stimulated genes,CREG)表达调控的分子机制,应用生物信息学方法,定位人CREG(hCREG)基因启动子并确定转录因子E2F1在hCREG启动子区的结合位点,PCR方法克隆并构建hCREG基因启动子绿色荧光报告基因载体,以hCREG启动子区E2F1结合位点为模板,化学合成E2F1寡聚脱氧核苷酸(ODN)和错配E2F1ODN,利用转录因子"诱骗(Decoy)"策略,用E2F1ODN转染体外培养的VSMCs以阻断E2F1与hCREG基因启动子区的结合,蛋白质印迹(Western blot)分析检测阻断前后细胞内hCREG蛋白、报告基因绿色荧光蛋白(green fluorescent protein,GFP)和平滑肌细胞分化标志蛋白SMα-actin表达变化.结果显示:分化表型HITASY细胞中E2F1表达下调伴出核转位,而增殖表型的HITASY细胞中E2F1蛋白表达明显增加且定位于核内.进一步应用FuGene6瞬时转染E2F1ODN和错配E2F1ODN于体外培养HITASY细胞中,蛋白质印迹分析发现,转染E2F1ODN后,HITASY细胞中hCREG、SMα-actin和GFP表达均较未阻断组及错配组细胞明显增加.上述研究结果证实,E2F1是hCREG基因转录的重要调控因子,能够直接结合于hCREG启动子区阻遏hCREG表达,参与hCREG蛋白对VSMCs表型转化的调控作用.     

E2F-1- and E2Ftr-mediated apoptosis: the role of DREAM and HRK

E2F-1-deleted mutant, 'truncated E2F' (E2Ftr, E2F-1[1-375]), lacking the carboxy-terminal transactivation domain, was shown to be more potent at inducing cancer cell apoptosis than wild-type E2F-1 (wtE2F-1; full-length E2F-1). Mechanisms by which wtE2F-1 and E2Ftr induce apoptosis, however, are not fully elucidated. Our study demonstrates molecular effects of pro-apoptotic BH3-only Bcl-2 family member Harakiri (Hrk) in wtE2F-1- and E2Ftr-induced melanoma cell apoptosis. We found that Hrk mRNA and Harakiri (HRK) protein expression was highly up-regulated in melanoma cells in response to wtE2F-1 and E2Ftr overexpression. HRK up-regulation did not require the E2F-1 transactivation domain. In addition, Hrk gene up-regulation and HRK protein expression did not require p53 in cancer cells. Hrk knockdown by Hrk siRNA was associated with significantly reduced wtE2F-1- and E2Ftr-induced apoptosis. We also found that an upstream factor, 'downstream regulatory element antagonist modulator' (DREAM), may be involved in HRK-mediated apoptosis in response to wtE2F-1 and E2Ftr overexpression. DREAM expression levels increased following wtE2F-1 and E2Ftr overexpression. Western blotting detected increased DREAM primarily in dimeric form. The homodimerization of DREAM resulting from wtE2F-1 and E2Ftr overexpression may contribute to the decreased binding activity of DREAM to the 3'-untranslated region of the Hrk gene as shown by electromobility shift assay. Results showed wtE2F-1- and E2Ftr-induced apoptosis is partially mediated by HRK. HRK function is regulated in response to DREAM. Our findings contribute to understanding the mechanisms that regulate wtE2F-1- and E2Ftr-induced apoptosis and provide insights into the further evaluation of how E2Ftr-induced apoptosis may be used for therapeutic gain.