The p33ING1b tumor suppressor cooperates with p53 to induce apoptosis in response to etoposide in human osteosarcoma cells

p33ING1b induces cell cycle arrest and stimulates DNA repair, apoptosis and chemosensitivity. The magnitude of some p33ING1b effects may be due to activation of the tumor suppressor p53. To investigate if the p33ING1b protein affected chemosensitivity of osteosarcoma cells, we overexpressed p33ING1b in p53+/+ U2OS cells or in p53-mutant MG63 cells, and then assessed for growth arrest and apoptosis after treatment with etoposide. p33ING1b increased etoposide-induced growth inhibition and apoptosis to a much greater degree in p53+/+ U2OS cells than in p53-mutant MG63 cells. Moreover, ectopic expression of p33ING1b markedly upregulated p53, p21WAF1 and bax protein levels and activated caspase-3 protein kinase in etoposide-treated U2OS cells. Together, our data indicate that p33ING1b prominently enhances etoposide-induced apoptosis through p53-dependent pathways in human osteosarcoma cells. p33ING1b may be an important marker and/or therapeutic target in the prevention and treatment of metastatic osteosarcoma.     

Nucleolar protein CSIG is required for p33ING1 function in UV-induced apoptosis

Cellular senescence-inhibited gene (CSIG) protein, a nucleolar protein with a ribosomal L1 domain in its N-terminus, can exert non-ribosomal functions to regulate biological processes, such as cellular senescence. Here, we describe a previously unknown function for CSIG: promotion of apoptosis in response to ultraviolet (UV) irradiation-induced CSIG upregulation. We identified p33ING1 as a binding partner that interacts with CSIG. After UV irradiation, p33ING1 increases its protein expression, translocates into the nucleolus and binds CSIG. p33ING1 requires its nucleolar targeting sequence region to interact with CSIG and enhance CSIG protein stability, which is essential for activation of downstream effectors, Bcl-2-associated X protein, to promote apoptosis. Thus, our data imply that p33ING1–CSIG axis functions as a novel pro-apoptotic regulator in response to DNA damage.     

The PCNA-associated factor KIAA0101/p15(PAF) binds the potential tumor suppressor product p33ING1b

The KIAA0101/p15(PAF)/OEATC-1 protein was initially isolated in a yeast two-hybrid screen for proliferating cell nuclear antigen (PCNA) binding partners, and was shown to bind PCNA competitively with the cell cycle regulator p21(WAF). PCNA is involved in DNA replication and damage repair. Using polyclonal antisera raised against a p15(PAF) fusion protein, we have shown that in a range of mammalian tumor and non-tumor cell lines the endogenous p15(PAF) protein localises to the nucleus and the mitochondria. Under normal conditions no co-localisation with PCNA could be detected, however following exposure to UV it was possible to co-immunoprecipitate p15(PAF) and PCNA from a number of cell lines, suggesting a UV-enhanced association of the two proteins. Overexpression of p15(PAF) in mammalian cells was also found to protect cells from UV-induced cell death. Based on similarities between the behaviour of p15(PAF) and the potential tumor suppressor product p33ING1b, we have further shown that these two proteins interact in the same complex in cell cultures. This suggests that p15(PAF) forms part of a larger protein complex potentially involved in the regulation of DNA repair, apoptosis and cell cycle progression.     

p33 Enhances UVB-Induced Apoptosis in Melanoma Cells

The biological functions of the tumor suppressor ING1 have been studied extensively in the past few years since it was cloned. It shares many biological functions with p53 and has been reported to mediate growth arrest, senescence, apoptosis, anchorage-dependent growth, chemosensitivity, and DNA repair. Some of these functions, such as cell cycle arrest and apoptosis, have been shown to be dependent on the activity of both ING1 and p53 proteins. Two recent reports by Scott and colleagues demonstrate that p33^ING1 (one of the ING1 isoforms) translocates to the nucleus and binds to PCNA upon UV irradiation. Here we report that p33^ING1 mediates UV-induced cell death in melanoma cells. We found that overexpression of p33^ING1 increased while the introduction of an antisense p33^ING1 plasmid reduced the apoptosis rate in melanoma cells after UVB irradiation. We also demonstrated that enhancement of UV-induced apoptosis by p33^ING1 required the presence of p53. Moreover, we found that p33^ING1 enhanced the expression of endogenous Bax and altered the mitochondrial membrane potential. Taken together, these observations strongly suggest that p33^ING1 cooperates with p53 in UVB-induced apoptosis via the mitochondrial cell death pathway in melanoma cells.     

过表达人p33ING1b促进UV诱导的衰老细胞凋亡

p33 ^ING1b是肿瘤抑制基因ING1的主要表达形式,已有的研究表明,p33^ING1b参与了细胞的生长抑制、凋亡、染色质重塑、DNA损伤修复、肿瘤抑制等.但是,它在细胞衰老过程中的作用目前还不清楚.本研究分析了p33 ^ING1b基因在细胞衰老过程中的表达情况.结果发现,无论在mRNA水平还是在蛋白水平,p33 ^ING1b在衰老细胞中的表达均降低.通过构建和包装含p33^ING1b基因的重组腺病毒,将p33 ^ING1b导入衰老细胞中使其过表达,结果显示,p33^ING1b的过表达明显促进UV诱导的衰老细胞凋亡,提示p33I^NG1b在衰老细胞中的表达下调与衰老细胞抗凋亡有关.     

腺病毒介导的p33ING1b过表达显著促进衰老细胞的DNA损伤修复

p33^ING1b是一个较晚发现的肿瘤抑制基因ING1的主要表达形式,自从被成功克隆以后得到了广泛的研究,已有的研究表明,p33^ING1b参与了细胞的生长抑制、凋亡、染色质重塑、DNA损伤修复、肿瘤抑制和细胞衰老等。但是它在细胞衰老过程中的作用特别是对衰老细胞DNA损伤修复的影响还没有被地阐明,在本研究中,我们首先用2BS细胞构建了细胞衰老模型,通过RT-PCR和Western blot技术证实p33^ING1b在衰老细胞中的表达水平是下调的,然后通过构建和包装包含p33^ING1b基因的腺病毒,将p33^ING1b导入年轻和衰老细胞中并使其过表达,用HCR（host cell reactivation）方法检测年轻细胞和衰老细胞DNA损伤修复能力。我们的实验首次表明,相对于年轻细胞,p33^ING1b的过表达使衰老细胞的DNA的损伤修复能力显著增加,这说明p33^ING1b在衰老细胞中的表达下调与衰老细胞DNA损伤修复能力的下降有关,也进一步证实了p33^ING1b在细胞衰老过程中起着十分重要的作用。     

NAD(P)H quinone oxidoreductase 1 inhibits the proteasomal degradation of the tumour suppressor p33(ING1b)

The tumour suppressor p33(ING1b) ((ING1b) for inhibitor of growth family, member 1b) is important in cellular stress responses, including cell-cycle arrest, apoptosis, chromatin remodelling and DNA repair; however, its degradation pathway is still unknown. Recently, we showed that genotoxic stress induces p33(ING1b) phosphorylation at Ser 126, and abolishment of Ser 126 phosphorylation markedly shortened its half-life. Therefore, we suggest that Ser 126 phosphorylation modulates the interaction of p33(ING1b) with its degradation machinery, stabilizing this protein. Combining the use of inhibitors of the main degradation pathways in the nucleus (proteasome and calpains), partial isolation of the proteasome complex, and in vitro interaction and degradation assays, we set out to determine the degradation mechanism of p33(ING1b). We found that p33(ING1b) is degraded in the 20S proteasome and that NAD(P)H quinone oxidoreductase 1 (NQO1), an oxidoreductase previously shown to modulate the degradation of p53 in the 20S proteasome, inhibits the degradation of p33(ING1b). Furthermore, ultraviolet irradiation induces p33(ING1b) phosphorylation at Ser 126, which, in turn, facilitates its interaction with NQO1.     

抑癌蛋白p33(ING1)对肿瘤作用的研究进展

p33(ING1)是生长抑制基因(ING1)编码的重要抑癌蛋白,具有抑制细胞生长﹑促进细胞老化﹑维持基因组稳定性、作用于细胞周期调控点等作用,其失活与肿瘤的发生、发展密切相关。本文就近年来有关p33(ING1)的结构、功能及其在肿瘤中的失活机制、临床应用前景等方面的研究进展进行了概述。     

miR-138转录可下调p33ING1在衰老细胞中的表达

p33ING1参与了多种生物学过程,包括细胞生长抑制、凋亡、DNA损伤修复、染色质重塑等．近来研究显示,p33在细胞衰老过程中表达降低,这可能与衰老细胞的抗凋亡有关．但p33在衰老细胞中表达下调的分子机理仍不清楚．我们发现,在衰老细胞中miR-138表达升高与p33基因的表达降低密切相关．以下实验结果支持如此结论：(1)与年轻细胞相比,带p33ING1 3′UTR 报告载体荧光素酶活性在衰老细胞中降低;突变3′UTR上的miR-138结合位点可升高报告载体荧光素酶在衰老细胞中的活性;(2)在衰老细胞中miR-138的表达升高;(3)在年轻细胞中,过表达miR-138不仅可抑制带p33ING1 3′UTR 报告载体荧光素酶活性,而且下调细胞内p33ING1基因mRNA和蛋白水平.与此相反,抑制miR-138活性可升高带p33ING1 3′UTR 报告载体荧光素酶活性,并且上调细胞内p33ING1基因mRNA和蛋白水平．这些结果表明,p33ING1基因是miR-138的靶基因;在衰老过程中,miR-138表达升高, 由此导致该基因的表达降低．     

ING1家族各异构体抑制HeLa细胞增殖的分子机制研究

ING1(inhibitor of growth 1)是一个候选抑癌基因家族,p47ING1a、p33ING1b和p24ING1c是其三种剪接异构体.通过MTT法和流式细胞术研究ING1a、ING1b和ING1c对HeLa细胞增殖的影响,结果发现三者均可将HeLa细胞阻滞于G0/G1期并抑制细胞生长.采用PCR方法构建ING1a和ING1b的PHD结构域缺失体1aΔC和1bΔC,进而使ING1a,ING1b、ING1c、1aΔC和1bΔC在HeLa细胞中过表达.采用Western blot检测上述HeLa细胞中p16INK4a、PTEN/p27Kip1和p53/p21Waf1的表达变化,结果发现ING1a、ING1b、ING1c和1aΔC均可促进p16INK4a的表达,其中ING1c的促进作用最为显著,1bΔC则略微抑制了p16INK4a的蛋白质表达.利用荧光素酶分析初步确定1aΔC可增强p16INK4a启动子活性而促进p16INK4a的转录,1bΔC则抑制了p16INK4a启动子活性.上述结果阐释了ING1家族各成员对HeLa细胞增殖的影响及其分子机制,从而确定了各异构体功能的异同及它们所调控的重要基因.首次发现除p53/p21Waf1通路外,ING1家族各异构体还可通过上调p16INK4a和PTEN的表达而抑制肿瘤细胞生长,并且ING1a的PHD结构域删除体可以增强p16INK4a的转录,这为研究ING1家族抑制肿瘤细胞生长的分子机制提供了新的线索.     

p53-, SIRT1-, and PARP-1-independent downregulation of p21WAF1 expression in nicotinamide-treated cells

Nicotinamide at mM concentration is a potent inhibitor of certain key molecules involved in cell survival, such as SIRT1 and PARP-1, and affects cell survival in various conditions in vivo and in vitro. However, the effect of an acute treatment of nicotinamide on gene expression has rarely been closely examined. In our study, the treatment of 10 mM nicotinamide downregulated p21WAF1 expression in various human cells including p53-negative or SIRT1-knockdown cells indicating gene regulation not mediated by p53 or SIRT1. Meanwhile, in the nicotinamide-treated cells, Sp1 activity and protein level was substantially reduced due to increased proteasome-mediated degradation. Our results indicate that nicotinamide treatment attenuates p21WAF1 expression through Sp1 downregulation, and suggest a possible involvement of nicotinamide metabolism in cellular gene expression.     

ING1 induces apoptosis through direct effects at the mitochondria

The ING family of tumor suppressors acts as readers and writers of the histone epigenetic code, affecting DNA repair, chromatin remodeling, cellular senescence, cell cycle regulation and apoptosis. The best characterized member of the ING family, ING1, interacts with the proliferating cell nuclear antigen (PCNA) in a UV-inducible manner. ING1 also interacts with members of the 14-3-3 family leading to its cytoplasmic relocalization. Overexpression of ING1 enhances expression of the Bax gene and was reported to alter mitochondrial membrane potential in a p53-dependent manner. Here we show that ING1 translocates to the mitochondria of primary fibroblasts and established epithelial cell lines in response to apoptosis inducing stimuli, independent of the cellular p53 status. The ability of ING1 to induce apoptosis in various breast cancer cell lines correlates well with its degree of translocation to the mitochondria after UV treatment. Endogenous ING1 protein specifically interacts with the pro-apoptotic BCL2 family member BAX, and colocalizes with BAX in a UV-inducible manner. Ectopic expression of a mitochondria-targeted ING1 construct is more proficient in inducing apoptosis than the wild type ING1 protein. Bioinformatic analysis of the yeast interactome indicates that yeast ING proteins interact with 64 mitochondrial proteins. Also, sequence analysis of ING1 reveals the presence of a BH3-like domain. These data suggest a model in which stress-induced cytoplasmic relocalization of ING1 by 14-3-3 induces ING1-BAX interaction to promote mitochondrial membrane permeability and represent a paradigm shift in our understanding of ING1 function in the cytoplasm and its contribution to apoptosis.     

Mitochondrial ribosomal protein L41 mediates serum starvation-induced cell-cycle arrest through an increase of p21(WAF1/CIP1)

Ribosomal proteins not only act as components of the translation apparatus but also regulate cell proliferation and apoptosis. A previous study reported that MRPL41 plays an important role in p53-dependent apoptosis. It also showed that MRPL41 arrests the cell cycle by stabilizing p27(Kip1) in the absence of p53. This study found that MRPL41 mediates the p21(WAF1/CIP1)-mediated G1 arrest in response to serum starvation. The cells were released from serum starvation-induced G1 arrest via the siRNA-mediated blocking of MRPL41 expression. Overall, these results suggest that MRPL41 arrests the cell cycle by increasing the p21(WAF1/CIP1) and p27(Kip1) levels under the growth inhibitory conditions.