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.     

ING2 recruits histone methyltransferase activity with methylation site specificity distinct from histone H3 lysines 4 and 9

p33ING2 belongs to the ING-gene family that is involved in tumor suppression, DNA repair, cell cycle regulation, and cellular senescence. Most functions are dependent on the tumor suppressor p53. p33ING2 was also shown to bind to trimethylated lysine 4 of histone H3. Here, we show that p33ING2 contains a transferable silencing function, which is independent of p53. p33ING2-mediated gene silencing is resistant to the HDAC-inhibitor trichostatin A indicating that p33ING2 uses a non-HDAC class I or II pathway for gene repression in reporter assays. In line with that we show that p33ING2 is associated with histone methyltransferase (HMT) activity in vitro and in vivo, methylating specifically histone H3. Interestingly, the specificity is distinct from the MeCP2-recruited HMT. Mutation or methylation of lysine 9, a mark well known for repression, abrogates histone methylation by MeCP2 but not by the p33ING2 complex. Instead, the ING2-associated HMT shows an increased methylation activity if lysine 9 is methylated. In contrast, mutation or methylation of lysine 4, a methylation preferentially detected at active genes, led to a reduction of the ING2-associated HMT. Notably, also p33ING1 recruits HMT activity suggesting a more general biochemical interaction between members of p33ING family and HMT activity. Deletion analyses revealed that the ING2 C-terminus recruits HMT activity, which correlates with silencing function.     

p33(ING1) 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.     

Results and prospects of the yeast three-hybrid system

The known members of inhibitor of growth (ING) gene family are considered as candidate tumor suppressor genes. ING4, a novel member of ING family, is recently reported to interact with tumor suppressor p53, p300 (a major component of histone acetyl transferase complexes), and p65(RelA) subunit of NF-κB. In this study, we investigated the cellular behaviors of HepG2 cells with exogenous ING4. Interestingly, the overexpression of ING4 negatively regulated the cell growth with significant G2/M arrest of cell cycle, and moreover, enhanced the cell apoptosis triggered by serum starvation in HepG2 cells. Furthermore, the exogenous ING4 could upregulate endogenous p21 and Bax in HepG2 cells, not in p53-deficient Saos-2 cells, suggesting that G2/M arrest induced by ING4 could be mediated by the increased p21 expression in a p53-dependent manner, although there is no significant increase of p53 expression in HepG2 cells. Moreover, HepG2 cells with exogenous ING4 could significantly increase cell death, as exposed to some DNA-damage agents, such as etoposide and doxorubicin, implying that ING4 could enhance chemosensitivity to certain DNA-damage agents in HepG2 cells.     

Gain of function properties of mutant p53 proteins at the mitotic spindle cell cycle checkpoint

Mutations in the p53 tumor suppressor gene locus predispose human cells to chromosomal instability. This is due in part to interference of mutant p53 proteins with the activity of the mitotic spindle and postmitotic cell cycle checkpoints. Recent data demonstrates that wild type p53 is required for postmitotic checkpoint activity, but plays no role at the mitotic spindle checkpoint. Likewise, structural dominant p53 mutants demonstrate gain-of-function properties at the mitotic spindle checkpoint and dominant negative properties at the postmitotic checkpoint. At mitosis, mutant p53 proteins interfere with the control of the metaphase-to-anaphase progression by up-regulating the expression of CKs1, a protein that mediates activatory phosphorylation of the anaphase promoting complex (APC) by Cdc2. Cells that carry mutant p53 proteins overexpress CKs1 and are unable to sustain APC inactivation and mitotic arrest. Thus, mutant p53 gain-of-function at mitosis constitutes a key component to the origin of chromosomal instability in mutant p53 cells.     

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.     

ING2 controls the G1 to S-phase transition by regulating p21 expression

ING2 (Inhibitor of Growth 2) is a candidate tumor suppressive protein frequently lost in human tumors. Recently, we have reported that ING2 downregulation impairs DNA replication forks progression and leads to genome instability. To better understand the tumor suppressive functions of ING2 and its role in the cell cycle, we downregulated its expression in cells and studied the consequences of this downregulation on the G1/S transition. We observed that the inhibition of ING2 expression accelerated the progression of cells from G1 to S phase, and was accompanied by a decrease of p21 expression. Moreover, we show that the regulation of p21 by ING2 is independent, of the tumor suppressive protein p53. Interestingly, this function seems to be unique for ING2 since its closest homologue ING1 does not regulate the G1/S transition. It has been suggested previously that ING2 may modulate the trimethylation of H3K4 at the promoter of p21. Accordingly, our results suggest that there may be a link between the regulation of the G1/S transition by ING2 and the level of H3K4Me3. All together, these results bring new information concerning the role of ING2 in the regulation of the cell cycle and suggest that it may play important roles in controlling several S phase checkpoints.     

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.     

Cloning of a novel gene (ING1L) homologous to ING1, a candidate tumor suppressor

The ING1 gene encodes p33(ING1), a putative tumor suppressor for neuroblastomas and breast cancers, which has been shown to cooperate with p53 in controlling cell proliferation. We have isolated a novel human gene, ING1L, that potentially encodes a PHD-type zinc-finger protein highly homologous to p33(ING1). Fluorescence in situ hybridization and radiation-hybrid analyses assigned ING1L to human chromosome 4. Both ING1 and ING1L are expressed in a variety of human tissues, but we found ING1L expression to be significantly more pronounced in tumors from several colon-cancer patients than in normal colon tissues excised at the same surgical sites. Although the significance of this observation with respect to carcinogenesis remains to be established, the data suggest that ING1L might be involved in colon cancers through interference with signal(s) transmitted through p53 and p33(ING1).     

ATM: the product of the gene mutated in ataxia-telangiectasia.

Ataxia-telangiectasia mutated (ATM) is the product of the gene mutated in the human genetic disorder ataxia-telangeictasia (A-T). It is a 370 kDa protein that is a member of the phosphatidyl inositol 3-kinases superfamily. A-T cells and those derived from Atm-/- mice are characterized by hypersensitivity to ionizing radiation and defective cell cycle checkpoints. Defects are observed at all cell cycle checkpoints in A-T cells post-irradiation including the G1/S interface where ATM plays an important role in the activation of the tumour suppressor gene product p53. Activation leads to the induction of p21/WAF1, inhibition of cyclin-dependent kinase activity, failure to phosphorylate key substrates such as the retinoblastoma protein and consequently G1 arrest. ATM also plays an important role in the regulation and surveillance of meiotic progression. Absence of ATM gives rise to a spectrum of defects including immunodeficiency, neurodegeneration, radiosensitivity and cancer predisposition. It is clear that a better definition of the role of ATM in DNA damage recognition, cell cycle control and cell signalling may assist in the treatment of the progressive neurodegeneration in this syndrome.     

The tumor suppressor ING1: structure and function.

The biological functions of the tumor suppressor ING1 have been studied extensively in the past 5 years since it was cloned. Of the three alternatively spliced forms of ING1, p24(ING1) has been the focus of much of past research. Information on the other currently known isoforms, p47(ING1), p32(ING1), and p27(ING1), has been lacking. ING1 shares many biological functions with p53. It has been reported to mediate growth arrest, senescence, apoptosis, anchorage-dependent growth, and chemosensitivity. 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. In this review, we will examine what is known about ING1 up to this point and clarify the cloning errors originating from the isolation of this gene.     

Radiation-induced genetic instability in vivo depends on p53 status

In response to ionizing radiation and other agents that damage DNA, the p53 tumor suppressor protein activates multiple cellular processes including cell cycle checkpoints and programmed cell death. Although loss of p53 function is associated with radiation-induced genetic instability in cell lines, it is not clear if this relationship exists in vivo. To study the role of p53 in maintenance of genetic stability in normal tissues following irradiation, we have measured mutant frequencies at the adenine phosphoribosyltransferase (Aprt) and hypothanine-guanine phosphoribosyltransferase (Hprt) loci and examined mechanisms of loss of heterozygosity (LOH) in normal T cells of p53-deficient, Aprt heterozygous mice that were subjected to whole-body irradiation with a single dose of 4Gy X-rays. The radiation-induced mutant frequency at both the Aprt and Hprt loci was elevated in cells from mice with different p53 genotypes. The radiation-induced elevation of p53-/- mice was significantly greater than that of p53+/- or p53+/+ mice and was caused by several different kinds of mutational events at the both chromosomal and intragenic levels. Most significantly, interstitial deletion, which occurs rarely in unirradiated mice, became the most common mechanism leading to LOH in irradiated p53 null mice. These observations support the idea that absence or reduction of p53 expression enhances radiation-induced tumorigenesis by increasing genetic instability at various loci, such as those for tumor suppressor genes.     

Nuclear localization signal of ING4 plays a key role in its binding to p53

ING4, a novel member of ING family, is recently reported to interact with tumor suppressor p53 and negatively regulate the cell growth with significant G2/M arrest of cell cycle in HepG2 cells through upregulation of p53-inducible gene p21. However, which region of ING4 could have contributed to the binding to p53 remains largely unclear. Herein, the GST-pulldown experiments revealed that the middle region of ING4, a potential bipartite nuclear localization signal (NLS), could be involved in the binding to p53. Furthermore, the interaction of ING4 to p53 was abrogated in vitro and in vivo when certain mutations or the entire deletion of the NLS domain occurred. More interestingly, the mutations of the NLS domain could alter the ING4 nuclear localization, disrupt the interaction of ING4 with p53, and even, deregulate the p53-inducible gene p21 in MCF-7 cells. All data indicated that the NLS domain of ING4 is essential for the binding of ING4 to p53 and the function of ING4 associated with p53.