ING5 activity in self-renewal of glioblastoma stem cells via calcium and follicle stimulating hormone pathways.

Stem cell-like brain tumor initiating cells (BTICs) cause recurrence of glioblastomas, with BTIC 'stemness' affected by epigenetic mechanisms. The ING family of epigenetic regulators (ING1-5) function by targeting histone acetyltransferase (HAT) or histone deacetylase complexes to the H3K4me3 mark to alter histone acetylation and subsequently, gene expression. Here we find that ectopic expression of ING5, the targeting subunit of HBO1, MOZ and MORF HAT complexes increases expression of the Oct4, Olig2 and Nestin stem cell markers, promotes self-renewal, prevents lineage differentiation and increases stem cell pools in BTIC populations. This activity requires the plant homeodomain region of ING5 that interacts specifically with the H3K4me3 mark. ING5 also enhances PI3K/AKT and MEK/ERK activity to sustain self-renewal of BTICs over serial passage of stem cell-like spheres. ING5 exerts these effects by activating transcription of calcium channel and follicle stimulating hormone pathway genes. In silico analyses of The Cancer Genome Atlas data suggest that ING5 is a positive regulator of BTIC stemness, whose expression negatively correlates with patient prognosis, especially in the Proneural and Classical subtypes, and in tumors with low SOX2 expression. These data suggest that altering histone acetylation status and signaling pathways induced by ING5 may provide useful clinical strategies to target tumor resistance and recurrence in glioblastoma.

LincRNA-p21 acts as a mediator of ING1b-induced apoptosis.

ING1b is a tumor suppressor that affects transcription, cell cycle control and apoptosis. ING1b is deregulated in disease, and its activity is closely linked to that of p53. In addition to regulating protein-coding genes, we found that ING1b also influences the expression of large intergenic non-coding RNAs (lincRNAs). In particular, lincRNA-p21 was significantly induced after DNA-damage stress or by ING1b overexpression. Furthermore, lincRNA-p21 expression in response to DNA damage was significantly attenuated in cells lacking ING1b. LincRNA-p21 is also a target of p53 and can trigger apoptosis in mouse cell models. We found that this function of lincRNA-p21 is conserved in human cell models. Moreover, ING1b and p53 could function independently to influence lincRNA-p21 expression. However, their effects become more additive under conditions of stress. In particular, ING1b regulates lincRNA-p21 levels by binding to its promoter and is required for induction of lincRNA-p21 by p53. The ability of ING1b to cause apoptosis is also impaired in the absence of lincRNA-p21. Surprisingly, deletion of the ING1b plant homeodomain, which allows it to bind histones and regulate chromatin structure, did not alter regulation of lincRNA-p21. Our findings suggest that ING1b induces lincRNA-p21 expression independently of histone 3 lysine 4 trimethylation mark recognition and that lincRNA-p21 functions downstream of ING1b. Thus, regulation at the level of lincRNA-p21 may represent the point at which ING1b and p53 pathways converge to induce apoptosis under specific stress conditions.

Defining the minimal peptide sequence of the ING1b tumour suppressor capable of efficiently inducing apoptosis.

The ING1b protein is a type-II tumour suppressor and stoichiometric member of the Sin3 histone deacetylase (HDAC) protein complex in which it acts to target HDAC activity to regulate chromatin structure. Altering ING1 levels by ectopic expression of ING1b in cancer cells promotes apoptosis, whereas altering levels by knockout in normal murine fibroblasts alters sensitivity to doxorubicin-induced apoptosis. We have identified a minimal region of ING1b capable of inducing levels of apoptosis in targeted cells as effectively as full-length ING1b, using transient overexpression of ING1b fragments followed by the Annexin V assay. We observed high levels of apoptosis in 14 of 14 cancer cell lines tested. Infecting triple-negative tumorigenic MDA-MB-468 breast cancer, U2OS or Saos-2 cells at multiplicities of infection (MOIs) ranging from 10 to 20 rapidly triggered apoptosis in ~80% of infected cells within 48 h. This was not due to the effects of virus, as infection at the same MOI with a control adenovirus expressing GFP was not effective in inducing apoptosis. When used at low MOIs, the ING1b fragment showed a cell-killing efficacy that was higher than native, full-length ING1b. Using a doxycycline-regulated inducible p53 expression system demonstrated that apoptosis induced by the ING1b fragment was p53 independent. Given the growing importance of combination therapies, we evaluated whether there was synergism between the ING1b fragment and HDAC inhibitors. Combination treatments with TSA, LBH 589 and SAHA reduced cancer cell survival by 3.9-4.7-fold as compared with single-drug treatment, and resulted in ~90% reduction in cell survival. Normalized isobologram analysis confirmed strong synergism between the ING1b fragment and drugs tested. These findings provide support for using ING1b-derived therapeutics as adjuvant treatments in combination with existing epigenetic therapies.

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 the14-3-3 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 by14-3-3 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 [corrected].

ING1b-inducible microRNA203 inhibits cell proliferation.

BACKGROUND: The ING family of type II tumour suppressors serve as both epigenetic 'readers' and target histone acetyl transferase (HAT) and histone deacetylase (HDAC) 'writers' of the epigenetic histone code. The ING1 protein has also been implicated in regulating microRNA (miRNA) levels. In this study, we identify a link between ING1b and the miRNA epigenetic network. METHODS: Primary fibroblasts infected with adenoviruses expressing GFP control or GFP plus ING1b were examined for alterations in miRNA profiles using a miRNA PCR array. Additional experiments confirmed specificity and consequences of altered miRNA expression. RESULTS: MicroRNAs miR-203, miR-375, miR-449b and miR-200c were increased by ING1b overexpression. Ectopic expression of miR-203 inhibited U2OS and MDA-MB-231 cancer cell growth, and induced G1 cell cycle arrest in U2OS cells as estimated by flow cytometry. Transfection with miR-203 inhibitor reversed the proliferation inhibition induced by ING1b in U2OS cells. CHIP assays showed that ING1b bound to the promoter of miR-203. Western blot analyses showed that CDK6, c-Abl and Src were downregulated by the transfection of miR-203. CONCLUSION: These results indicate that ING1b epigenetically regulates several miRNAs including miR-203. The several-fold increase in miR-203 by ING1b might inhibit cancer cell proliferation through coordinate downregulation of CDK6, c-Abl and Src.

The viral tropism of two distinct oncolytic viruses, reovirus and myxoma virus, is modulated by cellular tumor suppressor gene status.

Replication-competent oncolytic viruses hold great potential for the clinical treatment of many cancers. Importantly, many oncolytic virus candidates, such as reovirus and myxoma virus, preferentially infect cancer cells bearing abnormal cellular signaling pathways. Reovirus and myxoma virus are highly responsive to activated Ras and Akt signaling pathways, respectively, for their specificity for viral oncolysis. However, considering the complexity of cancer cell populations, it is possible that other tumor-specific signaling pathways may also contribute to viral discrimination between normal versus cancer cells. Because carcinogenesis is a multistep process involving the accumulation of both oncogene activations and the inactivation of tumor suppressor genes, we speculated that not only oncogenes but also tumor suppressor genes may have an important role in determining the tropism of these viruses for cancer cells. It has been previously shown that many cellular tumor suppressor genes, such as p53, ATM and Rb, are important for maintaining genomic stability; dysfunction of these tumor suppressors may disrupt intact cellular antiviral activity due to the accumulation of genomic instability or due to interference with apoptotic signaling. Therefore, we speculated that cells with dysfunctional tumor suppressors may display enhanced susceptibility to challenge with these oncolytic viruses, as previously seen with adenovirus. We report here that both reovirus and myxoma virus preferentially infect cancer cells bearing dysfunctional or deleted p53, ATM and Rb tumor suppressor genes compared to cells retaining normal counterparts of these genes. Thus, oncolysis by these viruses may be influenced by both oncogenic activation and tumor suppressor status.

Functional links between transcription, DNA repair and apoptosis.

DNA damage initiates damage response pathways, cell cycle arrest and apoptosis. These processes act in a concerted fashion and remain functionally linked through mechanisms not completely understood. Programmed cell death, referred to as apoptosis, is a tightly regulated phenomenon ensuring that cells that accumulate irreversible DNA damage do not replicate. Interestingly, hyperacetylation of histone proteins, which alters transcription patterns and appears linked to DNA repair, also induces apoptosis, suggesting that aspects of chromatin modification link these very distinct processes. Modulating chromatin structure in the absence of any DNA lesions also activates key DNA damage-signalling proteins, further supporting the role of higher-order chromatin structure in mediating stress responses. This review will present an overview of the epigenetic control of eukaryotic genomes by chromatin remodelling as it pertains to DNA damage and highlight the potential role of the ING PHD proteins in linking apoptosis and DNA repair to gene transcription.

Expression of p33ING1 mRNA and chemosensitivity in brain tumor cells.

BACKGROUND: Mutations and down-regulation of tumor suppressor genes can contribute to both tumorigenesis and chemotherapy resistance. The tumor suppressor p33ING1 has growth-inhibitory and pro-apoptotic effects recruiting p53 and it plays a role in DNA repair through interaction with PCNA. We questioned whether p33ING1 mRNA expression correlates with the chemosensitivity of brain tumor cells. MATERIALS AND METHODS: Various malignant brain tumor cell lines were examined for their sensitivity to cisplatin, doxorubicin, etoposide and the antimitotic agents vincristine and paclitaxel by MTT-cytotoxicity assays. p33ING1 mRNA expression was determined by RT-PCR. RESULTS: We found that, unlike other tumor types, ING1 levels were higher in glioma cell lines than in normal control cells. Medulloblastoma cells revealed the lowest ING1 expression of the lines tested. Comparing all cell lines, p33ING1 gene expression significantly (p = 0.028) correlated with resistance to vincristine (r2 = 0.87). CONCLUSION: Our results suggest that p33ING1 mRNA levels may be used to predict the chemosensitivity of brain tumor cells to vincristine.

Expression of novel ING variants is regulated by thyroid hormone in the Xenopus laevis tadpole.

The candidate tumor suppressor gene, ING1, encodes several protein isoforms as a result of alternative splicing that may possess agonistic and antagonistic roles in the control of cell proliferation and apoptosis. Recently a related gene, ING2, was isolated in human whose expression is increased in adenocarcinomas. Little is known about the cellular function and regulation of these ING family members, but the fact that ING proteins contain a plant homeodomain finger suggests that these proteins may modulate transcription factor-mediated pathways. To elucidate how ING may interact in different tissues to modulate function, we used amphibian metamorphosis as a model system in which a single stimulus, thyroid hormone (TH), initiates tissue-specific proliferation, differentiation, and apoptosis. We have isolated the first Xenopus laevis ING2 and demonstrate that transcript levels increase in response to TH treatment. We provide evidence for the existence of splice variants that are differentially expressed in tissues with different TH-induced fates. Western blots using an antibody directed against the highly conserved C-terminal end of ING proteins reveal a tissue-specific pattern of ING isoform expression in adult Xenopus tissues. Analyses of premetamorphic tadpole tissues show a TH-induced accumulation of ING proteins in tail, whereas the levels in the leg are not affected. This TH-induced accumulation is also observed in serum-free tail organ cultures and is prevented by inhibitors of tail apoptosis. Therefore, this work presents the first link between ING expression and a hormonally regulated nuclear transcription factor-mediated apoptotic response opening the possibility that ING family members may be involved in transducing the signal initiated by TH that determines cell fate.

UV-induced binding of ING1 to PCNA regulates the induction of apoptosis.

Previous studies have shown that UV-induced binding of p21(WAF1) to PCNA through the PCNA-interacting protein (PIP) domain in p21(WAF1) promotes a switch from DNA replication to DNA repair by altering the PCNA protein complex. Here we show that the p33(ING1b) isoform of the ING1 candidate tumour suppressor contains a PIP domain. UV rapidly induces p33(ING1b) to bind PCNA competitively through this domain, a motif also found in DNA ligase, the DNA repair-associated FEN1 and XPG exo/endonucleases, and DNA methyltransferase. Interaction of p33(ING1b) with PCNA occurs between a significant proportion of ING1 and PCNA, increases more than tenfold in response to UV and is specifically inhibited by overexpression of p21(WAF1), but not by p16(MTS1), which has no PIP sequence. In contrast to wild-type p33(ING1b), ING1 PIP mutants that do not bind PCNA do not induce apoptosis, but protect cells from UV-induced apoptosis, suggesting a role for this PCNA-p33(ING1b) interaction in eliminating UV-damaged cells through programmed cell death. These data indicate that ING1 competitively binds PCNA through a site used by growth regulatory and DNA damage proteins, and may contribute to regulating the switch from DNA replication to DNA repair by altering the composition of the PCNA protein complex.

DNA damage-inducible gene p33ING2 negatively regulates cell proliferation through acetylation of p53.

The p33ING1 protein is a regulator of cell cycle, senescence, and apoptosis. Three alternatively spliced transcripts of p33ING1 encode p47ING1a, p33ING1b, and p24ING1c. We cloned an additional ING family member, p33ING2/ING1L. Unlike p33ING1b, p33ING2 is induced by the DNA-damaging agents etoposide and neocarzinostatin. p33ING1b and p33ING2 negatively regulate cell growth and survival in a p53-dependent manner through induction of G(1)-phase cell-cycle arrest and apoptosis. p33ING2 strongly enhances the transcriptional-transactivation activity of p53. Furthermore, p33ING2 expression increases the acetylation of p53 at Lys-382. Taken together, p33ING2 is a DNA damage-inducible gene that negatively regulates cell proliferation through activation of p53 by enhancing its acetylation.

Genetic alterations of candidate tumor suppressor ING1 in human esophageal squamous cell cancer.

Overexpression of ING1, a candidate tumor suppressor gene, efficiently blocks cell growth or induces apoptosis in different experimental systems. ING1 maps to chromosome 13q33-34, and because loss of the terminal region of chromosome 13q has been implicated in esophageal squamous cell cancer (ESCC), we examined ESCC for genetic alterations of ING1. Among 31 informative cases of ESCC, 58.9% of the tumors showed allelic loss at chromosome 13q33-34, and we detected four tumor-specific missense nucleotide changes. These alterations were found within the PHD finger domain and nuclear localization motif of the ING1 and may be functionally involved in the development of ESCC. Because immunohistochemical study revealed that all of the ESCC samples showed loss of ING1 protein expression, genetic or epigenetic alterations that abrogate the normal function of ING1 may contribute to esophageal squamous cell carcinogenesis.

UV induces nucleolar translocation of ING1 through two distinct nucleolar targeting sequences.

The ING1 candidate tumor suppressor is downregulated in a variety of primary tumors and established cancer cell lines. Blocking its expression experimentally promotes unregulated growth in vitro and in vivo, using cell and animal models. Alternative splicing products encode proteins that localize to the nucleus, inhibit cell cycle progression and affect apoptosis in different model systems. Here we show that ING1 proteins translocate to the nucleolus 12-48 h after UV-induced DNA damage. When a small 50 amino acid portion of ING1 was fused to green fluorescent protein, the fusion protein was efficiently targeted to the nucleolus, indicating that ING1 possesses an intrinsic nucleolar targeting sequence (NTS). We mapped this activity to two distinct 4 amino acid regions, which individually direct fused heterologous proteins to the nucleolus. Overexpression of ING1 induced apoptosis of primary fibroblasts in the presence and absence of UV exposure. In contrast, NTS mutants of ING1 that were not targeted to the nucleolus did not efficiently induce apoptosis when overexpressed and instead protected cells from UV-induced apoptosis. Taken together, these results indicate that UV induces ING1 to translocate to the nucleolus and that this translocation may facilitate apoptosis.

Loss of functional caveolae during senescence of human fibroblasts.

Primary human fibroblasts have a finite replicative lifespan in culture that culminates in a unique state of growth arrest, termed senescence that is accompanied by distinct morphological and biochemical alterations. Senescent cell responses to extracellular stimuli are believed to be altered at a point after receptors are bound by ligand, leading to improper integration of the signals which initiate DNA replication. In this study we demonstrate that one of the key organizing membrane microdomains for receptor signaling, caveolae, are absent in senescent cells. A comparison of young and senescent cells indicated that senescent cells contained a higher total amount of caveolins 1 and 2 but had significantly less of both proteins in the caveolar fraction. Additionally, caveolar fractions from senescent cells completely lacked the tyrosine-kinase activity associated with functional caveolae. Furthermore, old cells had little caveolar protein exposed to the outer plasma membrane as estimated by using an in vivo biotinylation assay and no detectable caveolin 1 on the cell surface when processed for immunofluoresence and confocal microscopy. Together, these data suggest that a fundamental loss of signal integration at the plasma membrane of senescent cells is due to the loss of signaling competent caveolae.

Accelerated replicative senescence of the peripheral immune system induced by HIV infection.

OBJECTIVES: HIV induces rapid turnover of T lymphocytes but whether this leads to replicative senescence of CD4+ and CD8+ cells and contributes to AIDS symptoms is unclear. The aim of this study was to address this question by analyzing telomere length in blood cell populations as a measure of replicative history in a significant number of patients infected with HIV. DESIGN: Total peripheral blood mononuclear cells (PBMCs), CD4+ or CD8+ cells were isolated from blood collected from a total of 73 HIV patients and 27 controls. Samples were isolated to measure telomere length, telomerase activity and proliferative ability, and analyses were carried out in a blind experimental protocol. METHODS: PBMCs isolated on Ficoll-Hypaque gradients were washed and prepared for additional fractionation into CD4+ and CD8+ cells using antibody-bound magnetic beads. Total PBMCs, CD4+ and CD8+ cells were used for cell cycle analysis, for telomerase activity assays and were measured for telomere length using the terminal restriction fragment assay. RESULTS: Telomere analyses in this study show a clear (P < 0.0001) inverse relationship between telomere length and progression of immunosuppression, with HIV infection resulting in a five-fold or greater acceleration of aging of the circulating PBMC component of the immune system. Patients who are 37 years old showed telomere lengths similar to uninfected 75-year-olds. Telomere loss correlated well with progression of AIDS and with reduced proliferative ability of patient PBMCs but was unrelated to telomerase activity. Mean telomere length was shorter in both CD4+ and CD8+ cells, with three-fold higher rates of telomere loss for CD8+ lymphocytes. CONCLUSIONS: These data provide strong support for the occurrence of accelerated replicative aging of the peripheral immune system, possibly resulting in a loss of T cells leading to AIDS symptoms.

A panel of CAb antibodies recognize endogenous and ectopically expressed ING1 protein.

Nine monoclonal antibodies (MAbs) against human and rodent ING1 protein have been generated using an IL6-secreting mouse myeloma line. These antibodies are all effective in recognizing ING1 protein in ELISAs, Western blot assays, and by indirect immunofluorescence. Combining different CAb monoclonal antibodies in a Western blot assay also allows detection of the very low levels of endogenous ING1 found in fibroblast cells in culture and the identification of at least two isoforms of ING1 in normal human diploid fibroblasts and established brain cancer cell lines.

Three yeast proteins related to the human candidate tumor suppressor p33(ING1) are associated with histone acetyltransferase activities.

Three Saccharomyces cerevisiae proteins (Yng1/YOR064c, Yng2/YHR090c, and Pho23) and two Schizosaccharomyces pombe proteins (Png1/CAA15917 and Png2/CAA21250) share significant sequence identity with the human candidate tumor suppressor p33(ING1) in their C-terminal regions. The homologous regions contain PHD finger domains which have been implicated in chromatin-mediated transcriptional regulation. We show that GFP-Yng2, like human Ing1, is localized in the nucleus. Deletion of YNG2 results in several phenotypes, including an abnormal multibudded morphology, an inability to utilize nonfermentable carbon sources, heat shock sensitivity, slow growth, temperature sensitivity, and sensitivity to caffeine. These phenotypes are suppressed by expression of either human Ing1 or S. pombe Png1, suggesting that the yeast and human proteins are functionally conserved. Yng1- and Pho23-deficient cells also share some of these phenotypes. We demonstrated by yeast two-hybrid and coimmunoprecipitation tests that Yng2 interacts with Tra1, a component of histone acetyltransferase (HAT) complexes. We further demonstrated by coimmunoprecipitation that HA-Yng1, HA-Yng2, HA-Pho23, and HA-Ing1 are associated with HAT activities in yeast. Genetic and biochemical evidence indicate that the Yng2-associated HAT is Esa1, suggesting that Yng2 is a component of the NuA4 HAT complex. These studies suggest that the yeast Ing1-related proteins are involved in chromatin remodeling. They further suggest that these functions may be conserved in mammals and provide a possible mechanism for the human Ing1 candidate tumor suppressor.

A role for serine proteases in mediating phorbol ester-induced differentiation of HL-60 cells.

Treatment of human HL-60 promyelocytic leukemia cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) results in increases in proteolytic activity and maturation toward the monocytic lineage. To investigate the potential roles that different classes of proteases play in the monocytic differentiation of HL-60 cells, cells were treated with phorbol ester in the presence of various serine and cysteine protease inhibitors. The serine protease inhibitors 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), N-alpha-tosyl-phenylalanine chloromethyl ketone (TPCK), and N-alpha-tosyl-lysine chloromethyl ketone (TLCK) repressed a number of phenotypic markers of monocytic differentiation including surface expression of the CD11b integrin, cell aggregate formation, cell cycle exit, and cell death. CD11b was not detected at the cell surface by FACS analysis up to 24 h after induction of differentiation; however, both CD11b mRNA and protein were present. Downregulation of c-myc mRNA and upregulation of c-fos and egr-1 mRNA and protein, which normally occur during TPA-induced differentiation, were not affected by inclusion of the protease inhibitors. These data indicate that serine proteases specifically mediate many of the phenotypic aspects of TPA-induced monocytic differentiation but are not involved with the induction or repression of differentiation-sensitive transcription factors and suggest that serine protease activity is required for intracellular processing of CD11b.

Decreased expression of p33ING1 mRNA in lymphoid malignancies.

The ING1 is a newly cloned putative tumor-suppressor gene, which is involved in the p53 signaling pathway. We found decreased expression of ING1 mRNA in 4 of 5 T-cell lines and 5 of 11 B-cell lines including two Burkitt lymphomas and two myelomas. These observations suggest that decreased ING1 expression might play an important role in the development or progression of some lymphoid tumors. Polymerase chain reaction-SSCP and sequencing analyses found neither point mutations nor small deletions in the ING1 gene, suggesting that decreased expression is due to transcriptional or post-transcriptional mechanisms.