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.

Suppression of the novel growth inhibitor p33ING1 promotes neoplastic transformation.

Using a new strategy for tumour suppressor gene isolation based on subtractive hybridization and the subsequent selection of transforming 'genetic suppressor elements', we have cloned a novel gene called ING1 encoding a 33-kD protein (p33ING1) that displays characteristics of a tumour suppressor. Acute expression of transfected constructs encoding this gene inhibited cell growth while chronic expression of ING1 antisense constructs promoted cell transformation. Limited analyses of tumour cell lines show that mutation of the ING1 gene occurs in neuroblastoma cells and reduced expression was seen in some breast cancer cell lines. These results demonstrate that ING1 can act as a potent growth regulator in normal and in established cells and provide evidence for a role as a candidate tumour suppressor gene whose inactivation may contribute to the development of cancers.

Heat shock is lethal to fibroblasts microinjected with antibodies against hsp70.

Synthesis of a small group of highly conserved proteins in response to elevated temperature and other agents that induce stress is a universal feature of prokaryotic and eukaryotic cells. Although correlative evidence suggests that these proteins play a role in enhancing survival during and after stress, there is no direct evidence to support this in mammalian cells. To assess the role of the most highly conserved heat shock protein (hsp) family during heat shock, affinity-purified monoclonal antibodies to hsp70 were introduced into fibroblasts by needle microinjection. In addition to impairing the heat-induced translocation of hsp70 proteins into the nucleus after mild heat shock treatment, injected cells were unable to survive a brief incubation at 45 degrees C. Cells injected with control antibodies survived a similar heat shock. These results indicate that functional hsp70 is required for survival of these cells during and after thermal stress.

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.

Extension of the replicative life span of human diploid fibroblasts by inhibition of the p33ING1 candidate tumor suppressor.

Previous studies suggest that tumor suppressors may play significant roles in blocking the growth of cells during cellular senescence. We therefore studied the potential involvement of a novel growth inhibitor and candidate tumor suppressor gene called ING1, which we have cloned recently (I. Garkavtsev, A. Kazarov, A. Gudkov, and K. Riabowol, Nat. Genet. 14:415-420, 1996), in the process of cellular senescence. Our results show that the RNA and protein levels of ING1 were 8- to 10-fold higher in senescent cells than in young, proliferation-competent human diploid fibroblasts. Expression of the nuclear p33ING1 protein was regulated during the cell cycle, reaching maximal levels during DNA synthesis. Chronic expression of antisense ING1 RNA reproducibly resulted in extension of the proliferative life span of normal human fibroblasts by approximately seven population doublings.

Loss of serum response element-binding activity and hyperphosphorylation of serum response factor during cellular aging.

Human diploid fibroblasts undergo a limited number of population doublings in vitro and are used widely as a model of cellular aging. Despite growing evidence that cellular aging occurs as a consequence of altered gene expression, little is known about the activity of transcription factors in aging cells. Here, we report a dramatic reduction in the ability of proteins extracted from the nuclei of near-senescent fibroblasts to bind the serum response element which is necessary for serum-induced transcription of the c-fos gene. In contrast, the activities of proteins binding to the RNA polymerase core element, TATA, as well as to the cyclic AMP response element were maintained during cellular aging. While no major differences in the expression of the serum response factor (SRF) that binds the serum response element were seen between early-passage and late-passage cells, hyperphosphorylation of SRF was observed in near-senescent cells. Furthermore, removal of phosphatase inhibitors during the isolation of endogenous nuclear proteins restored the ability of SRF isolated from old cells to bind the SRE. These data, therefore, indicate that hyperphosphorylation of SRF plays a role in altering the ability of this protein to bind to DNA and regulate gene expression in senescent cells.

Increased expression of cyclin D2 during multiple states of growth arrest in primary and established cells.

Cyclin D2 is a member of the family of D-type cyclins that is implicated in cell cycle regulation, differentiation, and oncogenic transformation. To better understand the role of this cyclin in the control of cell proliferation, cyclin D2 expression was monitored under various growth conditions in primary human and established murine fibroblasts. In different states of cellular growth arrest initiated by contact inhibition, serum starvation, or cellular senescence, marked increases (5- to 20-fold) were seen in the expression levels of cyclin D2 mRNA and protein. Indirect immunofluorescence studies showed that cyclin D2 protein localized to the nucleus in G0, suggesting a nuclear function for cyclin D2 in quiescent cells. Cyclin D2 was also found to be associated with the cyclin-dependent kinases CDK2 and CDK4 but not CDK6 during growth arrest. Cyclin D2-CDK2 complexes increased in amounts but were inactive as histone H1 kinases in quiescent cells. Transient transfection and needle microinjection of cyclin D2 expression constructs demonstrated that overexpression of cyclin D2 protein efficiently inhibited cell cycle progression and DNA synthesis. These data suggest that in addition to a role in promoting cell cycle progression through phosphorylation of retinoblastoma family proteins in some cell systems, cyclin D2 may contribute to the induction and/or maintenance of a nonproliferative state, possibly through sequestration of the CDK2 catalytic subunit.

Multiple sequence elements of a single functional class are required for cyclic AMP responsiveness of the mouse c-fos promoter.

Agents that elevate the intracellular concentration of cyclic AMP (cAMP) rapidly and transiently induce expression of the c-fos proto-oncogene in BALB/c 3T3 cells. We show that the mouse c-fos promoter-enhancer region contains multiple elements that contribute to cAMP responsiveness of the promoter in transient expression assays. The most potent element was found to correspond to a previously mapped basal promoter element and protein-binding site located 65 base pairs upstream of the transcriptional initiation site. This element and two less potent sites contained a match to the cAMP response element (CRE) core sequence defined in several mammalian genes. The relative potencies of these elements corresponded with their relative affinities for cellular factors that bound to the CRE in vitro. Mutation of all three elements failed to abolish completely cAMP responsiveness of the c-fos promoter in the transient expression assay. However, we present evidence that this residual responsiveness may have been due to sequences present in vector DNA. Finally, we show, by using a new microinjection competition assay, that a double-stranded oligonucleotide carrying the major c-fos CRE is sufficient to block induction of the endogenous c-fos gene by cAMP. Therefore, induction of the endogenous gene requires positively acting cellular factors that interact with a single functional class of regulatory sites in the c-fos gene. Unrelated regulatory elements, such as the serum response element and putative AP-2 sites, are not by themselves sufficient to mediate the cAMP response.

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.

Microinjection of fos-specific antibodies blocks DNA synthesis in fibroblast cells.

Transcription of the protooncogene c-fos is increased greater than 10-fold within minutes of treatment of fibroblasts with serum or purified growth factors. Recent experiments with mouse 3T3 cell lines containing inducible fos antisense RNA constructs have shown that induced fos antisense RNA transcripts cause either a marked inhibition of growth in continuously proliferating cells or, conversely, a minimal effect except during the transition from a quiescent (G0) state into the cell cycle. Since intracellular production of large amounts of antisense RNA does not completely block gene expression, we microinjected affinity-purified antibodies raised against fos to determine whether and when during the cell cycle c-fos expression was required for cell proliferation. Using this independent method, we found that microinjected fos antibodies efficiently blocked serum-stimulated DNA synthesis when injected up to 6 to 8 h after serum stimulation of quiescent REF-52 fibroblasts. Furthermore, when fos antibodies were injected into asynchronously growing cells, a consistently greater number of cells was prevented from synthesizing DNA than when cells were injected with nonspecific immunoglobulins. Thus, whereas the activity of c-fos may be necessary for transition of fibroblasts from G0 to G1 of the cell cycle, its function is also required during the early G1 portion of the cell cycle to allow subsequent DNA synthesis.

Testing the in vivo role of protein kinase C and c-fos in neurite outgrowth by microinjection of antibodies into PC12 cells.

To define the molecular bases of growth factor-induced signal transduction pathways, antibodies known to block the activity of either protein kinase C (PKC) or the fos protein were introduced into PC12 cells by microinjection. The antibody against PKC significantly inhibited neurite outgrowth when scored 24 h after microinjection and exposure to nerve growth factor (NGF). Microinjection of antibodies to fos significantly increased the percentage of neurite-bearing cells after exposure to either NGF or basic fibroblast growth factor (bFGF) but inhibited the stimulation of DNA synthesis by serum, suggesting that in PC12 cells, fos is involved in cellular proliferation. Thus, activation of PKC is involved in the induction of neurite outgrowth by NGF, but expression of the fos protein, which is induced by both NGF and bFGF, is not necessary and inhibits neurite outgrowth.

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.

A novel candidate tumor suppressor, ING1, is involved in the regulation of apoptosis.

We have recently cloned a novel growth inhibitor and candidate tumor suppressor called p33ING1 (I. Garkavtsev et al., Nature Genet., 14: 415-420, 1996). Because some tumor suppressors participate in the regulation of apoptosis, we hypothesized that the ING1 gene may also play a role in this process. Our results show that p33ING1 levels increase upon the induction of apoptosis in P19 teratocarcinoma cells by serum deprivation. Elevated expression of ING1 in P19 and rodent fibroblast cells containing a tetracycline-controlled human c-myc gene enhanced the extent of serum starvation-induced apoptosis. This suggests that the pathway by which ING1 modulates cell death is synergistic with Myc-dependent apoptosis. Conversely, constitutive expression of an antisense construct of INGI conferred protection against apoptosis in these cells. These data support the idea that loss of proper ING1 function may facilitate tumorigenesis, in part, by reducing the cell's sensitivity to apoptosis.

Selective inhibition of telomerase activity during terminal differentiation of immortal cell lines.

Telomerase, the enzyme that maintains the ends of linear eukaryotic chromosomes, is active in human germ cells and in a majority of tumor tissues and immortalized cell lines. In contrast, most mature somatic cells and tissues contain low or undetectable telomerase activity, implying a stringent negative regulatory control mechanism. We report here that telomerase activity is dramatically inhibited during the terminal differentiation of HL-60 human promyelocytic leukemia cells to monocytic and granulocytic lineages. A loss of telomerase activity was seen in response to three different inducers of differentiation, was independent of differentiation-induced apoptosis, and occurred in the presence of unaltered expression of the RNA component of telomerase. Reduction in telomerase activity was also observed during the differentiation of murine F9 teratocarcinoma and C2C12 myoblast cells. In contrast, induced differentiation of murine p19 embryonal carcinoma and Neuro 2a neuroblastoma cells did not result in a loss of telomerase activity. These results are therefore consistent with the absence of telomerase activity in human somatic cells and the presence of telomerase activity in many somatic murine cells and tissues.

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.

The cdk2 kinase is required for the G1-to-S transition in mammalian cells.

In the cell cycle of fission and budding yeast, the p34cdc2/CDC28 kinase is required for both the G1-to-S and G2-to-M phase transitions. In vertebrates, the homologous p34cdc2 kinase is required for G2-to-M phase transitions but appears to be dispensable for DNA synthesis. We have investigated the function of a related kinase, p33cdk2, using serum-stimulated quiescent human fibroblasts. While the p33cdk2 protein was expressed at constant levels throughout the cell cycle, p33cdk2 kinase activity was first detected a few hours prior to the onset of DNA synthesis. Microinjection of anti-p33cdk2 antibodies blocked cells from entering S phase. Pre-adsorption of these antibodies with cdk2 protein abrogated their blocking effect suggesting that the G1 arrest caused by these antibodies is cdk2-specific. These results indicate that p33cdk2 is required for the G1-to-S phase transition in mammalian cells. We also show evidence to suggest that the cyclin E/p33cdk2 complex is likely to be required for entry into S phase since the timing of the cyclin E-associated kinase activity was coincident with that of p33cdk2 and preclearing of either component abolished the majority of the histone H1 kinase activity present in the lysates harvested from the late G1.

Suppression of ING1 expression in sporadic breast cancer.

Down regulation of the ING1 candidate tumour suppressor promotes growth in soft agar and focus formation in vitro and tumour formation in vivo. ING1 encodes a nuclear, cell cycle-regulated protein, overexpression of which efficiently blocks cell growth and is capable of inducing apoptosis in different experimental systems. Here we present the first report of ING1 mutation and expression analysis in a total of 452 cancer samples. One germline missense alteration and three germline silent alterations were detected in 377 primary breast cancers while marked (2 - 10-fold) decreases in ING1 mRNA expression were seen in 44% of primary breast cancers and in ten of ten breast cancer cell lines examined. Furthermore, the majority of breast cancers (58%) showing decreased ING1 expression had metastasized to regional lymph nodes whereas only 9% of cancers with elevated ING1 expression, compared to adjacent normal tissues, were metastatic. Thus, ING1 mutation is very rare in breast or ovarian cancers, however, repression of ING1 expression frequently accompanies tumour development of breast cancer.

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.

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.

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.