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The Inhibitor of Growth (ING) gene family encodes structurally related proteins that alter chromatin to regulate gene expression and cell growth. The initial member, ING1, has also been proposed to function as a tumor suppressor in human cancer based on its ability to suppress cell growth and transformation in vitro. Mouse Ing1 produces two proteins (p31 and p37) from differentially spliced transcripts. We have recently generated p37Ing1b-null mice and observed spontaneous follicular B-cell lymphomagenesis in this model to show that ING proteins can function in vivo as tumor suppressors. In this present report, we examine the role of p37Ing1b in the regulation of B-cell growth and explore the relationship between p37Ing1b and p53-mediated tumor suppression. Our results indicate that p37Ing1b inhibits the proliferation of B cells and follicular B cells regardless of p53 status, and loss of p53 greatly accelerates the rate of B-cell lymphomagenesis in p37Ing1b-null mice. However, in contrast to the highly penetrant follicular B-cell lymphomas observed in p37Ing1b-null mice, mice lacking both p37Ing1b and p53 typically present with aggressive diffuse large B-cell lymphomas (DLBL). Analysis of marker gene expression in p37Ing1b/p53 null tumors indicates that the double-null mice develop both nongerminal center and germinal center B-cell–like DLBL, and also documents up-regulation of nuclear factor-κB activity in p37Ing1b/p53-null B cells and B-cell tumors. These results confirm that p53 mutation is an important mechanistic step in the formation of diffuse large B-cell lymphomas and reveals a p53-independent role for Ing1b in suppressing B-cell tumorigenesis. [Cancer Res 2008;68(21):8705–14]

ING proteins have been proposed to alter chromatin structure and gene transcription to regulate numerous aspects of cell physiology, including cell growth, senescence, stress response, apoptosis, and transformation. ING1, the founding member of the inhibitor of growth family, encodes p37Ing1, a plant homeodomain (PHD) protein that interacts with the p53 tumor suppressor protein and seems to be a critical cofactor in p53-mediated regulation of cell growth and apoptosis. In this study, we have generated and analyzed p37Ing1-deficient mice and primary cells to further explore the role of Ing1 in the regulation of cell growth and p53 activity. The results show that endogenous levels of p37Ing1 inhibit the proliferation of p53-wild-type and p53-deficient fibroblasts, and that p53 functions are unperturbed in p37Ing1-deficient cells. In addition, loss of p37Ing1 induces Bax expression and increases DNA damage–induced apoptosis in primary cells and mice irrespective of p53 status. Finally, p37Ing1 suppresses the formation of spontaneous follicular B-cell lymphomas in mice. These results indicate that p53 does not require p37Ing1 to negatively regulate cell growth and offers genetic proof that Ing1 suppresses cell growth and tumorigenesis. Furthermore, these data reveal that p37Ing1 can negatively regulate cell growth and apoptosis in a p53-independent manner. [Cancer Res 2007;67(5):2054–61]

The ING family of proteins is involved in the regulation of diverse processes ranging from cell cycle and cellular senescence to apoptosis. These effects are most likely through activation of acetylation-dependent pathways that ultimately alter gene expression. Despite reports linking ING to p53 activation, the molecular basis of how ING activates p53 function has not been elucidated. In this study, we found that a subset of ING family members strongly repressed human alpha-fetoprotein (AFP) promoter activity but stimulated the p21(WAF1) promoter in parallel experiments in the same cell type, similar to the effects of p53. The p47(ING1a) isoform also repressed AFP promoter activity, but in contrast to other ING isoforms, it repressed the p21(WAF1) promoter. p47(ING3) up-regulated p21(WAF1) promoter activity, but it did not have any effect on the AFP promoter. ING1b and ING2 also repressed the AFP promoter in Hep3B p53-null cell lines, and p53 coexpression enhanced this transcriptional repression. Suppression of AFP gene transcription by ING was strongly dependent on AT-motifs that bind to the hepatocyte nuclear factor 1 (HNF1) transcription factor. Indeed, electrophoretic mobility shift assays confirmed that HNF1 binds to AT-motifs, but we found, surprisingly, that the ING1 complexes binding to these AT-motifs were devoid of HNF1 protein. Both ING1 and p53 were able to suppress AFP transcription and cause p21 induction; hSIR2, a negative regulator of the p53 protein, showed the opposite effects on the AFP promoter and, like HDAC1, repressed p21 promoter activity. In addition, we found that p33(ING1b) physically interacts with hSIR2, reverses its ability to induce the AFP promoter, and induces acetylation of p53 residues at Lys(373) and/or Lys(382). These findings provide novel evidence that p33(ING1b) represses AFP transcription by at least two mechanisms, one of which includes p53. The first is by binding to the AT-motif and excluding HNF1 binding while possibly targeting HAT activity to promoter regions, and the second is by increasing the levels of active, acetylated p53 via binding and inhibiting the ability of hSIR2 to deacetylate p53 protein.

ING1b is a candidate tumor suppressor that can stimulate the transcriptional activity of p53 and inhibit cell proliferation. The molecular basis of how ING1b activates p53 function remains unclear. Here we show that ING1b could stimulate the activity of p53 by increasing the level and stability of the p53 protein. The stabilization and activation of p53 by ING1b could be reversed by MDM2 in a dose-dependent manner. Conversely, ING1b could reverse the inhibition and degradation of p53 caused by MDM2 in a dose-dependent manner. Furthermore, ING1b and MDM2 bound to p53 in a mutually exclusive manner. In agreement with these observations, we found that similarly to MDM2, ING1b binds to the NH(2)-terminal region of p53. These data suggest a model in which ING1b disrupts the interaction between p53 and MDM2, leading to the stabilization of p53 and growth inhibition.

Recently, several novel human ING1 isoforms have been cloned. However,the biochemical functions and the involvement of these proteins in apoptosis remain uncharacterized. We have examined the apoptotic effects and biochemical functions of the two major human ING1 isoforms p47(ING1a) and p33(ING1b) in young and senescent human diploid fibroblasts induced to enter into apoptosis by diverse treatments. We have found that ING1 displayed isoform-, stimulus- and cell age-dependent apoptotic properties. We present evidence indicating that ING1 proteins bind to chromatin and are regulated in a manner related to their apoptotic properties. In agreement with previous reports, we have found that only young but not senescent fibroblasts were able to enter into apoptosis induced by growth factor deprivation. This effect was accompanied by up-regulation of endogenous p33(ING1b). Ectopic up-regulation of p33(ING1b), but not p47(ING1a), also induced apoptosis and sensitized young but not senescent cells to UV irradiation and hydrogen peroxide-mediated apoptosis. Cotransfection of p33(ING1b) and the tumor suppressor p53 increased the percentage of apoptotic cells yielded by either of these two proteins alone, in agreement with data from tumor cell models. Finally, we found that the chromatin binding affinity of p33(ING1b) was increased in senescent cells, which were resistant to apoptosis. Together, these data support the idea that the apoptotic functions of ING1 may be exerted by chromatin-related functions that are subject to cell age-dependent mechanisms of regulation.

The candidate tumor suppressor p33(ING1) plays an important role in inducinggrowth arrest at G(0)-G(1) phase of the cell cycle and/or promoting apoptosis in cancerous cells. p33(ING1) is reported to act as a transcriptional cofactor by associating with tumor suppressor p53, HAT, or histone deacetyltransferase, suggesting that p33(ING1) is involved in chromatin-mediated transcriptional regulation. However, the molecular mechanism of p33(ING1)-mediated transcriptional regulation is poorly understood. Here we analyzed expression profiles in mouse mammary epithelial cells (NMuMG) by using a cDNA microarray consisting of 2304 mouse cDNAs after inducing transformation with antisense inhibitor of growth 1 (ING1) in retrovirus vector. The subsequent confirmation of the altered expression levels of the selected genes by semiquantitative reverse transcription-PCR demonstrated that overexpression of the antisense ING1 stimulated expression of 14 genes, which included cyclin B1, 12-O-tetradecanoylphorbol-13-acetate-inducible sequence 11, proto-oncogene DEK, and osteopontin, whereas we have detected transcriptional repression of 5 genes, including TPT1. In addition, adenovirus-mediated overexpression of ING1 in NMuMG cells resulted in down-regulation of cyclin B1, 12-O-tetradecanoylphorbol-13-acetate-inducible sequence 11, DEK, and osteopontin, whereas the levels of TPT1 expression were increased. The further analysis using p53(-/-) SAOS2 cells showed that the p33(ING1)-induced cyclin B1 down-regulation was p53 dependent. Thus, our cDNA microarray analysis suggested that p33(ING1) targets the multiple genes, including proto-oncogene DEK and cyclin B1, at least some of which are regulated in a p53-dependent manner, in the cells undergoing cell growth or apoptosis.

We cloned mouse ING1 homologue (mINGh), an A1/Bfl-1-interacting protein, from mouse mammary glands using a yeast two-hybrid assay and unexpectedly found four splicing variants of mINGh by reverse transcription-PCR assay and sequence analysis. The alternative splicing variants were mINGh-S, mINGh-M, mINGh-L, and mINGh-L2 encoding 171, 248, 166, and 227 amino acids, respectively. Cell death of HC11 cells, induced by serum starvation, was enhanced by mINGhs, and the action of mINGhs was inhibited by A1 protein. These results indicate that A1 can inhibit cell death not only via the well known pathway related to the Bcl-2 family but also through direct interaction with mINGh in mammary epithelial cells.

The biological functions of the tumor suppressor, ING1, have been studied extensively in the last 5 years since it was cloned. It shares many biological functions with those of p53 and 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 study, we report that p33(ING1) (one of ING1 isoforms) is also involved in the modulation of DNA repair. We found that overexpression of p33(ING1) enhances repair of UV-damaged DNA and that p53 is required for the repair process. Furthermore, binding between ING1 and GADD45 has been detected. These observations suggest that p33(ING1) cooperates with p53 in nucleotide excision repair and that GADD45 may be one of its components.

We characterized the genomic structure of the human ING1 gene, a candidate tumor suppressor gene, and found that the gene has three exons. We also demonstrated that four mRNA variants were transcribed from three different promoter regions. Of 34 informative cases of head and neck squamous cell carcinoma, 68% of tumors showed loss of heterozygosity at chromosome 13q33-34, where the ING1 gene is located. Here we present the first report that three missense mutations and three silent changes were detected in the ING1 gene in 6 of 23 tumors with allelic loss at the 13q33-34 region. These missense mutations were found within the PHD finger domain and nuclear localization motif in ING1 protein, probably abrogating the normal function.

SEREX (serological analysis of recombinant tumor cDNA expression libraries) has been applied to several different tumor types and has led to the identification of a wide range of tumor antigens. In this study, a breast cancer library and a normal testicular library were analyzed using autologous and allogeneic breast cancer sera. Thirty genes were isolated, including 27 known genes and 3 previously unknown genes. Among the known genes, two cancer-testis (CT) antigens, NY-ESO-1 and SSX2, previously defined by SEREX analysis, were found. In addition, ING1, a candidate breast cancer suppressor gene, was isolated. This ING1 gene product was also recognized by 2 of 14 allogeneic sera from breast cancer patients but not 12 normal adult sera. Comparison of ING1 cDNA from normal and tumor tissues showed no mutation in the index breast cancer case and revealed the presence of at least three different mRNA transcripts with variable transcription initiation sites and exon usage. Tissue-specific expression of these transcripts was found in normal tissues and tumor cell line mRNAs. Furthermore, a novel gene, designated as ING2, sharing 76% nucleotide homology with ING1 was identified in the breast cancer cDNA library. The basis of the immunogenicity of ING1 and the biological role of ING1 and ING2 need further exploration.

The p53 tumor suppressor gene is an important target for the gene therapy of cancers, and clinical trials targeting this gene have been conducted. Some cancers, however, are refractory to p53 gene therapy. Therefore, it has been combined with other therapies, including chemotherapy and radiotherapy, to enhance the cytopathic effect of p53 induction. The p33ING1 gene cooperates with p53 to block cell proliferation. In this study, we investigated whether adenovirus (Adv)-mediated coinduction of p33ING1 and p53 enhances apoptosis in glioma cells (U251 and U-373 MG), which showed no genetic alterations but low expression levels of p33ING1. Although the single infection of Adv for p33ING1 (Adv-p33) at a multiplicity of infection (MOI) of 100, or Adv for p53 controlled by myelin basic protein (MBP) promoter (Adv-MBP-p53), a glioma-specific promoter, at a MOI of 50, did not induce apoptosis in U251 and U-373 MG glioma cells; coinfection of Adv-p33 and Adv-MBP-p53 at the same MOIs induced drastically enhanced apoptosis in both cell lines. Apoptosis was not induced in NGF-treated PC-12 cells infected with a high MOI (300) of Adv-p33 nor in those coinfected with Adv-p33 (100) and Adv-MBP-p53 (50). Coinfection of Adv-p33 and Adv-MBP-p53 demonstrated morphological mitochondrial damage during the initial stage of apoptosis, which likely led to apoptotic cell death. Our results indicate that this coinfection approach can be used as a modality for the gene therapy of gliomas, sparing damage to normal tissues.

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.