Search

The ING family of tumor suppressor proteins controls several cellular functions relevant to antitumor protection, such as cell cycle control, apoptosis, senescence, or migration. ING proteins are functionally linked to the p53 pathway, and they participate in transcriptional control via the recognition of histone marks and recruitment of protein complexes with chromatin-modifying activity to specific promoters. Here, we have investigated the global effect of ING1 in gene regulation through genome-wide analysis of expression profiles in primary embryonic fibroblasts deficient for the Ing1 locus. We find that Ing1 has a predominant role as transcriptional repressor in this setting, affecting the expression of genes involved in a variety of cellular functions. Within the subset of genes showing differential expression, we have identified DGCR8, a protein involved in the early steps of microRNA biogenesis. We show that ING1 binds to the DGCR8 promoter and controls its transcription through chromatin regulation. We also find that ING1 and DGCR8 can cooperate in restraining proliferation. In summary, this study reveals a novel connection between ING1 and a regulator of microRNA biogenesis and identifies new links between tumor suppressor proteins and the microRNA machinery.

Aim: To investigate the inhibitory effect of tumor suppressor p33(ING1b) and its synergy with p53 gene in hepatocellular carcinoma (HCC)., Methods: Recombinant sense and antisense p33(ING1b) plasmids were transfected into hepatoma cell line HepG2 with lipofectamine. Apoptosis, G0/G1 arrest, cell growth rate and cloning efficiency in soft agar of HepG2 were analyzed after transfection. In three hepatoma cell lines with different endogenous p53 gene expressions, the synergistic effect of p33(ING1b) with p53 was analyzed by flow cytometry and luciferase assay was performed to detect the activation of p53 downstream gene p21(WAF1/CIP1). In addition, the expression and mutation rates of p33(ING1b) in HCC tissues were measured by immunohistochemistry and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP)., Results: Overexpression of p33(ING1b) inhibited cell growth of HepG2, induced more apoptosis and protected cells from growth in soft agar. Combined transfer of p33(ING1b) and p53 gene promoted hepatoma cell apoptosis, G0/G1 arrest and elevated expression of p21(WAF1/CIP1). Immunostaining results showed co-localized P33(ING1b) with P53 protein in HCC tissues and there was a significant relation between protein expression rates of these two genes (P<0.01). Among 28 HCC samples, p33(ING1b) presented a low gene mutation rate (7.1%)., Conclusion: p33(ING1b) collaborates with p53 in cell growth inhibition, cell cycle arrest and apoptosis in HCC. Loss or inactivation of p33(ING1b) normal function may be an important mechanism for the development of HCC retaining wild-type p53.

p33ING1b is a candidate tumor suppressor gene and a nuclear protein. We investigated whether genetic and epigenetic mechanisms affect p33ING1b expression in ovarian cancer thus contributing toward its pathogenesis. A total of 111 ovarian cancers collected from Beijing and Hong Kong were used for this study. Weak or negative p33ING1b protein expression was demonstrated by immunohistochemistry on tissue microarray in 28/111 cases. Real-time quantitative RT-PCR also showed overall significant reduction of p33ING1b mRNA expression (P = 0.0137), with 53.1% (17/32) cases showing 2- to 5-fold reduction and absence of expression. The reduction of mRNA expression in cancer correlated with decreased p33ING1b protein expression (P < 0.0001). While no p33ING1b mutation was found, allelic loss at the p33ING1b locus was demonstrated in 25% (8/32) cases. The allelic loss profiles also showed statistical significant correlation with reduction of p33ING1b protein and mRNA expression (P = 0.031 and 0.030). Promoter methylation as assessed by methylation specific PCR was found in 23.9% (21/88) cases analyzed. Bisulfite sequencing results confirmed the p33ING1b promoter methylation status of these methylation positive cases. Statistical significant correlation between methylation and mRNA expression (P = 0.006) was demonstrated. Treatment with demethylating drug, 5'-aza-2'-deoxycytidine, resulted in dosage-dependent elevated mRNA expression of p33ING1b in ovarian cancer cell lines. This is the first study reporting epigenetic mechanism regulating the p33ING1b expression. Our findings support that genetic and epigenetic alteration of p33ING1b are likely to contribute towards the pathogenesis of ovarian cancers.

ING1 was identified as an inhibitor of growth and has been described as a tumor suppressor. Furthermore, the expression of ING1 is induced in senescent cells and antisense ING1 extends the proliferative life span of primary human fibroblasts. Cooperation of p33ING1 with p53 has been suggested to be an important function of ING1 in cell cycle control. Intriguingly, it has been shown that p33ING1 is associated with histone acetylation as well as with histone deacetylation function. Here we show that p33ING1 is a potent transcriptional silencer in various cell types. However, the silencing function is independent of the presence of p53. By use of deletion mutants two potent autonomous and transferable silencing domains were identified, but no evidence of an activation domain was found. The amino (N)-terminal silencing domain is sensitive to the histone deacetylase inhibitor trichostatin A (TSA) whereas the carboxy-terminal silencing function is resistant to TSA, suggesting that p33ING1 confers gene silencing through both HDAC-dependent and -independent mechanisms. Interestingly, the presence of oncogenic Ras, which is able to induce premature senescence, increases the p33ING1-mediated silencing function. Moreover, ING1-mediated silencing was reduced by coexpressing dominant-negative Ras or by treatment with the mitogen-activated protein kinase inhibitor PD98059 but not by treatment with SB203580, an inhibitor of the p38 pathway. In addition, we show that both silencing domains of ING1 are involved in cell cycle control, as measured by inhibition of colony formation of immortalized cells and by thymidine incorporation of primary human diploid fibroblasts (HDF). Interestingly, p33ING1 expression induces features of cellular senescence in HDFs.

Since the discovery of ING1 class II tumor suppressors in 1996, five different ING genes (ING1 to ING5) encoding proteins with highly conserved plant homeodomain (PHD) motifs and several splicing isoforms of the ING1 and ING2 gene have been identified. The ING family functions in DNA repair and apoptosis in response to UV damage through binding to proliferating cell nuclear antigen (PCNA); chromatin remodeling and regulation of gene expression through regulating and/or targeting histone acetyltransferase/deacetylase (HAT/HDAC) activities; binding targets of rare phosphatidylinositol phosphates (PtdInsPs) that function in DNA damage-initiated stress signaling; and regulating brain tumor angiogenesis through transcriptional repression of NF-KB-responsive genes. To elucidate the evolutionary history of ING proteins and summarize what is known about regions highly conserved in the ING family members, we have examined the sequences and phylogenetic relationships of ING proteins across taxonomically diverse organisms. We have identified novel ING family members in rats, frogs, fish, mosquitoes, fruit flies, worms, fungi, and plants. We have also clarified the naming and classification of ING proteins based on our phylogenetic analysis to allow better understanding of the ING protein family. Using sequence similarities, we have identified novel regions and motifs of unknown function that are conserved across family members. An evolutionary history for the ING family of PHD finger proteins is presented that indicates that five ING genes are present in vertebrates. Three of these may be paralogs of ING genes found in arthropods, whereas nematodes, fungi, and green plants contain ING genes that have general features of the vertebrate ING family.

Aim: To detect the expression of p33(ING1b) protein and the change of p33(ING1b) gene in pancreatic carcinoma and to evaluate the significance of p33(ING1b) in pancreatic cell carcinogenesis., Methods: Pathological specimens from pancreatic carcinoma and matched non-tumor pancreatic tissues were examined for p33(ING1b) expression and mutation by immunohistochemistry, polymerase chain reaction single-strand conformation polymorphisms (PCR-SSCP) and loss of heterozygosity (LOH)., Results: The rate of p33(ING1b) protein expression was 85% (34/40). A single germline missense mutation was detected in 1 of 40 tumors located at codon 215:TGC-TCC (Cys-Ser). Fourteen (60.9%) of 23 tumor samples showed LOH in all of the informative markers tested, but no mutation was detected in these tumors and only two of the informative tumors lacked expressions of p33(ING1b) protein., Conclusion: Mutation and loss of expression are not the main reasons for the disfunction of p33(ING1b) in pancreatic carcinoma, an abnormality at the level of chromosome and/or transcription may inhibit their normal functions, potentially contributing to pancreatic cell carcinogenesis.

Repression of gene transcription is mediated by histone deacetylases containing repressor-co-repressor complexes, which are recruited to promoters of target genes via interactions with sequence-specific transcription factors. The mammalian Sin3A co-repressor complex contains a core of at least seven proteins including the pRb-interacting protein RBP1 and a putative tumor suppressor p33(ING1b). By biochemical purification and mass spectrometry, we have identified a novel component p40 from this complex. p40 bears homology to both yeast Sds3, a component of yeast histone deacetylase complexes, and its mammalian homologue mSds3. The p40-associated complex purified from human cells shows a strong histone deacetylase activity. When tethered to a Gal-DNA binding domain, the Gal-p40 is able to significantly repress transcription of a Gal-luciferase promoter. Interestingly, database analysis reveals that p40 is also highly homologous to BRMS1, a breast carcinoma metastasis suppressor, and overexpression of p40 in human cells can significantly inhibit cell growth. Thus, our data indicate that p40 may be critically involved in transcription repression of cell growth-associated gene expression by recruiting the HDAC1 deacetylase complex.

ING1 has been identified as a novel candidate tumor suppressor gene using a genetic suppressor element (GSE) strategy. Ectopic expression of ING1 in mammalian cultured cells causes cell cycle arrest and apoptosis through a p53-dependent and/or p53-independent pathway. However, there has been no report on the prognostic significance of the ING1 expression level in human cancers, though the expression of the wild-type ING1 gene is significantly decreased in breast, lymphoid and gastric cancers as compared with their corresponding normal tissues. In order to explore the possible involvement of ING1 in tumorigenesis of neuroblastoma, we examined the expression levels of ING1 mRNA in 32 primary neuroblastomas by using a quantitative real-time PCR. ING1 mRNA was expressed independently of the disease stages. however, low levels of ING1 mRNA were significantly associated with a poor prognosis (log-rank test, p=0.017). Multivariate analysis showed that the expression level of ING1 was closely related to survival (p=0.020), even after controlling with age (p=0.008) or stage (p=0.025), while it was only marginally significant after controlling with TrkA expression (p=0.063). Mutation analysis revealed that there was no mutation or deletion of the ING1 gene except 1 silent mutation at codon 188 in primary neuroblastomas examined. Taken together, our results suggest for the first time that a decreased level of ING1 expression is a novel indicator of poor prognosis in advanced stages of neuroblastoma, and that ING1 may play a crucial role in genesis and progression of neuroblastoma.

Mutations of p53 tumor suppressor gene increase with tumor progression in colorectal cancers. In this study, we examined the expressions of p33ING1, p14ARF, MDM2 and p21WAF1 mRNA in 25 advanced colorectal cancers by quantitative RT-PCR method, and compared the expression levels of p33ING1, p14ARF, p21WAF1 and MDM2 in relation to p53 status in the tumors. Fifteen of 25 colorectal cancers (60%) showed abnormal accumulation of p53 protein in the nucleus, and the remaining 10 colorectal cancers (40%) were negative for p53 immunostaining. We found a G --> T transition (nonsense mutation) at the first nucleotide of codon 298 (exon 8) in one p53-negative case, and a frame shift mutation on exon 7 in another p53-negative case. In remaining eight p53-negative cases, there was no mutation in the entire open reading frame of p53 cDNA. Interestingly, in eight cases with p53 wild-type gene, 6 cases (75%) showed a marked down-regulation of p14ARF mRNA, and three cases (37.5%) over-expressed MDM2 mRNA. Only one case with wild-type p53 gene showed normal level expression of p53 regulatory-factors (p33ING1, p14ARF, and MDM2). Thus, p53 tumor suppressor pathway was disrupted in 24 of 25 colorectal cancers (96%).

Early studies of the inhibitor of growth 1 ( ING1) gene, the founding member of the ING tumor suppressor family, demonstrated that this gene plays an important role in apoptosis and cellular senescence. Four other related genes have since been identified and found to be involved in various biological activities, including cell cycle arrest, regulation of gene transcription, DNA repair and apoptosis. The biochemical functions of ING proteins as histone acetyltransferases and histone deacetylase co-factors ties this new tumor suppressor family to the regulation of transcription, cell cycle check-points, DNA repair and apoptosis. This review is aimed at summarizing the known biological functions of the ING tumor suppressors and the signalling pathways that they involve.

Epidemiological evidence indicates that ultraviolet radiation (UVR) is the primary environmental cause of the rapid increase in the incidence of human cutaneous melanoma observed in the past decades. However, the genetic changes caused by UVR that lead to melanoma formation remain unclear. The ING1 (inhibitor of growth 1) tumor suppressor plays an important role in cellular stress response to UVR. To further investigate whether ING1 is involved in melanoma development, we examined the mutational status of the ING1 gene in 46 human cutaneous melanoma biopsies and characterized the biological importance of ING1 mutations in nucleotide excision repair. Single-strand conformation polymorphism and DNA sequencing were used to detect the mutational status of the ING1 gene. The host-cell-reactivation assay and radioimmunoassay were used to determine the role of ING1 mutations in nucleotide excision repair. We show that 20% of the melanoma primaries contained missense mutations in the SAP30-interacting domain and PHD finger motif of the ING1 gene with the R102L and N260S alterations observed more than once. Furthermore, our data indicate that patients that harbor ING1 mutations in the tumors have a higher risk to die from the disease within 5 years (50%) compared to patients with no ING1 mutation (18%). Moreover, we demonstrated that mutations at codon 102 or 260 as well as deletion of the PHD finger motif are detrimental to p33ING1-mediated enhancement of DNA repair. Taken together, our data indicate that ING1 mutations abrogate its enhancement in nucleotide excision repair.

Background & Objective: As a new tumor suppressor gene, ING1 shared many biological functions with p53, such as cell cycle arrest, DNA repair, apoptosis, and chemosensitivity. The aim of this study was to investigate the effect of p33(ING1b) on chemosensitivity of osteosarcoma cells and its mechanism., Methods: p33(ING1b) was overexpressed in osteosarcoma cell line U2OS through transient transfection. After transfection, U2OS cells were treated with etoposide for 24 hours, then cell growth inhibitory rates were detected by trypan blue exclusion assay, and apoptosis was assessed using flow cytometry analysis and fluorescent microscopy. Furthermore, the protein expression of p53, p21(WAF1), MDM2 and Bax were determined by Western blot analysis., Results: After transient transfection with p33(ING1b) vector for 24 hours, U2OS cells were treated with 20 microg/ml VP-16 for 24 hours. The results showed that the cell growth inhibitory rates strongly increased [(63.1+/-5.1)%], and etoposide- induced apoptosis was increased(62.7%). Ectopic overexpression of p33(ING1b) increased the protein expression of p53 and strongly enhanced the expression of endogenous p21(WAF1) and Bax. Moreover, after transfection and treatment with 20 microg/ml VP-16, the protein expression of p53, p21(WAF1), and Bax strongly increased compared with other groups. The protein expression of MDM2 showed no significant difference., Conclusion: These observations suggest that p33(ING1b) up-regulates p53 protein, and cooperate with p53 in stimulating expression of p21(WAF1) and Bax gene, thus to enhance etoposide-induced apoptosis via p53-dependent pathways.

The ING1 family of proteins has been shown to have regulatory functions in oncogenesis, apoptosis, DNA repair and cell cycle regulation. Here we present the first report on LOH analysis of the ING1 locus, mutation analysis of the complete coding sequence including intron-exon boundaries and expression analysis of the different ING1 splice products and protein isoforms in primary brain tumours. No somatic ING1 mutations were detected. Semi-quantitative analysis revealed higher levels of p33ING1b RNA in benign than in malignant lesions. This correlation was significant in a subset of 37 astrocytic tumours WHO grades I to IV. ING1 protein isoforms p47ING1a, p33ING1b and p24ING1c were found to be expressed variably in this series. Our findings support a regulatory contribution of ING1 to the development or progression of brain tumours., (Copyright 2004 Wiley-Liss, Inc.)

The ING1 gene was originally cloned as a candidate tumor suppressor of human breast cancer, and recent studies suggest that ING1 proteins are involved in chromatin remodeling functions via physical association with both histone acetyltransferases (HATs) and histone deacetylases (HDACs). Both CREB binding protein (CBP) and the related p300 proteins show a marked preference for binding to complexes containing p33ING1b, one of the major ING1 isoforms, whereas HDAC immunocomplexes contain equal amounts of p33ING1b and p47ING1a. This observation is interesting, given that p33ING1b can selectively increase histone H3 and H4 acetylation when micro-injected into individual cells, whereas p47ING1a inhibits histone acetylation. We investigated whether p33ING1b modulated the transcriptional activity of estrogen receptor (ER)alpha. In cells transfected with increasing concentrations of a mammalian expression vector encoding p33ING1b, estrogen-induced ER alpha transcriptional activity was found to increase in a dose-dependent manner. As p33ING1b expression levels increased, transcription of an ER-responsive reporter gene by either estrogen-inducible full-length ER alpha or the activation function (AF) 1 deletion mutant was enhanced, while the AF2 deletion mutant was unaffected by the presence of p33ING1b. These results showed that p33ING1b enhanced estrogen-induced ER alpha activity through the AF2 domain. Our data demonstrate that p33ING1b acts like a coactivator for ER alpha and stimulates estrogen-induced ER alpha transcriptional activity consistent with a function for p33ING1b in chromatin remodeling.

The p33(ING1b) tumor suppressor protein plays a prominent role in cellular stress responses including cell cycle arrest, DNA repair, apoptosis, and chromatin remodeling. As the main product of the inhibitor of growth 1 (ING1) gene, p33(ING1b) is the most intensively studied protein of the ING family. So far, most ING1 antibodies have been raised against full-length proteins. Since all ING1 isoforms share an identical carboxyl-terminus, and commercially available ING1 antibodies often lack specificity, we sought to develop a polyclonal antibody capable of specifically recognizing the p33(ING1b) protein. Here, we describe the development and characterization of the p33(ING1b)-specific antibody.

Purpose: Clinical and experimental evidence suggest that the p33ING1b candidate tumor suppressor functionally cooperates with p53 in controlling biochemical and biological functions. Because p53 is frequently mutated in brain tumors and the ING1 locus maps to a site of which the loss is associated with gliomas, we analyzed the mutation and expression profiles of ING1B in human brain tumors. Here we present the first report of ING1 expression and mutation analyses in human brain tumor samples and malignant glioma cell lines., Experimental Design: Expression and mutation analyses of ING1B together with subcellular localization studies of ING1 proteins were performed on 29 brain tumor specimens and 6 human glioma cell lines., Results: A single point mutation (3.5%) was detected in the 29 brain tumor specimens analyzed. This missense mutation occurred in a sequence reported previously to confer nuclear translocation properties to p33ING1b. Interestingly, overexpression and subcellular mislocalization of p33ING1b were observed in all 29 of the brain tumor specimens and some glioma cell lines. In tumor samples, ING1 proteins aberrantly localized to the cytoplasm, and to a lesser extent, to the nucleus of glioma cells., Conclusions: Our data indicate that although mutations of ING1 seem to be infrequent in human brain tumors, deregulated expression and mislocalization of ING1 proteins, particularly the p33ING1b isoform, are common events in gliomas and glioblastomas.

Objective: To investigate the cooperation effect of p33(ING1b), coded protein of the novel tumor-suppressor gene ING1b, with p53 gene on the cell growth, cell cycle arrest, apoptosis and expression of the p53 downstream gene p21(WAF1/CIP1) in hepatocellular carcinoma., Methods: Recombinant plasmids pcDNA3-INGIb containing sense p33(ING1b) cDNA, and pCMV-wtp53 containing sense p53 gene, and were transfected into HepG2, PLC/PRF/5 and Hep3B hepatoma cell lines with different p53 endogenous expression status using lipofecTAMINE method. Recombinant plasmids pcDNA3-alphaING1b containing antisense p33(ING1b) cDNA, pCMV-alphawtp53 containing antisense p53 gene, and plain vector as control were transfected into the cells. Forty-eight hours after transfection, the apoptosis rates were detected and cell cycle arrest were analyzed by flow cytometry. The expression of p33(ING1b) and wtp53 proteins were detected by Western blot. The luciferase gene reporter plasmid drived by p21(WAF1/CIP1) promoter was also transfected into the cells to analyze the activation of p21(WAF1/CIP1) gene in hepatoma cells. Then 6% ethanol was added into the in DMEM culture medium and all of the above experiments were repeated., Results: After the p33(ING1b) gene was transfected, the apoptosis rate of HepG2 cells which express endogenous wtp53 was enhanced (22.53%), the number of cells arrested in G(0)/G(1) phase was increased (67.45%), and the activation of p21(WAF1/CIP1) promoter reached the highest level (13.08) (all P < 0.01). In the PLC/PRF/5 cells which express endogenous mutant p53, after combined p33(ING1b) and wtp53 gene transfection the percentage of cells arrested in G(0)/G(1) phase (78.16%) and the activation of p21(WAF1/CIP1) promoter (12.99) were higher than those in the experiment groups transfected with p33(ING1b) or wtp53 gene alone (both P < 0.01), however, the difference in apoptosis rate was not statistically significant (P > 0.05). After 6% ethanol treatment, apoptosis rate of PLC/PRF/5 cells transfected with combined p33(ING1b) and wtp53 gene was increased significantly (42.8%). The apoptosis rate and cell cycle arrest were not significantly different between the Hep3B cells with deletion of wtp53 which were transfected with p33(ING1b) and wtp53 gene and those of the control group (P > 0.05), however, the activity of the p21(WAF1/CIP1) promoter was activated in the combined transfectlion group (10.32, P < 0.01)., Conclusion: p33(ING1b) cooperate with wtp53 in the process of inhibiting hepatoma cell growth, inducing apoptosis, and activating p53 downstream gene p21(WAF1/CIP1), and after chemical inducing reagent treatment the cooperation between p33(ING1b) and wtp53 gene is enhanced.

ING1b can stimulate cell cycle arrest, repair, senescence, and apoptosis. The actions of ING1b are attributed to its activation of the tumor suppressor p53. Here we investigate the more subtle effects of ING1b on the cell cycle and DNA damage responses in the absence of p53. To this end, we have generated isogenic cell lines that expressed ING1b and p53 either individually or in combination under the control of inducible promoters. A five- to 10-fold induction of ING1b over the endogenous protein in a p53-null H1299 background slightly impairs proliferation by increasing the doubling time by approximately 10%. Significantly, ectopic expression of ING1b enhanced the G(2)/M DNA damage checkpoint induced by adriamycin. We demonstrated that the DNA damage-induced cell death mediated by the cooperation between ING1b and p53 was more prominent than by the individual proteins alone. In adriamycin-treated cells, p53 was stabilized and induced the expression of p21(CIP1/WAF1), but the expression of ING1b was not affected. The exact targets of ING1b in the p53-null background are not known, but we demonstrated that the transcriptional activities of other members of the p53 family, p63alpha and p73alpha, could be activated by ING1b. These data indicate that ING1 has a subtle antiproliferative effect even in the absence of p53, and ING1b enhances the DNA damage responses through p53-dependent and -independent mechanisms.

Purpose: We performed a clinical study on ING1b gene expression and p53 gene status in relation to p53 target genes., Experimental Design: Eighty-eight tumors from surgically treated non-small cell lung cancer (NSCLC) patients were studied. PCR-single-strand conformational polymorphism after sequencing was performed to investigate ING1 and p53 gene status. Quantitative reverse transcription-PCR was performed to evaluate the gene expression of ING1b, p21, and bax. The results of p21 and bax expression were confirmed with immunohistochemistry., Results: Only two carcinomas (2.3%) had nonmissense mutations of ING1b. Thirty-seven carcinomas (42.0%) had reduced ING1b gene expression. Thirty-seven carcinomas (42.0%) had mutations of p53. In total, 63 carcinomas (71.6%) had either reduced ING1b expression or mutant p53. The p21 gene expression ratio was significantly lower in the ING1b-reduced tumors than in the ING1b-positive tumors (P = 0.0029). Similarly, the bax gene expression ratio was significantly lower in the ING1b-reduced tumors than in the ING1b-positive tumors (P < 0.0001), and it was also significantly lower in tumors that had either reduced ING1b expression or mutant p53 than in tumors that had both positive ING1b expression and wild-type p53 (P = 0.0331)., Conclusions: Reduction of ING1b gene expression was associated with reduced p21 and bax gene expression in NSCLCs. The present study is the first clinical report to confirm the positive role of ING1b in regulating p21 and bax gene expression. ING1b might be one of the tumor suppressor genes that could play a role in carcinogenesis in NSCLC patients.

The ING1 gene was originally cloned as a candidate tumor suppressor of human breast cancer, and recent studies suggest that ING1 proteins are involved in chromatin remodeling functions via physical association with both histone acetyltransferases (HATs) and histone deacetylases (HDACs). In this study, we investigated whether p33(ING1b), one of the major ING1 isoforms, modulated the transcriptional activity of estrogen receptor (ER) alpha. In Cos-7 cells transfected with increasing concentrations of a mammalian expression vector encoding for p33(ING1b), estrogen-induced ERalpha transcriptional activity was found to increase in a dose-dependent manner. As p33(ING1b) expression levels increased, transcription of an ER-responsive reporter gene by either estrogen-inducible full-length ERalpha or activation function (AF) 1 deletion mutant was enhanced, while the AF2 deletion mutant was unaffected by the presence of p33(ING1b). These results showed that p33(ING1b) enhanced estrogen-induced ERalpha activity through the AF2 domain. Our data also demonstrated that the antiestrogens inhibited the transcriptional activity of ERalpha as stimulated by p33(ING1b). Furthermore, a weak physical association was observed between in vitro translated p33(ING1b) and ERalpha. Our data presented here demonstrate that p33(ING1b) acts like a coactivator for ERalpha and stimulates estrogen-induced ERalpha transcriptional activity consistent with a function for p33(ING1b) in chromatin remodeling.

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.

The inhibitor of growth (ING) genes (ING1-4) probably descend from tumour suppressor genes. ING1 was the first to be identified and later isolated using an approach to detect genes whose expression is suppressed in cancer. The others were isolated through homology and similarity searches in human and mouse databases. All members contain a plant homeodomain involved in macromolecule recognition. Apart from the extensively studied ING1, little is known about the number of transcripts encoded by the other members or their gene structure. ING1 encodes several differentially spliced mRNAs, which may produce a family of proteins. The most widely expressed protein isoform is p33(INGb1), which is involved in restriction of cell growth and proliferation, apoptosis, tumour anchorage independent growth, cellular senescence, maintenance of genomic stability, and modulation of cell cycle checkpoints. ING1 gene mutation is uncommon in cancer, although the subcellular localisation of p33(INGb1) may have an effect on its function. The p33(INGb1) cellular compartmental shift from the nucleus to the cytoplasm may cause loss of normal cellular function, and may play a central role in the pathogenesis of several cancers.

Background/aims: The inhibitor of growth gene 1 (ING1) is a modulator of cell cycle checkpoints, apoptosis, and cellular senescence. The most widely expressed ING1 isoform is p33(ING1b), which can modulate p53, a molecule that is frequently altered in breast cancer. Reduced ING1 mRNA expression has been observed in primary breast cancer expressing wild-type p53., Methods: p33(ING1b), p53, oestrogen receptor (ER), and progesterone receptor (PgR) expression was studied in 86 primary invasive breast cancers using immunohistochemistry., Results: Reduced nuclear expression of p33(ING1b) was found in cancer cells, both in intensity and the proportion of cells staining. This was associated with enhanced cytoplasmic p33(ING1b) expression in a proportion of cases. Analysis of several known biological factors indicated that high grade tumours were of larger size and more often negative for ER and PgR expression. However, larger tumours were more frequently p53 negative. These results provide evidence that p33(ING1b) alterations are associated with more poorly differentiated tumours. Positive correlations were found between nuclear p33(ING1b) expression and both ER and PgR expression., Conclusions: Optimum function of p53 is dependent on p33(ING1b) so that a reduction of nuclear p33(ING1b) expression, as seen in this series, would be predicted to compromise p53 function. This study showed that p33(ING1b) alterations were associated with more poorly differentiated tumours. Therefore, p33(ING1b) expression could be used as a marker of differentiation in invasive breast cancer. These results support the view that loss of p33(ING1b) may be an important molecular event in the differentiation and pathogenesis of invasive breast cancer.

Objective: To investigate the relationship between expressions of ING1 gene and genes of human telomerase reverse transcriptase (hTERT) and telomerase-associated protein 1 (hTP1) in human gliomas., Methods: The expressions of ING1 mRNA and p33(ING1) protein, hTERT mRNA and protein, and hTP1 mRNA and protein in seventy human glioma specimens with different malignant grades were studied using in situ hybridization and immunohistochemistry., Results: All of the 70 gliomas collected expressed hTP1 mRNA and protein and among them, 62 (88.6%) and 58 (82.9%) out of 70 expressed hTERT mRNA and protein respectively. The quantities of the four kinds of positive cells were correlated positively with one another (r = 0.758 - 0.882, P < 0.000 5), and all of them were significantly fewer in gliomas of WHO grade I - II than in grade III gliomas and the most in grade IV gliomas (P < 0.05 approximately 0.01). 66 (94.3%) and 62 (88.6%) out of 70 gliomas expressed ING1 mRNA and p33(ING1) protein respectively. The quantities of their positive cells were also correlated positively with each other (r = 0.831, P < 0.000 5), but the positive cells were more in gliomas of WHO grade I - II than in grade III gliomas and the fewest in grade IV gliomas (P < 0.01). The quantities of positive cells of ING1 mRNA and p33(ING1) protein were correlated negatively with those of hTERT mRNA and protein as well as hTP1 mRNA and protein respectively (r = -0.211 to -0.384, P < 0.05 approximately 0.001)., Conclusions: The results suggest that all of the parameters concerned are valuable in evaluating the biological behavior of gliomas. In glioma cells, overexpressions of hTERT and hTP1 genes might be significant in inhibiting the expression of ING1 gene. The abnormal expressions of the three genes play possibly the important roles in the development and malignant progression of gliomas.

The incidence of basal cell carcinoma is the highest among all human malignancies. Epidemiological evidence indicates that ultraviolet radiation is the primary environmental cause for the pathogenesis of basal cell carcinoma. However, the genetic changes caused by ultraviolet radiation that lead to basal cell carcinoma formation remain unclear. We and others have demonstrated that the ING1 (inhibitor of growth 1) tumour suppressor plays an important role in cellular stress response to ultraviolet irradiation, such as DNA repair and apoptosis. This study was designed to investigate whether ING1 is overexpressed and/or mutated in human basal cell carcinoma. Immunohistochemistry, single-strand conformation polymorphism, and DNA sequencing were used to determine the expression and mutational status of the ING1 gene in 54 basal cell carcinoma biopsies. Immunohistochemical staining demonstrated that ING1 is overexpressed in 25% (6/24) human basal cell carcinomas. Single-strand conformation polymorphism and DNA sequencing revealed that only 1 in 54 (1.8%) basal cell carcinoma primaries contained a missense mutation in the ING1 gene. The mutation is located in exon 2 and could thus potentially interfere with the structure of every ING1 isoforms and the functions of the PHD zinc finger motif. Our data indicate that overexpression and mutation of the ING1 gene are infrequent in human basal cell carcinoma.

The aim of this study was to characterize molecular alterations of the recently reported candidate tumor suppressor gene, ING1, and to explore the relationship between ING1 and p53 in a well-defined series of adenocarcinomas of the esophagogastric junction (AdEGJ). Polymerase chain reaction (PCR)-based assays were used to characterize ING1 and p53 alterations, relative to histologically normal esophageal mucosa. Two tumors were found to have ING1 mutations: one novel missense mutation (AGC(Ser)-->ATC(Ile)) at codon 147, and one silent mutation (TCG(Ser)-->TCA(Ser)) at codon 173. Reduced expression of the two major alternatively spliced ING1 messenger RNA variants, p47(ING1a) and p33(ING1b) was variable, but was reduced (1.2-10-fold) in 12 of 19 AdEGJs compared to normal esophageal epithelium. No association between p53 and ING1 alterations was apparent. We conclude that reduced ING1 expression is frequently associated with AdEGJ tumorigenesis, further supporting its role as a tumor suppressor gene, and that ING1 expression is independent of p53 status.

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.

Objective: To analyze the relationship of p33(ING1) gene expression and p33(ING1) exon-2 mutation to the pathogenesis, development and consequence of stomach cancer., Methods: Envision immunohistochemical method was utilized to detect the p33(ING1) expression in 103 specimens of stomach cancer, 36 specimens of stomach mucosal atypical hyperplasia, and 32 specimens of normal stomach mucosa. PCR-SSCP was utilized to detect p33(ING1) exon-2 mutation in stomach cancer tissues., Results: The p33(ING1) expression rate in stomach cancer was 54.4% (56/103), significantly lower than that in precarcinomatous tissues (94.4%, 34/36, P < 0.01) and that in normal tissues (100%, 32/32, P < 0.01). The p33(ING1) expression in stomach cancer was related to tumor growth, distant metastasis and tumor differentiation (all P < 0.05). p33(ING1) gene exon-2 mutation was detected in 3 cases of stomach cancer tissues (12%, 3/25), and not in other tissues by PCR-SSCP method., Conclusion: p33(ING1) low expression, and gene p33(ING1) exon-2 mutation may play an important role in the pathogenesis, development and consequence of stomach cancer.

Objective: To study effects of alternative transcripts of ING1 transfection on human cancer cell lines., Methods: p47/ING1A and p33/ING1B expression vehicles were constructed and introduced into a human breast cancer cell line MCF-7 and a human lung cancer cell line PAa, both expressing wild-type p53 protein. Growth characteristics of the transfectants and potentially related genes were analyzed., Results: The levels of p47/ING1A and p33/ING1B protein elevated respectively in tumor cells of MCF-7 and PAa after transfected with p47/ING1A and p33/ING1B, and the latter was much higher than that of the former. Ectopic overexpression of p33/ING1B effectively blocked tumor cell growth and arrested cells in the G(0) approximately G(1) phase of the cell cycle (P < 0.01), while p47/ING1A gave no effect on cell growth or cell cycle. Tumor cells overexpressing p33/ING1B contained more p21(WAF1) protein than that of the control cells, with undisturbed p53 protein level., Conclusions: Expression of two different transcripts of ING1 may have different effects on tumor cell growth. p33/ING1B may cooperate with p53 in stimulating expression of p21(WAF1) gene, thus to arrest cell cycle and to inhibit tumor cell growth. p33/ING1B may be considered to be a candidate as a partner of p53 in gene therapy.

The tumour suppressor ING1 shares many biological functions with p53, such as cell cycle arrest, DNA repair, apoptosis, and chemosensitivity. Since p53 inhibits invasion and angiogenesis of melanoma cells, we sought to investigate if p33ING1 (one of ING1 isoforms) is also involved in these biological processes. We first overexpressed p33ING1 in melanoma cells and assessed the protein levels in MMP-1, MMP-2, and MMP-9. Results from Western blot analysis showed no significant difference in these matrix metalloproteinase levels between cells transfected with vector, p33ING1, and antisense p33ING1. Wound healing assay was performed to examine if p33ING1 plays a role in migration and invasion. Results showed that there was no difference between vector, p33ING1, and antisense p33ING1 groups in melanoma cell migration across the wound. Western blot analysis also indicated that there is no difference in the levels of proteins which are directly involved in angiogenesis, such as VEGF, Flt-1, and Flk-1, between cells transfected with vector, p33ING1, and antisense p33ING1. Furthermore, functional studies indicated that cultured medium derived from p33ING1-transfected melanoma cells did not stimulate the growth of HUVEC cells, compared to controls, providing support to the lack of functional role of p33ING1 in angiogenesis. In conclusion, we demonstrate in vitro that p33ING1, unlike p53, does not play a role in angiogenesis and migration in melanoma cells.

Background: Inhibitor of growth-1 (ING1) is a new candidate for the tumour suppressor gene that encodes a 33k Da protein (p33(ING1)). While reduction of p33(ING1) is an important event in some human cancers, the expression of p33(ING1) in human hepatocellular carcinoma (HCC) remains to be examined. We evaluated p33(ING1) expression in various liver diseases including HCC., Methods: Expression of p33(ING1) was evaluated immunohistochemically not only in the normal liver (n = 5), but also in specimens of chronic hepatitis (n = 39) and HCC (n = 86). We also analysed the relationship between p33(ING1) expression and cyclin E kinase activity detected by autoradiography in 29 HCCs., Results: Expression of p33(ING1) was reduced in HCC, especially in moderately and poorly differentiated HCCs, and those at advanced stages. Furthermore, expression of p33(ING1) correlated inversely with cyclin E kinase activity., Conclusions: These data suggest that reduction of p33(ING1) may contribute to the process of malignant transformation, progression and dedifferentiation of HCC via an increase of cyclin E kinase activity.

The ING family of proteins are involved in chromatin remodelling, and bind to and affect the activity of histone acetyltransferase, histone deacetylase, and factor acetyltransferase protein complexes. Some family members affect transcription, including the expression of p53-inducible genes such as p21 and Bax, and ING2 induces p53 acetylation on a site implicated in the regulation of p53 activity. ING1 promotes DNA repair and interacts with proliferating cell nuclear antigen, thus linking DNA repair, apoptosis and chromatin remodelling. Here, we summarize what is known about the molecular interactions of ING1 family proteins and, based on these interactions, develop a model to better understand the impact of ING proteins on multiple biological processes.

Objective: To explore the significance of p33(ING1b) protein expression and p53 protein expression and investigate the mutation rate of ING1b gene in hepatocellular carcinoma (HCC)., Methods: Immunohistochemical method was employed to detect the expressions of p33(ING1b) and p53 proteins and the X antigen of hepatitis B virus (HBV) in 57 cases of HCC, 51 cases of surrounding non-tumor tissues surrounding HCC, and 12 cases of normal liver tissues; PCR-single strand conformation polymorphism (SSCP) was applied to reveal the mutation rates of two exons of ING1b gene in HCC. DNA fragments exhibiting abnormal shifting were cloned into PMD18-T vector and sequenced., Results: The positive rates of p33(ING1b) protein and of p53 protein were 42.1% and 57.9% respectively among the 57 cases of HCC. Both p33(ING1b) protein and p53 protein were positive in 19 cases (33.3%) of HCC tissues, and they were distributed in the same areas, mostly in the nucleus. The expression of p33(ING1b) was significantly correlated with that of p53 protein (r = 0.783, P < 0.01). The matched surrounding non-tumor tissues and normal liver tissues all showed negative p53 staining, while weak positive expression of p33(ING1b) was observed in 13 cases out of the 51 non-tumor tissues and 2 cases out of 12 normal liver tissues. Thirty-five cases, all with hepatocellular carcinoma, out of the 57 HCC cases showed positive HBV X protein (76.4%). The correlation of HBV infection with the expression of p33(ING1b) and p53 proteins was significant (P < 0.01). PCR-SSCP showed that the mutation rate of ING1b gene in HCC was 7.1% (2/28)., Conclusion: The expression of p33(ING1b) protein is significantly related to the expression of p53 protein in hepatocellular carcinoma; The existence of p33(ING1b) may have some other p53-independent pathway; Gene mutation was not the main reason that ING1b loses its tumor suppressing function in HCC. HBV infection has a close relationship with p33(ING1b) and p53 proteins expression.

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.

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.

ING1 proteins are nuclear, growth inhibitory, and regulate apoptosis in different experimental systems. Here we show that similar to their yeast homologs, human ING1 proteins interact with proteins associated with histone acetyltransferase (HAT) activity, such as TRRAP, PCAF, CBP, and p300. Human ING1 immunocomplexes contain HAT activity, and overexpression of p33(ING1b), but not of p47(ING1a), induces hyperacetylation of histones H3 and H4, in vitro and in vivo at the single cell level. p47(ING1a) inhibits histone acetylation in vitro and in vivo and binds the histone deacetylase HDAC1. Finally, we present evidence indicating that p33(ING1b) affects the degree of physical association between proliferating cell nuclear antigen (PCNA) and p300, an association that has been proposed to link DNA repair to chromatin remodeling. Together with the finding that human ING1 proteins bind PCNA in a DNA damage-dependent manner, these data suggest that ING1 proteins provide a direct linkage between DNA repair, apoptosis, and chromatin remodeling via multiple HAT.ING1.PCNA protein complexes.

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.

Background/aims: p33(ING1b) is a tumour suppressor protein involved in growth control and apoptosis. Suppression of p33(ING1b) expression is associated with the loss of cellular growth control and immortalisation, whereas its overexpression causes cell cycle arrest. Moreover, normal p33(ING1b) expression is essential for optimal function of p53. Acute lymphoblastic leukaemia (ALL) is the most common malignancy of childhood, accounting for one third of all childhood malignancies. A variety of cytogenetic abnormalities have been described but there is no single abnormality common to all cases. Deregulation of the TP53 pathway is a common genetic abnormality in human malignancies. However, TP53 mutations are uncommon in ALL. It is possible that alternative mechanisms of regulation of the TP53 apoptosis pathway, such as modulation of p33(ING1b) expression, may be important in ALL. The aim of this study was to assess the expression of p33(ING1b) in childhood ALL., Methods: One hundred and forty five patients with childhood ALL were investigated in this immunohistochemical study of the expression of p33(ING1b)., Results: Loss of nuclear expression of p33(ING1b) was seen in 78% of cases. This was associated with increased cytoplasmic expression of the protein. Kaplan Meier survival analysis demonstrated a trend towards a better prognosis for patients with tumours that had lost nuclear p33(ING1b)., Conclusion: These results suggest that the loss of nuclear p33(ING1b) expression may be an important molecular event in the pathogenesis of childhood ALL.

The protein product of the ING1 gene physically interacts with p53 and appears necessary for the role of p53 in growth inhibition/apoptosis. Alternative splicing of the ING1 gene produces three transcripts: p24/ING1-ALT1, p47/ING1-ALT2 and p33/ING1. A competitive RT-PCR, which determines the relative levels of these transcripts, was employed to study peripheral blood lymphocytes from 49 patients with haematological malignancies and five normal controls. Both groups expressed predominantly the p33/ING1 transcript, with low levels of p24/ING1 and p47/ING1. We screened the complete p33/ING1 transcript for sequence variations, by non-isotopic RNase cleavage assay (NIRCA); none were found. This study suggests that neither perturbation of alternative splicing, nor mutation of p33/ING1 plays a significant role in the development of haematological malignancies.

The tumour suppressor ING1 shares many biological functions with p53, such as cell cycle arrest, DNA repair, apoptosis, and chemosensitivity. Previous findings indicate that the isoform p24ING1 is capable of enhancing chemosensitivity in human fibroblasts. To investigate if the p33ING1 isoform is also involved in chemosensitivity, we overexpressed p33ING1 in melanoma cells and assessed for cell death after treatment with camptothecin. Results from the sulforhodamine B cell survival assay and flow cytometry analysis show no significant difference among cells transfected with vector, p33ING1, and antisense p33ING1. Furthermore, co-transfection of the p33ING1 and p53 constructs had no effect on the frequency of cell death, indicating that there is no synergistic effect between the two tumour suppressors in camptothecin-induced cell death in melanoma cells. This is in contrast to previously observed collaboration between p33ING1 and p53 in DNA repair and apoptosis. Taken together, we demonstrate that p33ING1 does not enhance camptothecin-induced cell death in melanoma cells.

We reported previously that mouse ING1 homolog (mINGh), localized in the nucleus, enhanced cell death in HC11 mouse mammary epithelial cells. Analysis of the mINGh amino acid sequences revealed the presence of potential nuclear localization signal (NLS) and plant homeodomain (PHD) finger DNA binding domain. In the present study, NLS site in mINGh was determined using different pieces of mutant mINGh proteins, which were fused to green fluorescent protein (GFP), and transfected into HC11 cells. NLS of mINGh was split into two parts consisting of amino acids KEKK and KKLK. Mutation in NLS sites of mINGh resulted in no enhancement of the cell death when over-expressed. These results indicated that mINGh contains NLS of bipartite type, which is essential for the regulation of cell death.

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.

Aims: Cutaneous malignant melanoma is an unpredictable neoplasm. Studies of cell cycle and proliferation-associated proteins may help in the understanding of the genesis of melanomas. The tumour suppressor gene TP53 has been shown to be involved in melanomas. However, the incidence of TP53 malfunction in cutaneous melanoma is unclear, and other regulators of cell cycle control are likely to be involved in both the development and progression of melanocytic neoplasia. A candidate is the ING1 gene, which co-operates with TP53 in growth suppression and apoptosis. Thus loss of ING1 function may have similar consequences to loss of TP53 function and may contribute to tumorigenesis. Therefore we have studied the expression of p33ING1b protein in cutaneous melanocytic neoplasia., Methods and Results: Sixty-seven melanocytic lesions were studied by immunohistochemistry for the expression of p33ING1b. In our series there was loss of nuclear p33ING1b expression in invasive malignant melanoma compared with normal cutaneous melanocytes or the melanocytes of benign melanocytic naevi. This was associated with an enhancement of cytoplasmic p33ING1b expression which was particularly prominent in invasive malignant melanoma., Conclusions: Cytoplasmic immunostaining for p33ING1b using MAb GN2 is strongly associated with 'activated' melanocytic lesions; therefore it is possible that this MAb could be of value in diagnostic practice. Furthermore, the reduction in p33ING1b expression and perhaps translocation from the nucleus to the cytoplasm may play a central role in the development and progression of melanomas.

Background: Epidemiological evidence indicates that exposure to ultraviolet (UV) radiation is directly linked to the increase of both incidence and mortality of melanoma. However, the genetic changes caused by UV radiation that lead to melanoma formation remain poorly understood. Recently, a potential tumour suppressor gene ING1 (inhibitor of growth 1) was shown to inhibit cell growth and induce apoptosis in the presence of p53. We have demonstrated that the expression of ING1 is induced after UV irradiation and that ING1 enhances the repair of UV-damaged DNA., Objectives: To investigate if ING1 plays a role in melanoma formation., Methods: We examined p33ING1 expression levels in 14 melanoma cell lines., Results: We found that p33ING1 is overexpressed at both mRNA and protein levels in melanoma cell lines compared with normal melanocytes. Single-strand conformation polymorphism (SSCP) analysis showed band shifting in two melanoma cell lines. DNA sequencing confirmed that there were nucleotide alterations in the ING1 gene in Sk-mel-24 and Sk-mel-110 cell lines. Two silent nucleotide alterations in exon 1a were detected in Sk-mel-110. In Sk-mel-24, the A-->G nucleotide alteration at codon 260 resulted in an amino acid change from Asn to Ser, while seven other nucleotide alterations were silent. To determine if the silent nucleotide alterations in these two melanoma cell lines were due to polymorphism, SSCP analysis of ING1 gene was performed in 25 healthy volunteers. No band shift was observed in the SSCP analysis, suggesting that the nucleotide alterations in the melanoma cell lines are unlikely to be due to polymorphism., Conclusions: Taken together, our data demonstrate that ING1 is overexpressed, but infrequently mutated, in melanoma cell lines.

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.

To investigate the effect of p33(ING1) on wild-type p53 gene therapy, T.Tn human esophageal carcinoma cells were stably transfected with p33(ING1) cDNA. Infection with Ad-p53 (recombinant adenovirus containing wild-type p53) into p33-transfected cells reduced cell viability, while infection with empty vector had little effect. This reduced viability was shown to be due to apoptotic cell death by the TUNEL (terminal deoxynucleotidyl transferase-mediated nick end-labeling) assay. Following infection with Ad-p53, levels of p53 were similar in p33-expressing cells and in the parental line. However, levels of p21 and Mdm2 were elevated in p33-transfected cells. Nonetheless, this enhanced expression of Mdm2 appeared to be ineffective in downregulating p53. Transient transfection with mutant Mdm2 prior to Ad-p53 infection provided a significant protection as compared with cells transfected with wild-type Mdm2. These results imply a synergistic effect between p33 and p53 in the induction of apoptosis of human esophageal carcinoma cells. A role for Mdm2 in this synergism is suggested.

p33(ING1) is a novel candidate tumor suppressor gene which is involved in the regulation of apoptosis. p33(ING1) interacts with p53 signaling pathway and regulates cellular growth. It has reported that the expression of p33(ING1) mRNA was decreased in lymphoid malignancies. We thus investigated the potential involvement of p33(ING1) abnormalities in myeloid leukemias. However, the levels of p33(ING1) transcript were almost equal in 3 AML cell lines and 10 fresh AML samples. In addition, neither point mutations nor deletions in p33(ING1) gene were found in myeloid leukemias. These results suggest that p33(ING1) may not be a major candidate tumor suppressor gene in myeloid leukemias., (Copyright 2002 Wiley-Liss, Inc.)

Sin3 is an evolutionarily conserved corepressor that exists in different complexes with the histone deacetylases HDAC1 and HDAC2. Sin3-HDAC complexes are believed to deacetylate nucleosomes in the vicinity of Sin3-regulated promoters, resulting in a repressed chromatin structure. We have previously found that a human Sin3-HDAC complex includes HDAC1 and HDAC2, the histone-binding proteins RbAp46 and RbAp48, and two novel polypeptides SAP30 and SAP18. SAP30 is a specific component of Sin3 complexes since it is absent in other HDAC1/2-containing complexes such as NuRD. SAP30 mediates interactions with different polypeptides providing specificity to Sin3 complexes. We have identified p33ING1b, a negative growth regulator involved in the p53 pathway, as a SAP30-associated protein. Two distinct Sin3-p33ING1b-containing complexes were isolated, one of which associates with the subunits of the Brg1-based Swi/Snf chromatin remodeling complex. The N terminus of p33ING1b, which is divergent among a family of ING1 polypeptides, associates with the Sin3 complex through direct interaction with SAP30. The N-terminal domain of p33 is present in several uncharacterized human proteins. We show that overexpression of p33ING1b suppresses cell growth in a manner dependent on the intact Sin3-HDAC-interacting domain.

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.

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., (Copyright 2001 Academic Press.)