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Rémy Pedeux - One of the best experts on this subject based on the ideXlab platform.
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Abstract B06: ING2 loss sensitizes KRAS-mutated NSCLC to WEE1 inhibition through regulation of CHK1 expression
Molecular Cancer Research, 2020Co-Authors: Charles Ricordel, Karl Riabowol, Jérôme Archambeau, Angela Chan, Benoît Desrues, Subash Thalappilly, Nancy A. Nixon, Gwyn Bebb, Rémy PedeuxAbstract:ING2 (Inhibitor of Growth 2) is a tumor-suppressor gene involved in chromatin acetylation whose loss has been observed in nearly 60% of NSCLC. The loss of ING2 expression has been associated with the progression of tumor cells from G1 to S phase, replication stress and genomic instability. The intra-S and the G2-M phase checkpoints are mainly regulated by the cell cycle tyrosine kinase WEE1. Therefore, we hypothesized that the loss of ING2 expression could sensitize NSCLC to the WEE1 inhibitor MK-1175, according to a synthetic lethality model. In silico analysis of the cancer gene dependency map (shRNAi Achilles library v.2016) was used to explore gene interactions with ING2. Antiproliferative effects of drugs were tested in vitro with AlamarBlue and MTT assays in a panel of NSCLC cells lines with varied mutational status. Multiple functional approaches were taken to decrease ING2 expression in RAS-mutated NSCLC cell lines (si-RNA, LNA-gapmer, and CrispR-Cas9). Pharmacologic inhibition of ING2 epigenetic functions was achieved using the HDAC inhibitor SAHA. Cell cycle and apoptosis analysis were performed using flow cytometry according to a BrdU-Pi and Annexin V-PI protocols, respectively. Compusyn software was used to model MK-1775 and SAHA drug interaction. NSCLC cell lines with mutant RAS that expressed high levels of ING2 protein were more resistant to the antiproliferative effect of MK-1775 than similar lines with low levels of ING2. Colony formation assays were consistent with these results. Multiple functional assays reproducibly showed that downregulation of ING2 expression increased MK-1775 sensitivity in vitro. Similar results were obtained with pharmacologic disruption of ING2 from the HDAC-SIN3A complex using SAHA, where disruption conferred a higher antiproliferative effect to MK-1775. Interestingly, combination treatment induced cell cycle progression and synergic cell death through apoptosis in RAS-mutated cell lines nondeficient for ING2. Since the synergistic effect of SAHA and WEE1 inhibitor was shown to be dependent on CHK1 activity in a previous report, we checked whether ING2 and CHK1 expression could be linked. Using public database and functional assays, we indeed find that ING2 downregulation represses CHK1 transcription through a mechanism that remains to be elucidated. Collectively, these findings demonstrate that inhibition of WEE1 by MK-1775 could represent a therapeutic approach to more effectively target ING2-deficient RAS-mutated NSCLC. Moreover, HDAC inhibitors targeting ING2 epigenetic functions synergize with MK-1775, possibly through regulation of CHK1 transcription. Consequently, this combination therapy could also be a promising strategy in ING2-proficient RAS-mutated NSCLC. Citation Format: Charles Ricordel, Subash Thalappilly, Jerome Archambeau, Angela Chan, Nancy Nixon, Benoit Desrues, Gwyn Bebb, Karl Riabowol, Remy Pedeux. ING2 loss sensitizes KRAS-mutated NSCLC to WEE1 inhibition through regulation of CHK1 expression [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B06.
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Exploiting ING2 Epigenetic Modulation as a Therapeutic Opportunity for Non-Small Cell Lung Cancer.
Cancers, 2019Co-Authors: Alice Blondel, Rémy Pedeux, Amine Benberghout, Charles RicordelAbstract:Non-small cell lung cancer (NSCLC) has been the leading cause of cancer-related death worldwide, over the last few decades. Survival remains extremely poor in the metastatic setting and, consequently, innovative therapeutic strategies are urgently needed. Inhibitor of Growth Gene 2 (ING2) is a core component of the mSin3A/Histone deacetylases complex (HDAC), which controls the chromatin acetylation status and modulates gene transcription. This gene has been characterized as a tumor suppressor gene and its status in cancer has been scarcely explored. In this review, we focused on ING2 and other mSin3A/HDAC member statuses in NSCLC. Taking advantage of existing public databases and known pharmacological properties of HDAC inhibitors, finally, we proposed a therapeutic model based on an ING2 biomarker-guided strategy.
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Focus-ING on DNA Integrity Implication of ING Proteins in Cell Cycle Regulation and DNA Repair Modulation
Cancers, 2019Co-Authors: Jérôme Archambeau, Alice Blondel, Rémy PedeuxAbstract:The ING family of tumor suppressor genes is composed of five members (ING1-5) involved in cell cycle regulation, DNA damage response, apoptosis and senescence. All ING proteins belong to various HAT or HDAC complexes and participate in chromatin remodeling that is essential for genomic stability and signaling pathways. The gatekeeper functions of the INGs are well described by their role in the negative regulation of the cell cycle, notably by modulating the stability of p53 or the p300 HAT activity. However, the caretaker functions are described only for ING1, ING2 and ING3. This is due to their involvement in DNA repair such as ING1 that participates not only in NERs after UV-induced damage, but also in DSB repair in which ING2 and ING3 are required for accumulation of ATM, 53BP1 and BRCA1 near the lesion and for the subsequent repair. This review summarizes evidence of the critical roles of ING proteins in cell cycle regulation and DNA repair to maintain genomic stability.
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Human ex vivo prostate tissue model system identifies ING3 as an oncoprotein
British Journal of Cancer, 2018Co-Authors: Urszula L Mcclurg, Arash Nabbi, Charles Ricordel, Svitlana Korolchuk, Stuart Mccracken, Rakesh Heer, Laura Wilson, Lisa M Butler, Bronwyn Kate Irving-hooper, Rémy PedeuxAbstract:Background: Although the founding members of the INhibitor of Growth (ING) family of histone mark readers, ING1 and ING2, were defined as tumour suppressors in animal models, the role of other ING proteins in cellular proliferation and cancer progression is unclear. Methods: We transduced ex vivo benign prostate hyperplasia tissues with inducible lentiviral particles to express ING proteins. Proliferation was assessed by H3S10^phos immunohistochemistry (IHC). The expression of ING3 was assessed by IHC on a human prostate cancer tissue microarray (TMA). Gene expression was measured by DNA microarray and validated by real-time qPCR. Results: We found that ING3 stimulates cellular proliferation in ex vivo tissues, suggesting that ING3 could be oncogenic. Indeed, ING3 overexpression transformed normal human dermal fibroblasts. We observed elevated levels of ING3 in prostate cancer samples, which correlated with poorer patient survival. Consistent with an oncogenic role, gene-silencing experiments revealed that ING3 is required for the proliferation of breast, ovarian, and prostate cancer cells. Finally, ING3 controls the expression of an intricate network of cell cycle genes by associating with chromatin modifiers and the H3K4^me3 mark at transcriptional start sites. Conclusions: Our investigations create a shift in the prevailing view that ING proteins are tumour suppressors and redefine ING3 as an oncoprotein.
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Human ex vivo prostate tissue model system identifies ING3 as an oncoprotein
British Journal of Cancer, 2018Co-Authors: Urszula L Mcclurg, Arash Nabbi, Charles Ricordel, Svitlana Korolchuk, Stuart Mccracken, Rakesh Heer, Laura Wilson, Lisa M Butler, Bronwyn Kate Irving-hooper, Rémy PedeuxAbstract:Background - Although the founding members of the INhibitor of Growth (ING) family of histone mark readers, ING1 and ING2, were defined as tumour suppressors in animal models, the role of other ING proteins in cellular proliferation and cancer progression is unclear. Methods - We transduced ex vivo benign prostate hyperplasia tissues with inducible lentiviral particles to express ING proteins. Proliferation was assessed by H3S10 immunohistochemistry (IHC). The expression of ING3 was assessed by IHC on a human prostate cancer tissue microarray (TMA). Gene expression was measured by DNA microarray and validated by real-time qPCR. Results - We found that ING3 stimulates cellular proliferation in ex vivo tissues, suggesting that ING3 could be oncogenic. Indeed, ING3 overexpression transformed normal human dermal fibroblasts. We observed elevated levels of ING3 in prostate cancer samples, which correlated with poorer patient survival. Consistent with an oncogenic role, gene-silencing experiments revealed that ING3 is required for the proliferation of breast, ovarian, and prostate cancer cells. Finally, ING3 controls the expression of an intricate network of cell cycle genes by associating with chromatin modifiers and the H3K4 mark at transcriptional start sites. Conclusions - Our investigations create a shift in the prevailing view that ING proteins are tumour suppressors and redefine ING3 as an oncoprotein.
Charles Ricordel - One of the best experts on this subject based on the ideXlab platform.
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ING2 tumor suppressive protein translocates into mitochondria and is involved in cellular metabolism homeostasis
Oncogene, 2021Co-Authors: Charles Ricordel, Audrey Mouche, Hervé Lena, Jérôme Archambeau, Alice Blondel, Laura Chaillot, Florence Jouan, Marie Tiercin, Agnès Burel, Benoît DesruesAbstract:ING2 (Inhibitor of Growth 2) is a tumor suppressor gene that has been implicated in critical biological functions (cell-cycle regulation, replicative senescence, DNA repair and DNA replication), most of which are recognized hallmarks of tumorigenesis occurring in the cell nucleus. As its close homolog ING1 has been recently observed in the mitochondrial compartment, we hypothesized that ING2 could also translocate into the mitochondria and be involved in new biological functions. In the present study, we demonstrate that ING2 is imported in the inner mitochondrial fraction in a redox-sensitive manner in human cells and that this mechanism is modulated by 14-3-3η protein expression. Remarkably, ING2 is necessary to maintain mitochondrial ultrastructure integrity without interfering with mitochondrial networks or polarization. We observed an interaction between ING2 and mtDNA under basal conditions. This interaction appears to be mediated by TFAM, a critical regulator of mtDNA integrity. The loss of mitochondrial ING2 does not impair mtDNA repair, replication or transcription but leads to a decrease in mitochondrial ROS production, suggesting a detrimental impact on OXPHOS activity. We finally show using multiple models that ING2 is involved in mitochondrial respiration and that its loss confers a protection against mitochondrial respiratory chain inhibition in vitro. Consequently, we propose a new tumor suppressor role for ING2 protein in the mitochondria as a metabolic shift gatekeeper during tumorigenesis.
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Abstract B06: ING2 loss sensitizes KRAS-mutated NSCLC to WEE1 inhibition through regulation of CHK1 expression
Molecular Cancer Research, 2020Co-Authors: Charles Ricordel, Karl Riabowol, Jérôme Archambeau, Angela Chan, Benoît Desrues, Subash Thalappilly, Nancy A. Nixon, Gwyn Bebb, Rémy PedeuxAbstract:ING2 (Inhibitor of Growth 2) is a tumor-suppressor gene involved in chromatin acetylation whose loss has been observed in nearly 60% of NSCLC. The loss of ING2 expression has been associated with the progression of tumor cells from G1 to S phase, replication stress and genomic instability. The intra-S and the G2-M phase checkpoints are mainly regulated by the cell cycle tyrosine kinase WEE1. Therefore, we hypothesized that the loss of ING2 expression could sensitize NSCLC to the WEE1 inhibitor MK-1175, according to a synthetic lethality model. In silico analysis of the cancer gene dependency map (shRNAi Achilles library v.2016) was used to explore gene interactions with ING2. Antiproliferative effects of drugs were tested in vitro with AlamarBlue and MTT assays in a panel of NSCLC cells lines with varied mutational status. Multiple functional approaches were taken to decrease ING2 expression in RAS-mutated NSCLC cell lines (si-RNA, LNA-gapmer, and CrispR-Cas9). Pharmacologic inhibition of ING2 epigenetic functions was achieved using the HDAC inhibitor SAHA. Cell cycle and apoptosis analysis were performed using flow cytometry according to a BrdU-Pi and Annexin V-PI protocols, respectively. Compusyn software was used to model MK-1775 and SAHA drug interaction. NSCLC cell lines with mutant RAS that expressed high levels of ING2 protein were more resistant to the antiproliferative effect of MK-1775 than similar lines with low levels of ING2. Colony formation assays were consistent with these results. Multiple functional assays reproducibly showed that downregulation of ING2 expression increased MK-1775 sensitivity in vitro. Similar results were obtained with pharmacologic disruption of ING2 from the HDAC-SIN3A complex using SAHA, where disruption conferred a higher antiproliferative effect to MK-1775. Interestingly, combination treatment induced cell cycle progression and synergic cell death through apoptosis in RAS-mutated cell lines nondeficient for ING2. Since the synergistic effect of SAHA and WEE1 inhibitor was shown to be dependent on CHK1 activity in a previous report, we checked whether ING2 and CHK1 expression could be linked. Using public database and functional assays, we indeed find that ING2 downregulation represses CHK1 transcription through a mechanism that remains to be elucidated. Collectively, these findings demonstrate that inhibition of WEE1 by MK-1775 could represent a therapeutic approach to more effectively target ING2-deficient RAS-mutated NSCLC. Moreover, HDAC inhibitors targeting ING2 epigenetic functions synergize with MK-1775, possibly through regulation of CHK1 transcription. Consequently, this combination therapy could also be a promising strategy in ING2-proficient RAS-mutated NSCLC. Citation Format: Charles Ricordel, Subash Thalappilly, Jerome Archambeau, Angela Chan, Nancy Nixon, Benoit Desrues, Gwyn Bebb, Karl Riabowol, Remy Pedeux. ING2 loss sensitizes KRAS-mutated NSCLC to WEE1 inhibition through regulation of CHK1 expression [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B06.
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Exploiting ING2 Epigenetic Modulation as a Therapeutic Opportunity for Non-Small Cell Lung Cancer.
Cancers, 2019Co-Authors: Alice Blondel, Rémy Pedeux, Amine Benberghout, Charles RicordelAbstract:Non-small cell lung cancer (NSCLC) has been the leading cause of cancer-related death worldwide, over the last few decades. Survival remains extremely poor in the metastatic setting and, consequently, innovative therapeutic strategies are urgently needed. Inhibitor of Growth Gene 2 (ING2) is a core component of the mSin3A/Histone deacetylases complex (HDAC), which controls the chromatin acetylation status and modulates gene transcription. This gene has been characterized as a tumor suppressor gene and its status in cancer has been scarcely explored. In this review, we focused on ING2 and other mSin3A/HDAC member statuses in NSCLC. Taking advantage of existing public databases and known pharmacological properties of HDAC inhibitors, finally, we proposed a therapeutic model based on an ING2 biomarker-guided strategy.
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Human ex vivo prostate tissue model system identifies ING3 as an oncoprotein
British Journal of Cancer, 2018Co-Authors: Urszula L Mcclurg, Arash Nabbi, Charles Ricordel, Svitlana Korolchuk, Stuart Mccracken, Rakesh Heer, Laura Wilson, Lisa M Butler, Bronwyn Kate Irving-hooper, Rémy PedeuxAbstract:Background: Although the founding members of the INhibitor of Growth (ING) family of histone mark readers, ING1 and ING2, were defined as tumour suppressors in animal models, the role of other ING proteins in cellular proliferation and cancer progression is unclear. Methods: We transduced ex vivo benign prostate hyperplasia tissues with inducible lentiviral particles to express ING proteins. Proliferation was assessed by H3S10^phos immunohistochemistry (IHC). The expression of ING3 was assessed by IHC on a human prostate cancer tissue microarray (TMA). Gene expression was measured by DNA microarray and validated by real-time qPCR. Results: We found that ING3 stimulates cellular proliferation in ex vivo tissues, suggesting that ING3 could be oncogenic. Indeed, ING3 overexpression transformed normal human dermal fibroblasts. We observed elevated levels of ING3 in prostate cancer samples, which correlated with poorer patient survival. Consistent with an oncogenic role, gene-silencing experiments revealed that ING3 is required for the proliferation of breast, ovarian, and prostate cancer cells. Finally, ING3 controls the expression of an intricate network of cell cycle genes by associating with chromatin modifiers and the H3K4^me3 mark at transcriptional start sites. Conclusions: Our investigations create a shift in the prevailing view that ING proteins are tumour suppressors and redefine ING3 as an oncoprotein.
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Human ex vivo prostate tissue model system identifies ING3 as an oncoprotein
British Journal of Cancer, 2018Co-Authors: Urszula L Mcclurg, Arash Nabbi, Charles Ricordel, Svitlana Korolchuk, Stuart Mccracken, Rakesh Heer, Laura Wilson, Lisa M Butler, Bronwyn Kate Irving-hooper, Rémy PedeuxAbstract:Background - Although the founding members of the INhibitor of Growth (ING) family of histone mark readers, ING1 and ING2, were defined as tumour suppressors in animal models, the role of other ING proteins in cellular proliferation and cancer progression is unclear. Methods - We transduced ex vivo benign prostate hyperplasia tissues with inducible lentiviral particles to express ING proteins. Proliferation was assessed by H3S10 immunohistochemistry (IHC). The expression of ING3 was assessed by IHC on a human prostate cancer tissue microarray (TMA). Gene expression was measured by DNA microarray and validated by real-time qPCR. Results - We found that ING3 stimulates cellular proliferation in ex vivo tissues, suggesting that ING3 could be oncogenic. Indeed, ING3 overexpression transformed normal human dermal fibroblasts. We observed elevated levels of ING3 in prostate cancer samples, which correlated with poorer patient survival. Consistent with an oncogenic role, gene-silencing experiments revealed that ING3 is required for the proliferation of breast, ovarian, and prostate cancer cells. Finally, ING3 controls the expression of an intricate network of cell cycle genes by associating with chromatin modifiers and the H3K4 mark at transcriptional start sites. Conclusions - Our investigations create a shift in the prevailing view that ING proteins are tumour suppressors and redefine ING3 as an oncoprotein.
Caren C. Helbing - One of the best experts on this subject based on the ideXlab platform.
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Modulation of Thyroid Hormone-Dependent Gene Expression in Xenopus laevis by INhibitor of Growth (ING) Proteins
2016Co-Authors: Caren C. Helbing, Mary J. Wagner, Katherine Pettem, Jill Johnston, Rachel A. Heimeier, Frank R. Jirik, Yun-bo Shi, Leon W. BrowderAbstract:Background: INhibitor of Growth (ING) proteins belong to a large family of plant homeodomain finger-containing proteins important in epigenetic regulation and carcinogenesis. We have previously shown that ING1 and ING2 expression is regulated by thyroid hormone (TH) during metamorphosis of the Xenopus laevis tadpole. The present study investigates the possibility that ING proteins modulate TH action. Methodology/Principal Findings: Tadpoles expressing a Xenopus ING2 transgene (TransING2) were significantly smaller than tadpoles not expressing the transgene (TransGFP). When exposed to 10 nM 3,5,39-triiodothyronine (T3), premetamorphic TransING2 tadpoles exhibited a greater reduction in tail, head, and brain areas, and a protrusion of the lower jaw than T3-treated TransGFP tadpoles. Quantitative real time polymerase chain reaction (QPCR) demonstrated elevated TH receptor b (TRb) and TH/bZIP transcript levels in TransING2 tadpole tails compared to TransGFP tadpoles while TRa mRNAs were unaffected. In contrast, no difference in TRa, TRb or insulin-like growth factor (IGF2) mRNA abundance was observed in the brain between TransING2 and TransGFP tadpoles. All of these transcripts, except for TRa mRNA in the brain, were inducible by the hormone in both tissues. Oocyte transcription assays indicated that ING proteins enhanced TR-dependent, T3-induced TRb gene promoter activity. Examination of endogenous T3-responsive promoters (TRb and TH/bZIP) in the tail by chromatin immunoprecipitation assays showed that ING proteins were recruited to TRE-containing regions in T3-dependent an
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Modulation of Thyroid Hormone-Dependent Gene Expression in Xenopus laevis by INhibitor of Growth (ING) Proteins
PloS one, 2011Co-Authors: Caren C. Helbing, Mary J. Wagner, Katherine Pettem, Rachel A. Heimeier, Frank R. Jirik, Yun-bo Shi, Jill E. Johnston, Nik Veldhoen, Leon W. BrowderAbstract:Background: INhibitor of Growth (ING) proteins belong to a large family of plant homeodomain finger-containing proteins important in epigenetic regulation and carcinogenesis. We have previously shown that ING1 and ING2 expression is regulated by thyroid hormone (TH) during metamorphosis of the Xenopus laevis tadpole. The present study investigates the possibility that ING proteins modulate TH action. Methodology/Principal Findings: Tadpoles expressing a Xenopus ING2 transgene (TransING2) were significantly smaller than tadpoles not expressing the transgene (TransGFP). When exposed to 10 nM 3,5,39-triiodothyronine (T3), premetamorphic TransING2 tadpoles exhibited a greater reduction in tail, head, and brain areas, and a protrusion of the lower jaw than T3treated TransGFP tadpoles. Quantitative real time polymerase chain reaction (QPCR) demonstrated elevated TH receptor b (TRb) and TH/bZIP transcript levels in TransING2 tadpole tails compared to TransGFP tadpoles while TRa mRNAs were unaffected. In contrast, no difference in TRa, TRb or insulin-like growth factor (IGF2) mRNA abundance was observed in the brain between TransING2 and TransGFP tadpoles. All of these transcripts, except for TRa mRNA in the brain, were inducible by the hormone in both tissues. Oocyte transcription assays indicated that ING proteins enhanced TR-dependent, T3-induced TRb gene promoter activity. Examination of endogenous T3-responsive promoters (TRb and TH/bZIP) in the tail by chromatin immunoprecipitation assays showed that ING proteins were recruited to TRE-containing regions in T3-dependent and independent ways, respectively. Moreover, ING and TR proteins coimmunoprecipitated from tail protein homogenates derived from metamorphic climax animals. Conclusions/Significance: We show for the first time that ING proteins modulate TH-dependent responses, thus revealing a novel role for ING proteins in hormone signaling. This has important implications for understanding hormone influenced disease states and suggests that the induction of ING proteins may facilitate TR function during metamorphosis in a tissuespecific manner.
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Multiple ING1 and ING2 genes in Xenopus laevis and evidence for differential association of thyroid hormone receptors and ING proteins to their promoters.
Biochimica et biophysica acta, 2007Co-Authors: Mary J. Wagner, Caren C. HelbingAbstract:ING (INhibitor of Growth) tumor suppressor proteins are epigenetic factors involved in numerous cellular processes including apoptosis in species ranging from yeast to humans. We recently isolated ING1 and ING2 transcript variants in Xenopus laevis and showed that these transcripts were differentially regulated by thyroid hormone (TH) during postembryonic development. However, no information exists regarding ING gene structure and how it relates to these differential responses to TH. To further investigate the regulation of ING genes by TH, we isolated ING1 and ING2 gene sequences and demonstrated that there are at least duplicate genes for each. The relationship between transcript variants and their responsiveness to TH were examined through promoter sequence and chromatin immunoprecipitation analyses on tail homogenates. Both TH receptors (TRs) differentially associated with ING1 and ING2 promoter regions with increased recruitment in the presence of TH. This occurred irrespective of gene transcript level response to this hormone. However, differential recruitment of RNA polymerase II corresponded well to transcript levels. ING proteins consistently associated with their own gene promoters except in the region generating the TH-inducible xING1b5 transcript. In this case, a substantial recruitment of TRβ in the absence ING proteins occurred. These data establish the TH-dependent recruitment of transcription factors to ING promoter regions and suggest that differential TR recruitment in response to TH may not be a sufficient indicator for modulating the expression of ING in the tail.
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Characterization of Inhibitor of Growth 2 tumor suppressor in Alligator mississippiensis, its conservation in Archosauria, and response to thyroid stimulating hormone
Comparative biochemistry and physiology. Part B Biochemistry & molecular biology, 2006Co-Authors: Caren C. Helbing, Nik Veldhoen, Kate Crump, Carmen M. Bailey, Satomi Kohno, Yue Song, Teresa A. Bryan, Dieldrich S. Bermudez, Juan Ausió, Louis J. GuilletteAbstract:Inhibitor of growth 2 (ING2) belongs to a family of tumor suppressors that are important regulators of a wide range of cellular processes including proliferation, apoptosis, and DNA repair. ING family members are found in yeast, plants, invertebrates and many vertebrate species. However, to date, ING has not been characterized in reptiles. Herein we describe the isolation of expressed ING2 sequence in the American alligator, Alligator mississippiensis, and compare this sequence with that isolated in the chicken. We identify features that are unique to these two representatives of the Archosaurs including conservation of specific amino acid residues and the absence of an adenylate residue in the 5' end of the nucleotide sequence relative to frogs and mammals. The latter feature results in an alteration of the coding potential leading to distinctive N-termini. Injection of juvenile alligators with thyroid stimulating hormone (TSH), which increases endogenous thyroid hormones, results in the modulation of ING2 transcript levels. Quantitative real time polymerase chain reaction analyses revealed a reduction in the steady-state levels of ING2 mRNA in the phallus/cliterophallus, lung, and liver by 48 h after TSH injection. ING2 expression in the thyroid gland, gonad, and heart was unaffected by TSH treatment. These data indicate that control of ING2 expression by the thyroid axis may be conserved among species and is tissue-dependent.
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Multiple variants of the ING1 and ING2 tumor suppressors are differentially expressed and thyroid hormone-responsive in Xenopus laevis
General and comparative endocrinology, 2005Co-Authors: Mary J. Wagner, Caren C. HelbingAbstract:The tumor suppressor candidate, inhibitor of growth (ING) is implicated in the control of apoptosis, cell cycle progression, chemosensitivity, and senescence. There are at least five different genes in mammals, ING1-ING5, and there is limited evidence that multiple transcript variants exist for ING1 that encode proteins with different functions. No variants have yet been reported for other ING genes. Here, we report the isolation of seven Xenopus laevis (x)ING1 and three xING2 transcript variants and give the first evidence for their independent regulation by thyroid hormone (TH). Comparison with mammalian genes reveals conservation in gene structure. xING1 and xING2 transcript variants are differentially expressed in adult tissues with the greatest number of variants expressed at high levels in brain, testis, and eye. During metamorphosis of the tadpole into a frog, the hindlimb, tail, and brain undergo growth, apoptosis, or remodeling, respectively. We show that xING1 and xING2 transcript variants are significantly reduced in the hindlimb while many variants increase in the tail. These transcript variants remain largely unchanged in the brain during this developmental period. By exposing premetamorphic tadpoles to TH, a precocious metamorphosis is induced. We identify specific variants whose steady state levels are significantly affected by TH at 24 and 48h of exposure. Although several of the variants show expression patterns reminiscent of that observed in natural metamorphosis, the results indicate that additional factors may be involved to influence the steady state transcript levels during development.
Michael Klocke - One of the best experts on this subject based on the ideXlab platform.
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the completely annotated genome and comparative genomics of the peptoniphilaceae bacterium str ING2 d1g a novel acidogenic bacterium isolated from a mesophilic biogas reactor
Journal of Biotechnology, 2017Co-Authors: Geizecler Tomazetto, Sarah Hahnke, Irena Maus, Daniel Wibberg, Alfred Pühler, Michael Klocke, Thomas Langer, Jochen Blom, Andreas SchlüterAbstract:Abstract The strictly anaerobic Peptoniphilaceae bacterium str. ING2-D1G (=DSM 28672 = LMG 28300) was isolated from a mesophilic laboratory-scale completely stirred tank biogas reactor (CSTR) continuously co-digesting maize silage, pig and cattle manure. Based on 16S rRNA gene sequence comparison, the closest described relative to this strain is Peptoniphilus obesi ph1 showing 91.2% gene sequence identity. The most closely related species with a validly published name is Peptoniphilus indolicus DSM 20464 T whose 16S rRNA gene sequence is 90.6% similar to the one of strain ING2-D1G. The genome of the novel strain was completely sequenced and manually annotated to reconstruct its metabolic potential regarding anaerobic digestion of biomass. The strain harbors a circular chromosome with a size of 1.6 Mb that contains 1466 coding sequences, 53 tRNA genes and 4 ribosomal RNA ( rrn ) operons. The genome carries a 28,261 bp prophage insertion comprising 47 phage-related coding sequences. Reconstruction of fermentation pathways revealed that strain ING2-D1G encodes all enzymes for hydrogen, lactate and acetate production, corroborating that it is involved in the acido- and acetogenic phase of the biogas process. Comparative genome analyses of Peptoniphilaceae bacterium str. ING2-D1G and its closest relative Peptoniphilus obesi ph1 uncovered rearrangements, deletions and insertions within the chromosomes of both strains substantiating a divergent evolution. In addition to genomic analyses, a physiological and phenotypic characterization of the novel isolate was performed. Grown in Brain Heart Infusion Broth with added yeast extract, cells were spherical to ovoid, catalase- and oxidase-negative and stained Gram-positive. Optimal growth occurred between 35 and 37 °C and at a pH value of 7.6. Fermentation products were acetate, butanoate and carbon dioxide.
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erratum to description of proteiniphilum saccharofermentans sp nov petrimonas mucosa sp nov and fermentimonas caenicola gen nov sp nov isolated from mesophilic laboratory scale biogas reactors and emended description of the genus proteiniphilum
International Journal of Systematic and Evolutionary Microbiology, 2016Co-Authors: Sarah Hahnke, Thomas Langer, Daniela E Koeck, Michael KlockeAbstract:Three novel, facultatively anaerobic bacteria of the family Porphyromonadaceae (phylum Bacteroidetes) were isolated from mesophilic laboratory-scale biogas reactors. The strains were Gram-negative rods. Optimal growth occurred between 35 and 45 °C and at pH 7.1–7.8. The main fermentation products were acetic and propionic acids. The predominant fatty acid in all strains was anteiso-C15 : 0, and the only respiratory quinone detected was menaquinone MK-8. 16S rRNA gene sequence comparison indicated that strains M3/6T and ING2-E5BT were most closely related to the type strain of Proteiniphilum acetatigenes, with sequence similarities of 97.3 and 94.5 %. Strain ING2-E5AT showed the closest affiliation to the type strain of Petrimonas sulfuriphila, with 97 % sequence identity. DNA–DNA hybridization of strain M3/6T and ING2-E5AT with the most closely related type strains showed 43.3–45.6 and 23.8–25.7 % relatedness, respectively, which supports the conclusion that both isolates represent novel species. Phylogenetic analysis and comparison of cellular fatty acid patterns indicated that strain ING2-E5BT cannot be classified as a member of any previously described genus. Therefore, because of the physiological, genotypic and chemotaxonomic differences, it is proposed to designate novel species within the genera Proteiniphilum and Petrimonas, Proteiniphilum saccharofermentans sp. nov. (type strain M3/6T = DSM 28694T = CECT 8610T = LMG 28299T) and Petrimonas mucosa sp. nov. (type strain ING2-E5AT = DSM 28695T = CECT 8611T), and a novel species of a new genus, Fermentimonas caenicola gen. nov., sp. nov. (type strain of Fermentimonas caenicola is ING2-E5BT = DSM 28696T = CECT 8609T = LMG 28429T). In addition, an emended description of the genus Proteiniphilum is provided.
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Description of Proteiniphilum saccharofermentans sp. nov., Petrimonas mucosa sp. nov. and Fermentimonas caenicola gen. nov., sp. nov., isolated from mesophilic laboratory-scale biogas reactors, and emended description of the genus Proteiniphilum.
International journal of systematic and evolutionary microbiology, 2016Co-Authors: Sarah Hahnke, Thomas Langer, Daniela E Koeck, Michael KlockeAbstract:Three novel, facultatively anaerobic bacteria of the family Porphyromonadaceae (phylum Bacteroidetes) were isolated from mesophilic laboratory-scale biogas reactors. The strains were Gram-negative rods. Optimal growth occurred between 35 and 45 °C and at pH 7.1-7.8. The main fermentation products were acetic and propionic acids. The predominant fatty acid in all strains was anteiso-C15 : 0, and the only respiratory quinone detected was menaquinone MK-8. 16S rRNA gene sequence comparison indicated that strains M3/6T and ING2-E5BT were most closely related to the type strain of Proteiniphilum acetatigenes, with sequence similarities of 97.3 and 94.5 %. Strain ING2-E5AT showed the closest affiliation to the type strain of Petrimonas sulfuriphila, with 97 % sequence identity. DNA-DNA hybridization of strain M3/6T and ING2-E5AT with the most closely related type strains showed 43.3-45.6 and 23.8-25.7 % relatedness, respectively, which supports the conclusion that both isolates represent novel species. Phylogenetic analysis and comparison of cellular fatty acid patterns indicated that strain ING2-E5BT cannot be classified as a member of any previously described genus. Therefore, because of the physiological, genotypic and chemotaxonomic differences, it is proposed to designate novel species within the genera Proteiniphilum and Petrimonas, Proteiniphilum saccharofermentans sp. nov. (type strain M3/6T = DSM 28694T = CECT 8610T = LMG 28299T) and Petrimonas mucosa sp. nov. (type strain ING2-E5AT = DSM 28695T = CECT 8611T), and a novel species of a new genus, Fermentimonas caenicola gen. nov., sp. nov. (type strain of Fermentimonas caenicola is ING2-E5BT = DSM 28696T = CECT 8609T = LMG 28429T). In addition, an emended description of the genus Proteiniphilum is provided.
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Complete genome sequence of the novel Porphyromonadaceae bacterium strain ING2-E5B isolated from a mesophilic lab-scale biogas reactor
Journal of biotechnology, 2014Co-Authors: Sarah Hahnke, Irena Maus, Daniel Wibberg, Geizecler Tomazetto, Alfred Pühler, Michael Klocke, Andreas SchlüterAbstract:In this study, the whole genome sequence of the mesophilic, anaerobic Porphyromonadaceae bacterium strain ING2-E5B (LMG 28429, DSM 28696) is reported. The new isolate belongs to the phylum Bacteroidetes and was obtained from a biogas-producing lab-scale completely stirred tank reactor (CSTR) optimized for anaerobic digestion of maize silage in co-fermentation with pig and cattle manure. The genome of strain ING2-E5B contains numerous genes encoding proteins and enzymes involved in the degradation of complex carbohydrates and proteinaceous compounds. Moreover, it possesses genes catalyzing the production of volatile fatty acids. Hence, this bacterium was predicted to be involved in hydrolysis and acidogenesis during anaerobic digestion and biomethanation.
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Complete genome sequence of Peptoniphilus sp. strain ING2-D1G isolated from a mesophilic lab-scale completely stirred tank reactor utilizing maize silage in co-digestion with pig and cattle manure for biomethanation
Journal of Biotechnology, 2014Co-Authors: Geizecler Tomazetto, Sarah Hahnke, Irena Maus, Daniel Wibberg, Alfred Pühler, Andreas Schlüter, Michael KlockeAbstract:The bacterium Peptoniphilus sp. strain ING2-D1G (DSM 28672), a mesophilic and obligate anaerobic bacterium belonging to the order Clostridiales was isolated from a biogas-producing lab-scale completely stirred tank reactor (CSTR) optimized for anaerobic digestion of maize silage in co-fermentation with pig and cattle manure. In this study, the whole genome sequence of Peptoniphilus sp. strain ING2-D1G, a new isolate potentially involved in protein breakdown and acidogenesis during biomass degradation, is reported. The chromosome of this strain is 1.6Mb in size and encodes genes predicted to be involved in the production of acetate, lactate and butyrate specifying the acidogenic metabolism of the isolate.
Curtis C. Harris - One of the best experts on this subject based on the ideXlab platform.
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ING Proteins as Potential Anticancer Drug Targets
Current drug targets, 2009Co-Authors: Motoko Unoki, Kensuke Kumamoto, Curtis C. HarrisAbstract:Recent emerging evidence suggests that ING family proteins play roles in carcinogenesis both as oncogenes and tumor suppressor genes depending on the family members and on cell status. Previous results from non-physiologic overexpression experiments showed that all five family members induce apoptosis or cell cycle arrest, thus it had been thought until very recently that all of the family members function as tumor suppressor genes. Therefore restoration of ING family proteins in cancer cells has been proposed as a treatment for cancers. However, ING2 knockdown experiments showed unexpected results: ING2 knockdown led to senescence in normal human fibroblast cells and suppressed cancer cell growth. ING2 is also overexpressed in colorectal cancer, and promotes cancer cell invasion through an MMP13 dependent pathway. Additionally, it was reported that ING2 has two isoforms, ING2a and ING2b. Although expression of ING2a predominates compared with ING2b, both isoforms confer resistance against cell cycle arrest or apoptosis to cancer cells, thus knockdown of both isoforms is critical to remove this resistance. Taken together, these results suggest that ING2 can function as an oncogene in some specific types of cancer cells, indicating restoration of this gene in cancer cells could cause cancer progression. Because knockdown of ING2 suppresses cancer cell invasion and induces apoptosis or cell cycle arrest, ING2 may be an anticancer drug target. In this brief review, we discuss possible clinical applications of ING2 with the latest knowledge of molecular targeted therapies.
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Reviewing the current classification of inhibitor of growth family proteins
Cancer science, 2009Co-Authors: Motoko Unoki, Kensuke Kumamoto, Seiichi Takenoshita, Curtis C. HarrisAbstract:Inhibitor of growth (ING) family proteins have been defined as candidate tumor suppressors for more than a decade. Recent emerging results using siRNA and knockout mice are expanding the previous understanding of this protein family. The results of ING1 knockout mouse experiments revealed that ING1 has a protective effect on apoptosis. Our recent results showed that ING2 is overexpressed in colorectal cancer, and induces colon cancer cell invasion through an MMP13-dependent pathway. Knockdown of ING2 by siRNA induces premature senescence in normal human fibroblast cells, and apoptosis or cell cycle arrest in various adherent cancer cells. Taken together, these results suggest that ING2 may also have roles in cancer progression and/or malignant transformation under some conditions. Additionally, knockdown of ING4 and ING5 by siRNA shows an inhibitory effect on the transition from G2/M to G1 phase and DNA replication, respectively, suggesting that these proteins may play roles during cell proliferation in some context. ING family proteins may play dual roles, similar to transforming growth factor-β, which has tumor suppressor-like functions in normal epithelium and also oncogenic functions in invasive metastatic cancers. In the present article, we briefly review ING history and propose a possible interpretation of discrepancies between past and recent data. (Cancer Sci 2009; 100: 1173–1179)
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A novel ING2 isoform, ING2b, synergizes with ING2a to prevent cell cycle arrest and apoptosis
FEBS letters, 2008Co-Authors: Motoko Unoki, Kensuke Kumamoto, Jiang Cheng Shen, Ana I. Robles, Zhi-ming Zheng, Curtis C. HarrisAbstract:We identified a novel inhibitor of growth family member 2 (ING2) isoform, ING2b, which shares exon 2 with ING2a, but lacks the N-terminal p53 binding region. Contrary to ING2a, ING2b's promoter has no p53 binding sites. Consistently, activation of p53 led to suppression of ING2a, leaving ING2b unaffected. Through isoform-specific targeting, we showed that ING2a knockdown suppressed cell growth only in the presence of p53, ING2b knockdown had no effect on cell growth, and knockdown of both induced cell cycle arrest and apoptosis independently of p53. ING2a and ING2b have compensatory roles that protect cells from cell cycle arrest and apoptosis and may be involved in development of chemotherapeutic resistance.
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Identification of a novel spice variant of ING2, ING2b
Cancer Research, 2008Co-Authors: Motoko Unoki, Graham Smyth, Jiang Cheng Shen, Zheng Zhi-ming, Curtis C. HarrisAbstract:2794 Previously, the ING2/ING1L gene, ING2a, was identified as a candidate tumor suppressor gene encoding a protein that modulates p53. Here we identified a novel splice variant of the gene that is transcribed from a different promoter that is located in intron 1 of the ING2a gene. Because both ING2a and ING2b are composed of two exons and share exon 2, only the N-terminal region encoded by a different exon 1 is different. Although we observed that both ING2a and ING2b inhibited cell growth by colony formation assay, only ING2a induced p21 promoter activity, and ING2b competed with this effect, suggesting that ING2b may have a dominant negative effect on ING2a. We also found that ING2a binds to mSin3A more strongly than ING2b; on the other hand ING2b binds to Brg1 more strongly than ING2a, suggesting that the major roles of ING2a and ING2b may be slightly different. Because the effect of ING2a and ING2b on cell growth is almost the same, the splice variants might compensate or promote each other’s function.
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nutlin 3a activates p53 to both down regulate inhibitor of growth 2 and up regulate mir 34a mir 34b and mir 34c expression and induce senescence
Cancer Research, 2008Co-Authors: Kensuke Kumamoto, Seiichi Takenoshita, Makoto Nagashima, Kaori Fujita, Izumi Horikawa, Taro Yamashita, Elisa A Spillare, Ettore Appella, Jun Yokota, Curtis C. HarrisAbstract:Nutlin-3, an MDM2 inhibitor, activates p53, resulting in several types of cancer cells undergoing apoptosis. Although p53 is mutated or deleted in ∼50% of all cancers, p53 is still functionally active in the other 50%. Consequently, nutlin-3 and similar drugs could be candidates for neoadjuvant therapy in cancers with a functional p53. Cellular senescence is also a phenotype induced by p53 activation and plays a critical role in protecting against tumor development. In this report, we found that nutlin-3a can induce senescence in normal human fibroblasts. Nutlin-3a activated and repressed a large number of p53-dependent genes, including those encoding microRNAs. mir-34a, mir-34b , and mir-34c , which have recently been shown to be downstream effectors of p53-mediated senescence, were up-regulated, and inhibitor of growth 2 ( ING2 ) expression was suppressed by nutlin-3a treatment. Two candidates for a p53-DNA binding consensus sequence were found in the ING2 promoter regulatory region; thus, we performed chromatin immunoprecipitation and electrophoretic mobility shift assays and confirmed p53 binding directly to those sites. In addition, the luciferase activity of a construct containing the ING2 regulatory region was repressed after p53 activation. Antisense knockdown of ING2 induces p53-independent senescence, whereas overexpression of ING2 induces p53-dependent senescence. Taken together, we conclude that nutlin-3a induces senescence through p53 activation in normal human fibroblasts, and p53-mediated mir34a, mir34b , and mir34c up-regulation and ING2 down-regulation may be involved in the senescence pathway. [Cancer Res 2008;68(9):3193–203]