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Aberrantly Expressed Genes and miRNAs in Slow Transit Constipation Based on RNA-Seq Analysis. BIOMED RESEARCH INTERNATIONAL 2018; 2018:2617432. [PMID: 30186855 PMCID: PMC6112260 DOI: 10.1155/2018/2617432] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/12/2018] [Revised: 06/26/2018] [Accepted: 07/18/2018] [Indexed: 02/06/2023]
Abstract
Background This study aims to identify the key genes and miRNAs in slow transit constipation (STC). Methods MRNA and miRNA expression profiling were obtained. Differentially expressed genes (DEGs) and miRNAs were identified followed by the regulatory network construction. Functional annotation analysis and protein-protein interaction (PPI) network were conducted. The electronic validation was performed. Results Hsa-miR-2116-3p, hsa-miR-3622a-5p, hsa-miR-424-5p, and hsa-miR-1273-3p covered most DEGs. HLA-DRB1, HLA-DRB5, C3, and ICAM were significantly involved in staphylococcus aureus infection. The PPI network generated several hub proteins including ZBTB16, FBN1, CCNF, and CDK1. Electronic validation of HLA-DRB1, PTGDR, MKI67, BIRC5, CCNF, and CDK1 was consistent with the RNA-sequencing analysis. Conclusion Our study might be helpful in understanding the pathology of STC at the molecular level.
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Kumar R, Yu F, Zhen YH, Li B, Wang J, Yang Y, Ge HX, Hu PS, Xiu J. PD-1 blockade restores impaired function of ex vivo expanded CD8 + T cells and enhances apoptosis in mismatch repair deficient EpCAM +PD-L1 + cancer cells. Onco Targets Ther 2017; 10:3453-3465. [PMID: 28761354 PMCID: PMC5516878 DOI: 10.2147/ott.s130131] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
BACKGROUND Adoptive T cell therapy has been proven to be a promising modality for the treatment of cancer patients in recent years. However, the increased expression of inhibitory receptors could negatively regulate the function and persistence of transferred T cells which mediates T cell anergy, exhaustion, and tumor regression. In this study, we investigated increased cytotoxic activity after the blockade of PD-1 for effective immunotherapy. METHODS The cytotoxic function of expanded CD8+ CTLs and interactions with tumor cells investigated after blocking of PD-1. Ex vivo expanded CD8+ CTLs were co-cultured with mismatch repair (MMR) stable or deficient (high microsatellite instability [MSI-H]) EpCAM+ tumor cells. The levels of IFN-γ and GrB were detected by enzyme-linked immunosorbent spot assay. Flow cytometry and confocal microscopy were used to assess CD107a mobilization, cytosolic uptake, and cell migration. RESULTS A dramatic increase in PD-1 expression on the surface of CD8+ CTLs during ex vivo expansion was observed. PD-1 level was downregulated by approximately 40% after incubation of the CD8+ CTLs with monoclonal antibody which enhanced the secretion of IFN-γ, GrB, and CD107a. Additionally, PD-1 blockade enhanced cell migration and cytosolic exchange between CD8+ CTLs and MMR deficient (MSI-H) EpCAM+PD-L1+ tumor cells. CONCLUSION The blockade of PD-1 enhanced the cytotoxic efficacy of CD8+ CTLs toward MMR deficient tumor cells. In conclusion, we propose that blocking of PD-1 during the expansion of CD8+ CTLs may improve the clinical efficacy of cell-based adoptive immunotherapy.
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Affiliation(s)
- Rajeev Kumar
- Clinical Research Centre, The Affiliated Hospital of Guizhou Medical University, Guiyang, People's Republic of China.,Cancer Immunology and Immunotherapy Centre, The Affiliated Hospital of Guizhou Medical University, Guiyang, People's Republic of China
| | - Fang Yu
- Clinical Research Centre, The Affiliated Hospital of Guizhou Medical University, Guiyang, People's Republic of China
| | - Yuan-Huan Zhen
- Department of Colorectal Surgery, The Affiliated Hospital of Guizhou Medical University, Guiyang, People's Republic of China
| | - Bo Li
- Cancer Immunology and Immunotherapy Centre, The Affiliated Hospital of Guizhou Medical University, Guiyang, People's Republic of China
| | - Jun Wang
- Clinical Research Centre, The Affiliated Hospital of Guizhou Medical University, Guiyang, People's Republic of China
| | - Yuan Yang
- Clinical Research Centre, The Affiliated Hospital of Guizhou Medical University, Guiyang, People's Republic of China.,Cancer Immunology and Immunotherapy Centre, The Affiliated Hospital of Guizhou Medical University, Guiyang, People's Republic of China
| | - Hui-Xin Ge
- Department of Surgery, The Affiliated Cancer Hospital of Guizhou Medical University, Guiyang, People's Republic of China
| | - Ping-Sheng Hu
- Clinical Research Centre, The Affiliated Hospital of Guizhou Medical University, Guiyang, People's Republic of China.,Cancer Immunology and Immunotherapy Centre, The Affiliated Hospital of Guizhou Medical University, Guiyang, People's Republic of China
| | - Jin Xiu
- Clinical Research Centre, The Affiliated Hospital of Guizhou Medical University, Guiyang, People's Republic of China.,Cancer Immunology and Immunotherapy Centre, The Affiliated Hospital of Guizhou Medical University, Guiyang, People's Republic of China
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Ma Y, Chen Y, Petersen I. Expression and promoter DNA methylation of MLH1 in colorectal cancer and lung cancer. Pathol Res Pract 2017; 213:333-338. [PMID: 28214209 DOI: 10.1016/j.prp.2017.01.014] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/02/2016] [Revised: 01/16/2017] [Accepted: 01/17/2017] [Indexed: 02/06/2023]
Abstract
AIMS Aberrant DNA methylation is a common molecular feature in human cancer. The aims of this study were to analyze the methylation status of MLH1, one of the DNA mismatch repair (MMR) genes, in human colorectal and lung cancer and to evaluate its clinical relevance. METHODS The expression of MLH1 was analyzed in 8 colorectal cancer (CRC) and 8 lung cancer cell lines by real-time RT-PCR and western blotting. The MLH1 protein expression was evaluated by immunohistochemistry on tissue microarrays including 121 primary CRC and 90 lung cancer patient samples. In cancer cell lines, the methylation status of MLH1 promoter and exon 2 was investigated by bisulfite sequencing (BS). Methylation-specific-PCR (MSP) was used to evaluate methylation status of MLH1. RESULTS The expression of MLH1 mRNA was detected in 8 CRC cell lines as well as normal colonic fibroblast cells CCD-33Co. At protein levels, MLH1 was lost in one CRC cell line HCT-116 and normal cells CCD-33Co. No methylation was found in the promoter and exon 2 of MLH1 in CRC cell lines. MLH1 was expressed in 8 lung cancer cell lines at both mRNA and protein levels. Compared to cancer cells, normal bronchial epithelial cells (HBEC) had lower expression of MLH1 protein. In primary CRC, 54.5% of cases exhibited positive staining, while 47.8% of lung tumors were positive for MLH1 protein. MSP analysis showed that 58 out of 92 (63.0%) CRC and 41 out of 73 (56.2%) lung cancer exhibited MLH1 methylation. In CRC, the MLH1 methylation was significantly associated with tumor invasion in veins (P=0.012). However, no significant links were found between MLH1 expression and promoter methylation in both tumor entities. CONCLUSIONS MLH1 methylation is a frequent molecular event in CRC and lung cancer patients. In CRC, methylation of MLH1 could be linked to vascular invasiveness.
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Affiliation(s)
- Yunxia Ma
- Institute of Pathology, University Hospital Jena, Friedrich Schiller University Jena, Ziegelmuehlenweg 1, 07740 Jena, Germany
| | - Yuan Chen
- Institute of Pathology, University Hospital Jena, Friedrich Schiller University Jena, Ziegelmuehlenweg 1, 07740 Jena, Germany
| | - Iver Petersen
- Institute of Pathology, University Hospital Jena, Friedrich Schiller University Jena, Ziegelmuehlenweg 1, 07740 Jena, Germany.
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Torchy MP, Hamiche A, Klaholz BP. Structure and function insights into the NuRD chromatin remodeling complex. Cell Mol Life Sci 2015; 72:2491-507. [PMID: 25796366 PMCID: PMC11114056 DOI: 10.1007/s00018-015-1880-8] [Citation(s) in RCA: 149] [Impact Index Per Article: 14.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2014] [Revised: 03/02/2015] [Accepted: 03/04/2015] [Indexed: 01/09/2023]
Abstract
Transcription regulation through chromatin compaction and decompaction is regulated through various chromatin-remodeling complexes such as nucleosome remodeling and histone deacetylation (NuRD) complex. NuRD is a 1 MDa multi-subunit protein complex which comprises many different subunits, among which histone deacetylases HDAC1/2, ATP-dependent remodeling enzymes CHD3/4, histone chaperones RbAp46/48, CpG-binding proteins MBD2/3, the GATAD2a (p66α) and/or GATAD2b (p66β) and specific DNA-binding proteins MTA1/2/3. Here, we review the currently known crystal and NMR structures of these subunits, the functional data and their relevance for biomedical research considering the implication of NuRD subunits in cancer and various other diseases. The complexity of this macromolecular assembly, and its poorly understood mode of interaction with the nucleosome, the repeating unit of chromatin, illustrate that this complex is a major challenge for structure-function relationship studies which will be tackled best by an integrated biology approach.
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Affiliation(s)
- Morgan P. Torchy
- Department of Integrated Structural Biology, Centre for Integrative Biology (CBI), Institute of Genetics and of Molecular and Cellular Biology (IGBMC), 1 rue Laurent Fries, Illkirch, France
- Centre National de la Recherche Scientifique (CNRS) UMR 7104, Illkirch, France
- Institut National de la Santé et de la Recherche Médicale (INSERM) U964, Illkirch, France
- Université de Strasbourg, Strasbourg, France
| | - Ali Hamiche
- Department of Integrated Structural Biology, Centre for Integrative Biology (CBI), Institute of Genetics and of Molecular and Cellular Biology (IGBMC), 1 rue Laurent Fries, Illkirch, France
- Centre National de la Recherche Scientifique (CNRS) UMR 7104, Illkirch, France
- Institut National de la Santé et de la Recherche Médicale (INSERM) U964, Illkirch, France
- Université de Strasbourg, Strasbourg, France
| | - Bruno P. Klaholz
- Department of Integrated Structural Biology, Centre for Integrative Biology (CBI), Institute of Genetics and of Molecular and Cellular Biology (IGBMC), 1 rue Laurent Fries, Illkirch, France
- Centre National de la Recherche Scientifique (CNRS) UMR 7104, Illkirch, France
- Institut National de la Santé et de la Recherche Médicale (INSERM) U964, Illkirch, France
- Université de Strasbourg, Strasbourg, France
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Dominguez-Valentin M, Therkildsen C, Da Silva S, Nilbert M. Familial colorectal cancer type X: genetic profiles and phenotypic features. Mod Pathol 2015; 28:30-6. [PMID: 24743215 DOI: 10.1038/modpathol.2014.49] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2013] [Revised: 01/27/2014] [Accepted: 01/27/2014] [Indexed: 12/19/2022]
Abstract
Heredity is a major cause of colorectal cancer, but although several rare high-risk syndromes have been linked to disease-predisposing mutations, the genetic mechanisms are undetermined in the majority of families suspected of hereditary cancer. We review the clinical presentation, histopathologic features, and the genetic and epigenetic profiles of the familial colorectal cancer type X (FCCTX) syndrome with the aim to delineate tumor characteristics that may contribute to refined diagnostics and optimized tumor prevention.
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Affiliation(s)
- Mev Dominguez-Valentin
- 1] HNPCC-Register, Clinical Research Centre, Hvidovre Hospital, Copenhagen University, Copenhagen, Denmark [2] Institute of Clinical Sciences, Department of Oncology, Lund University, Lund, Sweden
| | - Christina Therkildsen
- HNPCC-Register, Clinical Research Centre, Hvidovre Hospital, Copenhagen University, Copenhagen, Denmark
| | - Sabrina Da Silva
- Lady Davis Institute for Medical Research and Segal Cancer Centre, Sir Mortimer B. Davis-Jewish General Hospital, Department of Otolaryngology-Head and Neck Surgery, McGill University, Montreal, QC, Canada
| | - Mef Nilbert
- 1] HNPCC-Register, Clinical Research Centre, Hvidovre Hospital, Copenhagen University, Copenhagen, Denmark [2] Institute of Clinical Sciences, Department of Oncology, Lund University, Lund, Sweden
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Li CF, Huang HY, Wu WR, Liang SS, Chen YL, Chen LR, Peng YT, Lee HC, Shiue YL. Clinical aggressiveness of myxofibrosarcomas associates with down-regulation of p12CDK2AP1: prognostic implication of a putative tumor suppressor that induces cell cycle arrest and apoptosis via mitochondrial pathway. Ann Surg Oncol 2014; 21 Suppl 4:S711-S720. [PMID: 24889487 DOI: 10.1245/s10434-014-3825-0] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2014] [Indexed: 01/06/2023]
Abstract
BACKGROUND Attenuated endogenous protein levels of cyclin-dependent kinase 2 associated protein 1 (p12(CDK2AP1)) and its active homodimer p25(CDK2AP1) were found in myxofibrosarcoma-derived cell lines. Clinical and biological significances of this putative tumor suppressor in myxofibrosarcoma were studied. METHODS Plasmids carrying the CDK2AP1 gene and small hairpin RNA interference (shRNAi) targeting CDK2AP1 were transfected into NMFH-1 and/or OH931 cells to evaluate the effects on the CDK2, active caspase 3 (CASP3), cleaved-CASP8 and -CASP9 levels, cell cycle regulation, and/or apoptotic responses. Immunostaining of p12(CDK2AP1) was interpretable in 102 primary myxofibrosarcomas and correlated with clinicopathological variables, CDK2, Ki-67 and active CASP3 protein levels, and disease-specific survival. RESULTS Exogenous expression of p12(CDK2AP1) in NMFH-1 and OH931 cells significantly induced G0/G1 cell cycle arrest and down-regulated CDK2 protein level. In NMFH-1 cells, these aspects were reversed by shRNAi targeting CDK2AP1 gene. Increased active CASP3 and cleaved-CASP9, but not -CASP8, were detected after CDK2AP1 overexpression, suggesting the cellular apoptosis were induced through the mitochondrial pathway. Immunostains of p12(CDK2AP1) were aberrantly decreased in 56.9 % of cases; positively and negatively correlated with protein levels of CDK2 (p = 0.023), Ki-67 (p = 0.001) and active CASP3 (p < 0.001), respectively. Following by high histological grades, p12(CDK2AP1) down-regulation was predictive of worse disease-specific survival in univariate (p = 0.003) and multivariate (p = 0.004) analyses. CONCLUSIONS Through down-regulation of CDK2, high p12(CDK2AP1) level induced cell cycle arrest and the mitochondrial-dependent apoptotic pathway. Low p12(CDK2AP1) level represents a poor prognostic factor in patients with myxofibrosarcoma.
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Affiliation(s)
- Chien-Feng Li
- Department of Pathology, Chi Mei Medical Center, Tainan, Taiwan
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Dominguez-Valentin M, Therkildsen C, Veerla S, Jönsson M, Bernstein I, Borg A, Nilbert M. Distinct gene expression signatures in lynch syndrome and familial colorectal cancer type x. PLoS One 2013; 8:e71755. [PMID: 23951239 PMCID: PMC3741139 DOI: 10.1371/journal.pone.0071755] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2013] [Accepted: 07/02/2013] [Indexed: 02/01/2023] Open
Abstract
Introduction Heredity is estimated to cause at least 20% of colorectal cancer. The hereditary nonpolyposis colorectal cancer subset is divided into Lynch syndrome and familial colorectal cancer type X (FCCTX) based on presence of mismatch repair (MMR) gene defects. Purpose We addressed the gene expression signatures in colorectal cancer linked to Lynch syndrome and FCCTX with the aim to identify candidate genes and to map signaling pathways relevant in hereditary colorectal carcinogenesis. Experimental design The 18 k whole-genome c-DNA-mediated annealing, selection, extension, and ligation (WG-DASL) assay was applied to 123 colorectal cancers, including 39 Lynch syndrome tumors and 37 FCCTX tumors. Target genes were technically validated using real-time quantitative RT-PCR (qRT-PCR) and the expression signature was validated in independent datasets. Results Colorectal cancers linked to Lynch syndrome and FCCTX showed distinct gene expression profiles, which by significance analysis of microarrays (SAM) differed by 2188 genes. Functional pathways involved were related to G-protein coupled receptor signaling, oxidative phosphorylation, and cell cycle function and mitosis. qRT-PCR verified altered expression of the selected genes NDUFA9, AXIN2, MYC, DNA2 and H2AFZ. Application of the 2188-gene signature to independent datasets showed strong correlation to MMR status. Conclusion Distinct genetic profiles and deregulation of different canonical pathways apply to Lynch syndrome and FCCTX and key targets herein may be relevant to pursue for refined diagnostic and therapeutic strategies in hereditary colorectal cancer.
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Sun M, Zheng J, Xue H, Jiang Y, Li C, Li J, Jin W, Shen M, Yang X, Ni Q. Silencing P12CDK²AP¹ with a lentivirus promotes HaCaT cell proliferation. Mol Med Rep 2012; 7:471-5. [PMID: 23229879 DOI: 10.3892/mmr.2012.1205] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2012] [Accepted: 11/15/2012] [Indexed: 11/06/2022] Open
Abstract
The tumor suppressor P12CDK2AP1 negatively regulates cyclin-dependent kinase 2 (CDK2) activities and suppresses DNA replication. Notably, P12CDK2AP1 is known to be downregulated in head and neck squamous cell carcinomas (HNSCCs). Silencing of specific gene expression by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) using expression vectors and retroviruses has become a powerful tool for the genetic analysis of mammalian cells. In the present study, we utilized lentivirus‑mediated shRNA for functional gene knockdown in normal human skin keratinocytes (HaCaT) cells in order to assess the potential role of P12CDK2AP1 in HNSCCs. Lentivirus‑mediated RNA interference (RNAi) effectively reduced endogenous P12CDK2AP1 expression in HaCaT cells and significantly promoted HaCaT cell proliferation in vitro. Lentiviral vectors have the ability to infect dividing and non-dividing cells as well as to achieve long‑term multilineage gene expression. Thus, additional studies are needed to investigate the use of such vectors as a therapeutic tool for the delivery of siRNAs.
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Affiliation(s)
- Moyi Sun
- Department of Oral and Maxillofacial Surgery, School of Stomatology, Fourth Military Medical University, Xi'an, Shaanxi 710032, P.R. China.
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Zhou W, Guan X, Wang L, Liao Y, Huang J. p12(CDK2-AP1) inhibits breast cancer cell proliferation and in vivo tumor growth. J Cancer Res Clin Oncol 2012; 138:2085-93. [PMID: 22828875 DOI: 10.1007/s00432-012-1286-z] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2012] [Accepted: 07/02/2012] [Indexed: 12/11/2022]
Abstract
PURPOSE p12(CDK2-AP1) is a growth suppressor that negatively regulates cyclin-dependent kinase 2 (CDK2) activities and shows to interfere in DNA replication. Here, we aim to elucidate the role of p12(CDK2-AP1) in breast cancer progression. METHODS Expression of p12(CDK2-AP1) protein was examined in 60 pairs of breast cancer specimens and adjacent non-tumor tissues using immunohistochemistry assay. Loss-of-function and gain-of-function analysis was performed on MCF-7 and MDA-MB-231 breast cancer cells. Routine assays including MTT, colony formation, flow cytometry, and tumorigenesis in nude mice were performed and cell cycle regulators were analyzed. RESULTS p12(CDK2-AP1) was found to be significantly downregulated in 60 breast cancer tissues compared to corresponding non-tumorous tissues. The proliferation and colony formation ability was inhibited in cells that transduced with p12(CDK2-AP1) over-expression lentivirus, but enhanced in cells that transduced with p12(CDK2-AP1) RNAi lentivirus. p12(CDK2-AP1) over-expression led to G0/G1 phase arrest in the cell cycle and caused expression changes of cell cycle-related genes (CDK2, CDK4, p16(Ink4A), p21(Cip1/Waf1)). Furthermore, p12(CDK2-AP1) over-expression inhibited in vivo tumor growth in immunodeficiency mice, supporting an inhibitory role for p12(CDK2-AP1) in breast cancer development. CONCLUSIONS As a cell cycle regulator, p12(CDK2-AP1) is involved in the development of breast cancer and maybe a potential therapeutic candidate to suppress tumorigenicity in breast cancer.
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Affiliation(s)
- Weibing Zhou
- Department of Oncology, Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha, 410008, China
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Wu LC, Chen YL, Wu WR, Li CF, Huang HY, Lee SW, Chang SL, Lin CY, Chen YH, Hsu HP, Lu PJ, Shiue YL. Expression of cyclin-dependent kinase 2-associated protein 1 confers an independent prognosticator in nasopharyngeal carcinoma: a cohort study. J Clin Pathol 2012; 65:795-801. [PMID: 22791769 DOI: 10.1136/jclinpath-2012-200893] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/04/2022]
Abstract
BACKGROUND AND AIM Low expression of cyclin-dependent kinase 2-associated protein (CDK2AP1) is associated with tumour progression in oral and oesophageal carcinomas, but is not well studied in patients with head and neck cancer and nasopharyngeal carcinoma (NPC). METHODS A rabbit anti-human CDK2AP1 polyclonal antibody was prepared. Immunoblotting of CDK2AP1 was examined in three cell lines and immunoexpression was retrospectively assessed in biopsies of 124 consecutive NPC patients without initial distant metastasis and treated with consistent guidelines. RESULTS Higher CDK2AP1 expression level was identified in dysplastic oral keratinocytes, compared with two NPC-derived HONE-1 and TW01 cell lines. Low expression of CDK2AP1 (50.8%) was correlated with advanced nodal status (p=0.002) and American Joint Committee on Cancer (AJCC) stage (p=0.004). In multivariate analyses, low CDK2AP1 expression emerged as an independent prognosticator for worse disease-specific survival (DSS; p=0.037) and local recurrence-free survival (LRFS; p=0.042), along with AJCC stage III-IV (p=0.034, DSS; p=0.029, LRFS). CONCLUSIONS Low CDK2AP1 expression is common and associated with adverse prognosticators, conferring tumour aggressiveness through cycle cycle, cell growth or apoptosis cellular processes.
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Affiliation(s)
- Li-Ching Wu
- Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan
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The insulin receptor substrate 1 (IRS1) in intestinal epithelial differentiation and in colorectal cancer. PLoS One 2012; 7:e36190. [PMID: 22558377 PMCID: PMC3338610 DOI: 10.1371/journal.pone.0036190] [Citation(s) in RCA: 52] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2010] [Accepted: 04/01/2012] [Indexed: 12/13/2022] Open
Abstract
Colorectal cancer (CRC) is associated with lifestyle factors that affect insulin/IGF signaling, of which the insulin receptor substrate 1 (IRS1) is a key transducer. We investigated expression, localization and pathologic correlations of IRS1 in cancer-uninvolved colonic epithelium, primary CRCs with paired liver metastases and in vitro polarizing Caco2 and HT29 cells. IRS1 mRNA and protein resulted higher, relative to paired mucosa, in adenomas of familial adenomatous polyposis patients and in CRCs that overexpressed c-MYC, ß-catenin, InsRß, and IGF1R. Analysis of IRS1 immunostaining in 24 cases of primary CRC with paired colonic epithelium and hepatic metastasis showed that staining intensity was significantly higher in metastases relative to both primary CRC (P<0.01) and colonic epithelium (P<0.01). Primary and metastatic CRCs, compared to colonic epithelium, contained significantly higher numbers of IRS1-positive cells (P = 0.013 and P = 0.014, respectively). Pathologic correlations in 163 primary CRCs revealed that diffuse IRS1 staining was associated with tumors combining differentiated phenotype and aggressive markers (high Ki67, p53, and ß-catenin). In Caco 2 IRS1 and InsR were maximally expressed after polarization, while IGF1R was highest in pre-polarized cells. No nuclear IRS1 was detected, while, with polarization, phosphorylated IRS1 (pIRS1) shifted from the lateral to the apical plasma membrane and was expressed in surface cells only. In HT29, that carry mutations constitutively activating survival signaling, IRS1 and IGF1R decreased with polarization, while pIRS1 localized in nuclear spots throughout the course. Overall, these data provide evidence that IRS1 is modulated according to CRC differentiation, and support a role of IRS1 in CRC progression and liver metastatization.
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Ertekin A, Aramini JM, Rossi P, Leonard PG, Janjua H, Xiao R, Maglaqui M, Lee HW, Prestegard JH, Montelione GT. Human cyclin-dependent kinase 2-associated protein 1 (CDK2AP1) is dimeric in its disulfide-reduced state, with natively disordered N-terminal region. J Biol Chem 2012; 287:16541-9. [PMID: 22427660 DOI: 10.1074/jbc.m112.343863] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
CDK2AP1 (cyclin-dependent kinase 2-associated protein 1), corresponding to the gene doc-1 (deleted in oral cancer 1), is a tumor suppressor protein. The doc-1 gene is absent or down-regulated in hamster oral cancer cells and in many other cancer cell types. The ubiquitously expressed CDK2AP1 protein is the only known specific inhibitor of CDK2, making it an important component of cell cycle regulation during G(1)-to-S phase transition. Here, we report the solution structure of CDK2AP1 by combined methods of solution state NMR and amide hydrogen/deuterium exchange measurements with mass spectrometry. The homodimeric structure of CDK2AP1 includes an intrinsically disordered 60-residue N-terminal region and a four-helix bundle dimeric structure with reduced Cys-105 in the C-terminal region. The Cys-105 residues are, however, poised for disulfide bond formation. CDK2AP1 is phosphorylated at a conserved Ser-46 site in the N-terminal "intrinsically disordered" region by IκB kinase ε.
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Affiliation(s)
- Asli Ertekin
- Center for Advanced Biotechnology and Medicine and Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, USA
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Wong DTW, Kim JJ, Khalid O, Sun HH, Kim Y. Double edge: CDK2AP1 in cell-cycle regulation and epigenetic regulation. J Dent Res 2011; 91:235-41. [PMID: 21865592 DOI: 10.1177/0022034511420723] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
Cancer research has been devoted toward an understanding of the molecular regulation and functional significance of cell-cycle regulators in the pathogenesis and development of cancers. Cyclin-dependent Kinase 2-associated Protein 1 (CDK2AP1) is one such cell-cycle regulator, originally identified as a growth suppressor and a prognostic marker for human oral/head and neck cancers. Functional importance and the molecular mechanism of CDK2AP1-mediated cell-cycle regulation have been documented over the years. Recent progress has shown that CDK2AP1 is a competency factor in embryonic stem cell differentiation. Deletion of CDK2AP1 leads to early embryonic lethality, potentially through altered differentiation capability of embryonic stem cells. More intriguingly, CDK2AP1 exerts its effect on stem cell maintenance/differentiation through epigenetic regulation. Cancer cells and stem cells share common cellular characteristics, most prominently in maintaining high proliferative potential through an unconventional cell-cycle regulatory mechanism. Cross-talk between cellular processes and molecular signaling pathways is frequent in any biological system. Currently, it remains largely elusive how cell-cycle regulation is mechanistically linked to epigenetic control. Understanding the molecular mechanism underlying CDK2AP1-mediated cell-cycle regulation and epigenetic control will set an example for establishing a novel and effective molecular link between these two important regulatory mechanisms.
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Affiliation(s)
- D T W Wong
- UCLA School of Dentistry and Dental Research Institute, 10833 Le Conte Ave., 73-017 CHS, Los Angeles, CA 90095, USA
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Zheng J, Xue H, Wang T, Jiang Y, Liu B, Li J, Liu Y, Wang W, Zhang B, Sun M. miR-21 downregulates the tumor suppressor P12 CDK2AP1 and stimulates cell proliferation and invasion. J Cell Biochem 2011; 112:872-80. [PMID: 21328460 DOI: 10.1002/jcb.22995] [Citation(s) in RCA: 62] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
The present study was undertaken to investigate the regulation of P12(CDK2AP1) by miRNAs. A conserved target site for miR-21 within the CDK2AP1-3'-UTR at nt 349-370 was predicted by bioinformatics software and an inverse correlation of miR-21 and CDK2AP1 protein was observed. Highly specific amplification and quantification of miR-21 was achieved using real-time RT-PCR. Transfection of HaCaT cells with pre-miR-21 significantly suppressed a luciferase reporter including the CDK2AP1-3'-UTR, whereas transfection of Tca8113 with anti-miR-21 increased activity of this reporter. This was abolished when a construct mutated at the miR-21/nt 349-370 target site was used instead. Anti-miR-21-transfected Tca8113 cells showed an increase of CDK2AP1 protein and reduced proliferation and invasion. Resected primary tumors and tumor-free surgical margins of 18 patients with head and neck squamous cell carcinomas demonstrated an inverse correlation between miR-21 and P12(CDK2AP1). This study shows that P12(CDK2AP1) is downregulated by miR-21 and that miR-21 promotes proliferation and invasion in cultured cells.
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Affiliation(s)
- Jun Zheng
- Department of Oral and Maxillofacial Surgery, School of Stomatology, Fourth Military Medical University, 145 West Changle Road, Xi'an 710032, China
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15
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Zolochevska O, Figueiredo ML. Cell-cycle regulators cdk2ap1 and bicalutamide suppress malignant biological interactions between prostate cancer and bone cells. Prostate 2011; 71:353-67. [PMID: 20812223 DOI: 10.1002/pros.21249] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/02/2010] [Accepted: 07/22/2010] [Indexed: 11/06/2022]
Abstract
INTRODUCTION We examined whether the novel cell-cycle regulator cdk2-associated protein 1 (p12(cdk2ap1) or cdk2ap1), recently shown to regulate prostate cancer cell cycle and apoptosis, could have the capacity to reduce invasiveness and/or reduce malignant biological interactions between prostate cancer and bone cells. We also examined whether combining two cell-cycle arrest stimuli, cdk2ap1 plus bicalutamide (or casodex, CDX), could help enhance inhibition of prostate cancer cell phenotypes. METHODS We stably expressed cdk2ap1 in prostate cancer cell lines using lentiviral vectors, as well as several different co-culture assays to quantify cellular invasion, migration, and the effect of the treatments on interaction with the bone microenvironment. RESULTS We have determined that cdk2ap1 can further augment the effects of CDX on cell-cycle arrest, growth inhibition, and cellular invasion. Using a coculture model, we observed that either cdk2ap1 or cdk2ap1/CDX combination were able to reduce chemotaxis towards osteoblasts, and also reduce the osteoblastic proliferative response to prostate cancer. Also modified by cdk2ap1 and CDX were several signaling pathways associated with prostate cancer/bone crosstalk mechanisms involved in prostate cancer progression. CONCLUSIONS These results suggest that either cdk2ap1 or the cdk2ap1/CDX combination hold promise in regulating prostate cancer growth and malignant phenotypes, and potentially also in reducing procarcinogenic interactions with a bone microenvironment model, restoring malignant phenotypes and signaling to a more benign state.
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Affiliation(s)
- Olga Zolochevska
- Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana 70803, USA
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16
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Spruijt CG, Bartels SJJ, Brinkman AB, Tjeertes JV, Poser I, Stunnenberg HG, Vermeulen M. CDK2AP1/DOC-1 is a bona fide subunit of the Mi-2/NuRD complex. MOLECULAR BIOSYSTEMS 2010; 6:1700-6. [PMID: 20523938 DOI: 10.1039/c004108d] [Citation(s) in RCA: 50] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
The Mi-2/NuRD (NUcleosome Remodeling and histone Deacetylase) chromatin remodeling complex is a large heterogeneous multiprotein complex associated with transcriptional repression. Here we apply a SILAC based quantitative proteomics approach to show that all known Mi-2/NuRD complex subunits co-purify with Cyclin Dependent Kinase 2 Associated Protein1 (CDK2AP1), also known as Deleted in Oral Cancer 1 (DOC-1). DOC-1 displays in vitro binding affinity for methylated DNA as part of the meCpG binding MBD2/NuRD complex. In luciferase reporter assays, DOC-1 is a potent repressor of transcription. Finally, immunofluorescence experiments reveal co-localization between MBD2 and DOC-1 in mouse NIH-3T3 nuclei. Collectively, these results indicate that DOC-1 is a bona fide subunit of the Mi-2/NuRD chromatin remodeling complex.
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Affiliation(s)
- Cornelia G Spruijt
- Department of Physiological Chemistry and Cancer Genomics Centre, University Medical Center Utrecht, Utrecht, The Netherlands
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17
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Yagi K, Akagi K, Hayashi H, Nagae G, Tsuji S, Isagawa T, Midorikawa Y, Nishimura Y, Sakamoto H, Seto Y, Aburatani H, Kaneda A. Three DNA methylation epigenotypes in human colorectal cancer. Clin Cancer Res 2009; 16:21-33. [PMID: 20028768 DOI: 10.1158/1078-0432.ccr-09-2006] [Citation(s) in RCA: 184] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
PURPOSE Whereas the CpG island methylator phenotype (CIMP) in colorectal cancer associates with microsatellite instability (MSI)-high and BRAF-mutation(+), the existence of an intermediate-methylation subgroup associated with KRAS-mutation(+) is controversial, and suitable markers for the subgroup have yet to be developed. Our aim is to clarify DNA methylation epigenotypes of colorectal cancer more comprehensively. EXPERIMENTAL DESIGN To select new methylation markers on a genome-wide scale, we did methylated DNA immunoprecipitation-on-chip analysis of colorectal cancer cell lines and re-expression array analysis by 5-aza-2'-deoxycytidine/Trichostatin A treatment. Methylation levels were analyzed quantitatively in 149 colorectal cancer samples using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Colorectal cancer was epigenotyped by unsupervised two-way hierarchical clustering method. RESULTS Among 1,311 candidate silencing genes, 44 new markers were selected and underwent quantitative methylation analysis in colorectal cancer samples together with 16 previously reported markers. Colorectal cancer was clustered into high-, intermediate-, and low-methylation epigenotypes. Methylation markers were clustered into two major groups: group 1 showing methylation in high-methylation epigenotype, and group 2 showing methylation in high- and intermediate-methylation epigenotypes. A two-step marker panel deciding epigenotypes was developed with 95% accuracy: the 1st panel consisting of three group-1 markers (CACNA1G, LOX, SLC30A10) to extract high-methylation epigenotype, and the 2nd panel consisting of four group-2 markers (ELMO1, FBN2, THBD, HAND1) and SLC30A10 again to divide the remains into intermediate- and low-methylation epigenotypes. The high-methylation epigenotype correlated significantly with MSI-high and BRAF-mutation(+) in concordance with reported CIMP. Intermediate-epigenotype significantly correlated with KRAS-mutation(+). KRAS-mutation(+) colorectal cancer with intermediate-methylation epigenotype showed significantly worse prognosis. CONCLUSIONS Three methylation epigenotypes exist in colorectal cancer, and suitable classification markers have been developed. Intermediate-methylation epigenotype with KRAS-mutation(+) correlated with worse prognosis.
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Affiliation(s)
- Koichi Yagi
- Genome Science Division, Department of Gastrointestinal Surgery, Research Center for Advanced Science and Technology, Translational Systems Biology and Medicine Initiative, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
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Zolochevska O, Figueiredo ML. Cell cycle regulator cdk2ap1 inhibits prostate cancer cell growth and modifies androgen-responsive pathway function. Prostate 2009; 69:1586-97. [PMID: 19585490 DOI: 10.1002/pros.21007] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Abstract
BACKGROUND We evaluated the effect of expressing the cell cycle regulator cdk2ap1, downregulated in prostate cancer cell lines, in inhibiting prostate cancer cell growth. METHODS Expression of cdk2ap1 using a tet-inducible lentiviral system modified growth rate, induced cell cycle arrest and apoptosis and reduced the invasive ability of prostate cancer cell lines, as assayed by cell viability, cell cycle profiling, Caspase 3/7 detection, and matrigel invasion assays. We examined the effect of expressing cdk2ap1 on gene expression profiles of cytokine, invasion, apoptotic, and androgen response pathways using quantitative real-time PCR, and used androgen-responsive reporter gene assays, and methylation-sensitive PCR to examine the mechanism of cdk2ap1 interaction with androgen-responsive pathways. RESULTS The expression of cdk2ap1 correlated with a reduction in cellular growth, irrespective of inhibition or stimulation of androgen receptor (AR) signaling pathways. Cell cycle arrest, increased apoptosis, and a reduction in invasiveness phenotypes were observed upon cdk2ap1 expression. Enhanced demethylation at the AR promoter, AR expression increases, and enhanced AR transcriptional activity correlated with cdk2ap1 expression. CONCLUSIONS Our findings support a novel concept by which cell cycle inhibitor genes can impact prostate cancer phenotypes by restoring a tumor suppressive function to androgen-responsive pathways and this function may involve modulation of a subset of functions of the AR.
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Affiliation(s)
- Olga Zolochevska
- Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana 70803, USA
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19
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Cao J, Tang M, Li WL, Xie J, Du H, Tang WB, Wang H, Chen XW, Xiao H, Li Y. Upregulation of Activator Protein-4 in Human Colorectal Cancer With Metastasis. Int J Surg Pathol 2008; 17:16-21. [PMID: 18480385 DOI: 10.1177/1066896908315813] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
The aim of this study is to investigate the expression of AP-4, VEGF, and MMP-9 genes in human colorectal cancer. The expression pattern of activator protein-4 in 160 colorectal cancer compared with 32 colorectal adenomas and 32 normal colorectal tissues is demonstrated by tissue microarray-immunohistochemistry and real-time reverse transcriptase—polymerase chain reaction. Apoptosis status using terminal deoxynucleotidyl transferase—mediated dUTP nick-end labeling by comparing activator protein-4 positive versus activator protein-4 negative colorectal cancer is also assessed. The messenger RNA levels of vascular endothelial growth factor and matrix metalloproteinase-9 expression in activator protein-4 positive and negative colorectal cancer were measured using real-time reverse transcriptase— polymerase chain reaction. The activator protein-4 expression in normal colorectal tissue, adenoma, and adenocarcinoma were 4 of 32, 8 of 32, and 78 of 160, respectively. It is shown that the activator protein-4 expression was significantly correlated with the progression of colorectal cancer ( P < .01) and differentiation and lymph node metastasis ( P < .01). Our results also presented that the activator protein-4 expression was associated with the expression of matrix metalloproteinase-9 and vascular endothelial growth factor in the advanced colorectal cancer.
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Affiliation(s)
- Jie Cao
- Department of Gastrointestinal Surgery, Affiliated Guangzhou First Municipal People's Hospital, Guangzhou Medical College, Guangzhou, China.
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20
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Shin J, Yuan Z, Fordyce K, Sreeramoju P, Kent TS, Kim J, Wang V, Schneyer D, Weber TK. A del T poly T (8) mutation in the 3′ untranslated region (UTR) of the CDK2-AP1 gene is functionally significant causing decreased mRNA stability resulting in decreased CDK2-AP1 expression in human microsatellite unstable (MSI) colorectal cancer (CRC). Surgery 2007; 142:222-7. [PMID: 17689689 DOI: 10.1016/j.surg.2007.04.002] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2007] [Revised: 04/04/2007] [Accepted: 04/06/2007] [Indexed: 11/22/2022]
Abstract
BACKGROUND We have previously published results indicating that decreased expression of CDK2-AP1 in MSI human colorectal cancer is associated with deletion mutations in the poly (T) 8 repeat sequence within the 3'-UTR of the CDK2-AP1 gene. In this study, we test the hypothesis that the del T mutation results in decreased CDK2-AP1 expression by causing reduced mRNA stability. METHODS We introduced wild-type and mutant 3'-UTR sequences fused to a green fluorescent protein (GFP) gene separately into human CRC cell lines and quantified the expression of the GFP gene. Native CDK2-AP1 mRNA stability was measured in human CRC cell lines, using an actinomycin D assay and the mRNA structure folding software mfold 3.2. RESULTS Mutant GFP-3'-UTR samples demonstrated significantly reduced GFP expression compared with wild-type GFP-3'-UTR as measured by both FACS and real-time PCR. Both the actinomycin D assay and mfold software demonstrated significantly reduced mRNA stability for the del T poly (T) 8 transcript compared with the wild type. CONCLUSIONS In summary, these novel results support our hypotheses that the del T poly (T) 8 observed in the 3'-UTR of the CDK2-AP1 gene in human MSI CRC is functionally significant and results in decreased CDK2-AP1 expression. The results also indicate the mechanism of this decreased expression is caused at least in part by decreased mRNA stability.
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Affiliation(s)
- Joongho Shin
- Department of Surgery and Molecular Genetics, Albert Einstein College of Medicine, New York, NY, USA
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21
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Cardoso J, Boer J, Morreau H, Fodde R. Expression and genomic profiling of colorectal cancer. Biochim Biophys Acta Rev Cancer 2006; 1775:103-37. [PMID: 17010523 DOI: 10.1016/j.bbcan.2006.08.004] [Citation(s) in RCA: 60] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2006] [Revised: 08/11/2006] [Accepted: 08/12/2006] [Indexed: 12/13/2022]
Abstract
Colorectal cancer still represents a paradigm for the elucidation of the cellular, genetic and molecular mechanisms that underly solid tumor initiation, progression to malignancy, and metastasis to distal organ sites. The relative ease with which pathological specimens can be obtained by either surgery or endoscopy from different stages of tumor progression has facilitated the application of omics technologies to allow the genome-wide analysis both at the RNA (gene expression) and DNA (aneuploidy) levels. Here, we have reviewed the multiplicity of studies appeared to date in the scientific literature on the expression and genomic analysis of colorectal cancer, and attempted an integration of the profiling data generated and made available in the public domain. This approach is likely to pinpoint specific chromosomal loci and the corresponding genes which (i) play rate-limiting roles in colorectal cancer, (ii) represent putative diagnostic and prognostic markers for the accurate prediction of clinical outcome and response to treatment, and (iii) encompass potential therapeutic targets. Moreover, cross-species data mining and integration of the human colorectal cancer profiles with those obtained from mouse models of intestinal tumorigenesis will even more contribute to the elucidation of highly conserved pathways and cellular functions underlying malignancy in the GI tract. Notwithstanding the above promises, tumor heterogeneity, limited cohort sizes, and methodological differences among experimental and bioinformatic approaches still poses main obstacles towards the optimal utilization and integration of omics profiles.
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Affiliation(s)
- J Cardoso
- Department of Pathology, Josephine Nefkens Institute, Erasmus University Medical Center, 3000CA Rotterdam, The Netherlands
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22
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Le Guezennec X, Vermeulen M, Brinkman AB, Hoeijmakers WAM, Cohen A, Lasonder E, Stunnenberg HG. MBD2/NuRD and MBD3/NuRD, two distinct complexes with different biochemical and functional properties. Mol Cell Biol 2006; 26:843-51. [PMID: 16428440 PMCID: PMC1347035 DOI: 10.1128/mcb.26.3.843-851.2006] [Citation(s) in RCA: 265] [Impact Index Per Article: 13.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
The human genome contains a number of methyl CpG binding proteins that translate DNA methylation into a physiological response. To gain insight into the function of MBD2 and MBD3, we first applied protein tagging and mass spectrometry. We show that MBD2 and MBD3 assemble into mutually exclusive distinct Mi-2/NuRD-like complexes, called MBD2/NuRD and MBD3/NuRD. We identified DOC-1, a putative tumor suppressor, as a novel core subunit of MBD2/NuRD as well as MBD3/NuRD. PRMT5 and its cofactor MEP50 were identified as specific MBD2/NuRD interactors. PRMT5 stably and specifically associates with and methylates the RG-rich N terminus of MBD2. Chromatin immunoprecipitation experiments revealed that PRMT5 and MBD2 are recruited to CpG islands in a methylation-dependent manner in vivo and that H4R3, a substrate of PRMT, is methylated at these loci. Our data show that MBD2/NuRD and MBD3/NuRD are distinct protein complexes with different biochemical and functional properties.
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Affiliation(s)
- Xavier Le Guezennec
- Department of Molecular Biology, NCMLS M850/3.79, Radboud University, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands
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23
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Kruhøffer M, Jensen JL, Laiho P, Dyrskjøt L, Salovaara R, Arango D, Birkenkamp-Demtroder K, Sørensen FB, Christensen LL, Buhl L, Mecklin JP, Järvinen H, Thykjaer T, Wikman FP, Bech-Knudsen F, Juhola M, Nupponen NN, Laurberg S, Andersen CL, Aaltonen LA, Ørntoft TF. Gene expression signatures for colorectal cancer microsatellite status and HNPCC. Br J Cancer 2005; 92:2240-8. [PMID: 15956967 PMCID: PMC2361815 DOI: 10.1038/sj.bjc.6602621] [Citation(s) in RCA: 61] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023] Open
Abstract
The majority of microsatellite instable (MSI) colorectal cancers are sporadic, but a subset belongs to the syndrome hereditary nonpolyposis colorectal cancer (HNPCC). Microsatellite instability is caused by dysfunction of the mismatch repair (MMR) system that leads to a mutator phenotype, and MSI is correlated to prognosis and response to chemotherapy. Gene expression signatures as predictive markers are being developed for many cancers, and the identification of a signature for MMR deficiency would be of interest both clinically and biologically. To address this issue, we profiled the gene expression of 101 stage II and III colorectal cancers (34 MSI, 67 microsatellite stable (MSS)) using high-density oligonucleotide microarrays. From these data, we constructed a nine-gene signature capable of separating the mismatch repair proficient and deficient tumours. Subsequently, we demonstrated the robustness of the signature by transferring it to a real-time RT-PCR platform. Using this platform, the signature was validated on an independent test set consisting of 47 tumours (10 MSI, 37 MSS), of which 45 were correctly classified. In a second step, we constructed a signature capable of separating MMR-deficient tumours into sporadic MSI and HNPCC cases, and validated this by a mathematical cross-validation approach. The demonstration that this two-step classification approach can identify MSI as well as HNPCC cases merits further gene expression studies to identify prognostic signatures.
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Affiliation(s)
- M Kruhøffer
- Molecular Diagnostic Laboratory, Department of Clinical Biochemistry, Aarhus University Hospital, Brendstrupgaardsvej 100, DK-8200, Aarhus, Denmark
| | - J L Jensen
- Department of Statistics, Aarhus University, Aarhus, Denmark
| | - P Laiho
- Department of Medical Genetics, Haartman Institute, University of Helsinki, Helsinki, Finland
| | - L Dyrskjøt
- Molecular Diagnostic Laboratory, Department of Clinical Biochemistry, Aarhus University Hospital, Brendstrupgaardsvej 100, DK-8200, Aarhus, Denmark
| | - R Salovaara
- Department of Medical Genetics, Haartman Institute, University of Helsinki, Helsinki, Finland
| | - D Arango
- Department of Medical Genetics, Haartman Institute, University of Helsinki, Helsinki, Finland
| | - K Birkenkamp-Demtroder
- Molecular Diagnostic Laboratory, Department of Clinical Biochemistry, Aarhus University Hospital, Brendstrupgaardsvej 100, DK-8200, Aarhus, Denmark
| | - F B Sørensen
- Institute of Pathology, Aarhus University Hospital, Brendstrupgaardsvej 100, DK-8200, Aarhus, Denmark
| | - L L Christensen
- Molecular Diagnostic Laboratory, Department of Clinical Biochemistry, Aarhus University Hospital, Brendstrupgaardsvej 100, DK-8200, Aarhus, Denmark
| | - L Buhl
- Institute of Pathology, Aarhus University Hospital, Brendstrupgaardsvej 100, DK-8200, Aarhus, Denmark
| | - J-P Mecklin
- Department of Pathology, Jyväskylä Central Hospital, Jyväskylä, Helsinki, Finland
| | - H Järvinen
- University Central Hospital, Haartman Institute, University of Helsinki, Helsinki, Finland
| | - T Thykjaer
- AROS Applied Biotechnology ApS, Research Park Skejby. Aarhus. Denmark
| | - F P Wikman
- Molecular Diagnostic Laboratory, Department of Clinical Biochemistry, Aarhus University Hospital, Brendstrupgaardsvej 100, DK-8200, Aarhus, Denmark
| | - F Bech-Knudsen
- Department of Surgery, Aarhus University Hospital, Brendstrupgaardsvej 100, DK-8200, Aarhus, Denmark
| | - M Juhola
- Department of Pathology, Jyväskylä Central Hospital, Jyväskylä, Helsinki, Finland
| | - N N Nupponen
- Department of Medical Genetics, Haartman Institute, University of Helsinki, Helsinki, Finland
| | - S Laurberg
- Department of Surgery, Aarhus University Hospital, Brendstrupgaardsvej 100, DK-8200, Aarhus, Denmark
| | - C L Andersen
- Molecular Diagnostic Laboratory, Department of Clinical Biochemistry, Aarhus University Hospital, Brendstrupgaardsvej 100, DK-8200, Aarhus, Denmark
| | - L A Aaltonen
- Department of Medical Genetics, Haartman Institute, University of Helsinki, Helsinki, Finland
| | - T F Ørntoft
- Molecular Diagnostic Laboratory, Department of Clinical Biochemistry, Aarhus University Hospital, Brendstrupgaardsvej 100, DK-8200, Aarhus, Denmark
- Molecular Diagnostic Laboratory, Department of Clinical Biochemistry, Aarhus University Hospital, Brendstrupgaardsvej 100, DK-8200, Aarhus, Denmark. E-mail:
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Figueiredo ML, Kim Y, St John MAR, Wong DTW. p12CDK2-AP1 gene therapy strategy inhibits tumor growth in an in vivo mouse model of head and neck cancer. Clin Cancer Res 2005; 11:3939-48. [PMID: 15897596 DOI: 10.1158/1078-0432.ccr-04-2085] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
PURPOSE To test the potential of p12(CDK2-AP1) (p12), a cell cycle regulator and cyclin-dependent kinase-2-associating protein commonly down-regulated in head and neck squamous cell carcinoma ( approximately 70%), as a gene therapy in inhibiting head and neck squamous cell carcinoma growth in vivo. EXPERIMENTAL DESIGN We addressed the effect of p12 expression on tumor growth by using a well-established squamous cell carcinoma VII/SF floor of mouth xenograft mouse model. The effect of therapy on tumor growth was determined for: (a) no treatment, (b) PBS, (c) vehicle (1,2-dioleoyloxy-3-trimethylammonium propane:cholesterol liposomes / 5% dextrose), (d) empty vector controls, and (e) p12-encoding vector experimental groups. RESULTS p12 gene therapy significantly induced antitumor effects as compared with controls, including (a) size and weight of p12-treated tumors decreased by 51% to 72% compared with all controls (P < 0.02), (b) tumor growth rate post-therapy was inhibited by 55% to 64% compared with empty vector controls (P < 0.0001), and (c) p12 expression was higher in p12-treated than controls (P < 0.002) by two-tailed t test analyses. Mechanistically, p12 treatment affected cell turnover kinetics as assessed by apoptotic and cell proliferation indices. p12 therapy significantly increased terminal nucleotidyl transferase-mediated nick end labeling (P < 0.05) and morphology-based apoptotic indices (P < 0.05) as well as significantly decreased Ki-67 cell proliferation indices (P < 0.001) compared with controls, resulting in a net cell turnover reduction in p12-treated tumors. CONCLUSIONS We show that this novel therapeutic modality can significantly induce antitumor responses in vivo. These results support a role for p12 as a novel tumor growth suppressor gene therapy and suggest that optimization and/or combination with current therapies may hold considerable promise in preparation for clinical trials.
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Affiliation(s)
- Marxa L Figueiredo
- Laboratory of Head and Neck Cancer Research, School of Dentistry and Dental Research Institute, and Department of Head and Neck Surgery, University of California Los Angeles, Los Angeles, California, USA
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25
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Kim Y, Ohyama H, Patel V, Figueiredo M, Wong DT. Mutation of Cys105 inhibits dimerization of p12CDK2-AP1 and its growth suppressor effect. J Biol Chem 2005; 280:23273-9. [PMID: 15840587 DOI: 10.1074/jbc.m412929200] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
p12(CDK2-AP1) (p12) is a CDK2-associated protein that negatively regulates its kinase activity. Growth arrest of normal diploid cells by contact inhibition resulted in an induction of p27(kip1) and reduction of CDK2 levels. Interestingly, we observed concomitantly in growth-arrested cells, there was a reduction of nuclear p12 and the appearance of a nuclear 25-kDa molecule (p25) recognized by anti-p12 polyclonal antibody. Biochemical analysis showed that bacterial His-tagged p12 could be converted into a dimeric p25 in a reducing agent-dependent manner, and mutating the only cysteine residue of p12 (Cys(105) --> Ala(105)) abolished the dimerization. Transient transfection of wild type p12 into U2OS cells showed a reducing agent-sensitive dimerization that was also abolished by the C105A mutation. Furthermore, reduction of p12 expression by a short interfering RNA resulted in a parallel reduction of p25. These data supports the possibility that p25 is a homodimeric form of p12 through the cysteine residue. More interestingly, transient transfection of p12 (C105A) into the normal diploid lung fibroblast CCD18LU cells resulted in a reduction of the growth-inhibitory effect of p12 and abolished the inhibitory effect of p12 on CDK2 kinase activity. In addition, we found that the C105A mutation did not alter nuclear localization of p12, but it prevented association with CDK2. Taken together, our data suggest that p12 forms a nuclear homodimers in contact inhibited normal diploid cells and dimerization of p12 is a necessary process for the growth inhibition effect by p12.
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Affiliation(s)
- Yong Kim
- School of Dentistry and Dental Research Institute, UCLA, Los Angeles, California 90095, USA
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26
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Yuan Z, Gaba AG, Kent TS, Bennett A, Miller A, Weber TK. Modulation of CDK2-AP1 (p12DOC−1) expression in human colorectal cancer. Oncogene 2005; 24:3657-68. [PMID: 15806176 DOI: 10.1038/sj.onc.1208378] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
We have previously demonstrated an association between microsatellite instability and decreased CDK2-AP1 (p12(DOC-1)) expression in human colorectal cancer (CRC) cell lines. In those same studies, induction of CDK2-AP1 expression promoted both cell cycle arrest and apoptosis. The goals of our present study were to better understand the mechanisms leading to reduced CDK2-AP1 expression in microsatellite unstable (MSI) CRC and to study further the effect of CDK2-AP1 modulation on cell proliferation and apoptosis utilizing RNA interference (RNAi) techniques. We used direct sequencing to screen for mutations of the poly (T)8 microsatellite-like region in the 3' end of the CDK2-AP1 gene in 24 CRC cell lines. We then utilized an in vitro human mismatch repair (MMR) recombinant system to assess for correction of the mutation and changes in CDK2-AP1 expression secondary to hMLH1 transfection. We also investigated the effect of CDK2-AP1 modulation in four settings: (1) native CDK2-AP1 absence, (2) endogenous CDK2-AP1 expression, (3) RNAi-induced CDK2-AP1 inhibition and (4) induced CDK2-AP1 over expression. The mutation - del T poly (T)8 - at the 3' end of the CDK2-AP1 gene was found in 3/12 (25%) of MSI CRC cell lines, but in none of the microsatellite stable samples (0/12). Interestingly, when wild-type MMR protein - MLH1 - was induced in an in vitro human recombinant system, the del T poly (T)8 mutation was reversed and CDK2-AP1 expression increased. RNAi-mediated CDK2-AP1 inhibition was associated with decreased apoptosis and increased cell proliferation in CDK2-AP1-non deficient CRC cell lines. We conclude that mutations in the microsatellite-like sequence of the CDK2-AP1 gene in MSI CRC are associated with decreased CDK2-AP1 expression. In addition, modulation of CDK2-AP1 expression in human CRC alters cell proliferation and apoptosis.
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Affiliation(s)
- Ziquan Yuan
- Department of Molecular Genetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Ullmann 707, New York, NY 10461, USA
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Kim Y, McBride J, Zhang R, Zhou X, Wong DT. p12(CDK2-AP1) mediates DNA damage responses induced by cisplatin. Oncogene 2005; 24:407-18. [PMID: 15543230 DOI: 10.1038/sj.onc.1208222] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
We examined the biological role of p12(CDK2-AP1) in cisplatin-mediated responses by using murine ES p12(CDK2-AP1) knockout clones generated by a targeted disruption of murine p12(CDK2-AP1). Homozygous knockout clones showed an increased cellular proliferation along with an increase in S and a decrease in G2/M phase populations. Interestingly, ES p12(CDK2-AP1) knockout clones showed a resistance to cisplatin treatment along with an increased DNA repair activity assessed by host cell reactivation assay using a cisplatin-damaged reporter DNA and a significant reduction of apoptosis upon cisplatin treatment. By using stable p12(CDK2-AP1) short interfering RNA (siRNA) clones from human normal oral keratinocytes, we confirmed that downregulation of p12(CDK2-AP1) resulted in a resistance to cisplatin. More interestingly, cisplatin treatment resulted in a reduction of CDK2 kinase activity in control clones, but p12(CDK2-AP1) knockout clones showed a sustained CDK2 kinase activity. These data suggest that p12(CDK2-AP1) plays a role in cisplatin-mediated cellular responses by modulating CDK2 activity. These data further suggest p12(CDK2-AP1) is a potential gene therapeutic agent for oral/head and neck cancer in conjunction with DNA-damaging agents such as cisplatin.
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Affiliation(s)
- Yong Kim
- School of Dentistry and Dental Research Institute, University of California-Los Angeles, Los Angeles, CA 90095, USA
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Abstract
DNA microarray technology permits simultaneous analysis of thousands of DNA sequences for genomic research and diagnostics applications. Microarray technology represents the most recent and exciting advance in the application of hybridization-based technology for biological sciences analysis. This review focuses on the classification (oligonucleotide vs. cDNA) and application (mutation-genotyping vs. gene expression) of microarrays. Oligonucleotide microarrays can be used both in mutation-genotyping and gene expression analysis, while cDNA microarrays can only be used in gene expression analysis. We review microarray mutation analysis, including examining the use of three oligonucleotide microarrays developed in our laboratory to determine mutations in RET, beta-catenin and K-ras genes. We also discuss the use of the Affymetrix GeneChip in mutation analysis. We review microarray gene expression analysis, including the classifying of such studies into four categories: class comparison, class prediction, class discovery and identification of biomarkers.
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Affiliation(s)
- Il-Jin Kim
- Korean Hereditary Tumor Registry, Cancer Research Institute and Cancer Research Center, Seoul National University, Seoul, Korea
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Banerjea A, Ahmed S, Hands RE, Huang F, Han X, Shaw PM, Feakins R, Bustin SA, Dorudi S. Colorectal cancers with microsatellite instability display mRNA expression signatures characteristic of increased immunogenicity. Mol Cancer 2004; 3:21. [PMID: 15298707 PMCID: PMC514528 DOI: 10.1186/1476-4598-3-21] [Citation(s) in RCA: 128] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2004] [Accepted: 08/06/2004] [Indexed: 01/14/2023] Open
Abstract
Background Colorectal cancers displaying high-degree microsatellite instability (MSI-H) have an improved prognosis compared to microsatellite stable (MSS) cancers. The observation of pronounced lymphocytic infiltrates suggests that MSI-H cancers are inherently more immunogenic. We aimed to compare the gene expression profiles of MSI-H and MSS cancers to provide evidence for an activated immune response in the former. Results We analysed tissue from 133 colorectal cancer patients with full consent and Local Ethics Committee approval. Genomic DNA was analysed for microsatellite instability in BAT-26. High-quality RNA was used for microarray analysis on the Affymetrix® HG-U133A chip. Data was analysed on GeneSpring software version 6.0. Confirmatory real-time RT-PCR was performed on 28 MSI-H and 26 MSS cancers. A comparison of 29 MSI-H and 104 MSS cancers identified 2070 genes that were differentially expressed between the two groups [P < 0.005]. Significantly, many key immunomodulatory genes were up-regulated in MSI-H cancers. These included antigen chaperone molecules (HSP-70, HSP-110, Calreticulin, gp96), pro-inflammatory cytokines (Interleukin (IL)-18, IL-15, IL-8, IL-24, IL-7) and cytotoxic mediators (Granulysin, Granzyme A). Quantitative RT-PCR confirmed up-regulation of HSP-70 [P = 0.016], HSP-110 [P = 0.002], IL-18 [P = 0.004], IL-8 [0.002] and Granulysin [P < 0.0001]. Conclusions The upregulation of a large number of genes implicated in immune response supports the theory that MSI-H cancers are immunogenic. The novel observation of Heat Shock Protein up-regulation in MSI-H cancer is highly significant in light of the recognised roles of these proteins in innate and antigen-specific immunogenicity. Increased mRNA levels of pro-inflammatory cytokines and cytotoxic mediators also indicate an activated anti-tumour immune response.
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Affiliation(s)
- Ayan Banerjea
- Centre for Academic Surgery, Barts and The London Queen Mary School of Medicine and Dentistry. The Royal London Hospital, Whitechapel, London, E1 1BB, UK
| | - Shafi Ahmed
- Centre for Academic Surgery, Barts and The London Queen Mary School of Medicine and Dentistry. The Royal London Hospital, Whitechapel, London, E1 1BB, UK
| | - Rebecca E Hands
- Centre for Academic Surgery, Barts and The London Queen Mary School of Medicine and Dentistry. The Royal London Hospital, Whitechapel, London, E1 1BB, UK
| | - Fei Huang
- Department of Pharmacogenomics, Bristol Myers Squibb, 311 Pennington-Rocky Hill Road, Pennington, NJ 08534, USA
| | - Xia Han
- Department of Pharmacogenomics, Bristol Myers Squibb, 311 Pennington-Rocky Hill Road, Pennington, NJ 08534, USA
| | - Peter M Shaw
- Department of Pharmacogenomics, Bristol Myers Squibb, 311 Pennington-Rocky Hill Road, Pennington, NJ 08534, USA
| | - Roger Feakins
- Institute of Pathology, The Royal London Hospital, Whitechapel, London, E1 1BB, UK
| | - Stephen A Bustin
- Centre for Academic Surgery, Barts and The London Queen Mary School of Medicine and Dentistry. The Royal London Hospital, Whitechapel, London, E1 1BB, UK
| | - Sina Dorudi
- Centre for Academic Surgery, Barts and The London Queen Mary School of Medicine and Dentistry. The Royal London Hospital, Whitechapel, London, E1 1BB, UK
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Sotsky Kent T, Yuan Z, Miller A, Weber TK. Deleted in oral cancer-1 expression upregulates proapoptosis elements in microsatellite-unstable human colorectal cancer. Ann Surg Oncol 2004; 11:192-6. [PMID: 14761923 DOI: 10.1245/aso.2004.03.056] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
BACKGROUND We previously reported differential expression of the growth suppressor, deleted in oral cancer-1 (DOC-1), in microsatellite-unstable (MSI+) versus microsatellite-stable colorectal cancer (CRC) cell lines. MSI+ CRC cell lines demonstrated decreased DOC-1 expression and decreased apoptosis. Transfection of wild-type DOC-1 into an MSI+ cell line (SW48) resulted in increased apoptosis. We undertook our current experiment to identify specific elements modulated by DOC-1 expression that result in increased apoptosis. METHODS SW48 is an MSI+ CRC cell line that does not constitutively express DOC-1. SW48 was suspended in culture medium and incubated to 60% confluence. Half the plates were transfected with cytomegalovirus (CMV)-DOC-1. At 30 hours, RNA and protein were isolated with Trizol. Complementary DNA microarray was performed to compare SW48(CMV-DOC-1) with SW48, which lacks DOC-1. Signal intensity was analyzed by GenePix Pro 3.0 software. Expression ratios </=.67 and >/ 1.5 were considered significant. Poor-quality spots were flagged and excluded from analysis. Real-time polymerase chain reaction was performed to determine DOC-1 levels in both cell lines. RESULTS Successful transfection of DOC-1 was confirmed by real-time polymerase chain reaction and by Western blot. Microarray revealed significant differential expression of DOC-1, as expected. Increased DOC-1 expression in SW48(CMV-DOC-1) was associated with significantly increased expression of proapoptosis components of the caspase cascade (CASP7, CASP9) and bcl2/bax pathway (BNIP3, BNIP3L, BID). CONCLUSIONS DOC-1 expression promotes apoptosis by upregulation of specific elements of the caspase cascade and bcl2/bax pathways. DOC-1 therefore deserves further study as a candidate for the therapeutic modulation of apoptosis in MSI+ CRC.
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Affiliation(s)
- Tara Sotsky Kent
- Departments of Surgery, Albert Einstein College of Medicine/Montefiore Medical Center, Bronx, New York, USA
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