Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 22
Filter
Add more filters










Publication year range
1.
Breast Cancer Res Treat ; 157(3): 447-59, 2016 06.
Article in English | MEDLINE | ID: mdl-27250000

ABSTRACT

Current evidence suggests that the majority of DCIS lesions do not progress to invasive carcinoma, and overtreatment of DCIS is a significant problem. We previously reported an 8-gene signature that differentiated microdissected low-grade (LG) DCIS lesions with and without associated stromal invasion, based on differential DNA copy number changes detected by quantitative (q) PCR. The current study was undertaken to validate our candidate breast cancer invasion gene panel in a larger series of non-microdissected LG DCIS cases, and to investigate its potential utility in intermediate-grade (IG) and high-grade (HG) DCIS. Representative paraffin blocks were selected from 267 resected DCIS cases with 5-15 years of follow-up (139 pure DCIS ["PD"] and 128 mixed DCIS with associated invasion ["MD"]). These included 171 LG, 46 IG and 50 HG DCIS cases. Gene copy number changes were determined by qPCR, and their differential distribution in the PD and MD subgroups was evaluated. As an alternate platform, we employed immunohistochemistry (IHC). Novel IHC assays were developed for all eight candidate genes, and increased or reduced protein expression was manually scored. Separate multi-gene models were developed for qPCR and IHC to distinguish progressing and non-progressing DCIS lesions. By qPCR analysis, a panel of six genes, as well as CELSR1 alone (a potential invasion suppressor), differentiated PD and MD cases in LG and IG, but not in HG DCIS. By IHC, a panel of three genes, as well as GRAP2 alone (a potential invasion promoter), also distinguished PD and MD cases in LG and IG, but not in HG DCIS. The combination of CELSR1 (by qPCR) and GRAP2 (by IHC) had the best discriminatory power (p = 0.00004). Assays testing either or both of these genes have the potential to become important adjuncts for choosing appropriate treatment for LG/IG DCIS patients.


Subject(s)
Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Breast Neoplasms/pathology , Carcinoma, Intraductal, Noninfiltrating/pathology , DNA Copy Number Variations , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cadherins/genetics , Cadherins/metabolism , Carcinoma, Intraductal, Noninfiltrating/genetics , Carcinoma, Intraductal, Noninfiltrating/metabolism , Disease Progression , Female , Gene Expression Regulation, Neoplastic , Humans , Immunohistochemistry , Microdissection , Neoplasm Grading , Neoplasm Invasiveness , Nuclear Receptor Co-Repressor 2/genetics , Nuclear Receptor Co-Repressor 2/metabolism , Nuclear Receptor Subfamily 4, Group A, Member 1/genetics , Nuclear Receptor Subfamily 4, Group A, Member 1/metabolism , Prognosis
2.
Oncol Lett ; 9(4): 1772-1778, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25789040

ABSTRACT

Cancer and embryo expression protein 65 (CEP65) is a centrosomal protein that is expressed at relatively high levels in embryonic tissue and different cancerous tissues, but its role in tumorigenesis remains unknown. In the present study, CEP65 was stably expressed in AGS gastric cancer cells. CEP65 was found to promote cell growth in the MTT assay and to enhance cell migration and invasion in Transwell chamber assays. To validate results from the in vitro experiments, CEP65 was stably expressed in BICR-H1 breast cancer cells through adenovirus-mediated transduction. By inoculating BICR-H1 cells on chick chorioallantoic membrane (CAM), it was found that CEP65 promotes cell growth on the CAM and increases cell metastasis to the lungs of the chicken. By utilizing a xenograft severe combined immunodeficiency mouse model, CEP65 was also found to accelerate BICR-H1 cell growth and metastasis to the lungs. Furthermore, it was shown that CEP65 increases matrix metalloproteinase (MMP)2 activity in zymographic assays, however, microarray screening and reverse transcription polymerase chain reaction validation revealed that CEP65 had no effect on the expression levels of MMP2 or MMP9, but decreased the expression levels of metastasis-associated genes, TIMP2, RAP and VTN. Taken together, the results of the present study demonstrated the oncogenic function of CEP65 in promoting cancer cell growth and metastasis.

3.
Cancer Res ; 73(2): 496-501, 2013 Jan 15.
Article in English | MEDLINE | ID: mdl-23204232

ABSTRACT

Point mutations at Arg132 of the cytoplasmic NADP(+)-dependent isocitrate dehydrogenase 1 (IDH1) occur frequently in gliomas and result in a gain of function to produce the "oncometabolite" D-2-hydroxyglutarate (D-2HG). The mutated IDH1 allele is usually associated with a wild-type IDH1 allele (heterozygous) in cancer. Here, we identify 2 gliomas that underwent loss of the wild-type IDH1 allele but retained the mutant IDH1 allele following tumor progression from World Health Organization (WHO) grade III anaplastic astrocytomas to WHO grade IV glioblastomas. Intratumoral D-2HG was 14-fold lower in the glioblastomas lacking wild-type IDH1 than in glioblastomas with heterozygous IDH1 mutations. To characterize the contribution of wild-type IDH1 to cancer cell D-2HG production, we established an IDH1-mutated astrocytoma (IMA) cell line from a WHO grade III anaplastic astrocytoma. Disruption of the wild-type IDH1 allele in IMA cells by gene targeting resulted in an 87-fold decrease in cellular D-2HG levels, showing that both wild-type and mutant IDH1 alleles are required for D-2HG production in glioma cells. Expression of wild-type IDH1 was also critical for mutant IDH1-associated D-2HG production in the colorectal cancer cell line HCT116. These insights may aid in the development of therapeutic strategies to target IDH1-mutated cancers.


Subject(s)
Brain Neoplasms/genetics , Glioma/genetics , Glutarates/metabolism , Isocitrate Dehydrogenase/genetics , Mutation , Astrocytoma/genetics , Brain Neoplasms/metabolism , Cell Line, Tumor , Genotype , Glioblastoma/genetics , Glioma/metabolism , Humans , Isocitrate Dehydrogenase/metabolism
4.
Oncotarget ; 3(8): 774-82, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22885298

ABSTRACT

Frequent somatic hotspot mutations in isocitrate dehydrogenase 1 (IDH1) have been identified in gliomas, acute myeloid leukemias, chondrosarcomas, and other cancers, providing a likely avenue for targeted cancer therapy. However, whether mutant IDH1 protein is required for maintaining IDH1 mutated tumor cell growth remains unknown. Here, using a genetically engineered inducible system, we report that selective suppression of endogenous mutant IDH1 expression in HT1080, a fibrosarcoma cell line with a native IDH1(R132C) heterozygous mutation, significantly inhibits cell proliferation and decreases clonogenic potential. Our findings offer insights into changes that may contribute to the inhibition of cell proliferation and offer a strong preclinical rationale for utilizing mutant IDH1 as a valid therapeutic target.


Subject(s)
Fibrosarcoma/genetics , Isocitrate Dehydrogenase/genetics , Isocitrate Dehydrogenase/metabolism , Cell Line, Tumor , Cell Proliferation , Fibrosarcoma/pathology , Humans , Mutation , RNA Interference , RNA, Small Interfering
5.
Genome Res ; 22(12): 2339-55, 2012 Dec.
Article in English | MEDLINE | ID: mdl-22899282

ABSTRACT

Monoallelic point mutations of the NADP(+)-dependent isocitrate dehydrogenases IDH1 and IDH2 occur frequently in gliomas, acute myeloid leukemias, and chondromas, and display robust association with specific DNA hypermethylation signatures. Here we show that heterozygous expression of the IDH1(R132H) allele is sufficient to induce the genome-wide alterations in DNA methylation characteristic of these tumors. Using a gene-targeting approach, we knocked-in a single copy of the most frequently observed IDH1 mutation, R132H, into a human cancer cell line and profiled changes in DNA methylation at over 27,000 CpG dinucleotides relative to wild-type parental cells. We find that IDH1(R132H/WT) mutation induces widespread alterations in DNA methylation, including hypermethylation of 2010 and hypomethylation of 842 CpG loci. We demonstrate that many of these alterations are consistent with those observed in IDH1-mutant and G-CIMP+ primary gliomas and can segregate IDH wild-type and mutated tumors as well as those exhibiting the G-CIMP phenotype in unsupervised analysis of two primary glioma cohorts. Further, we show that the direction of IDH1(R132H/WT)-mediated DNA methylation change is largely dependent upon preexisting DNA methylation levels, resulting in depletion of moderately methylated loci. Additionally, whereas the levels of multiple histone H3 and H4 methylation modifications were globally increased, consistent with broad inhibition of histone demethylation, hypermethylation at H3K9 in particular accompanied locus-specific DNA hypermethylation at several genes down-regulated in IDH1(R132H/WT) knock-in cells. These data provide insight on epigenetic alterations induced by IDH1 mutations and support a causal role for IDH1(R132H/WT) mutants in driving epigenetic instability in human cancer cells.


Subject(s)
DNA Methylation , Heterozygote , Isocitrate Dehydrogenase/genetics , Mutation , Alleles , Blotting, Western , Chromatin Immunoprecipitation/methods , Down-Regulation , Epigenomics , Gene Expression Profiling , Gene Expression Regulation , Gene Silencing , Genetic Loci , HCT116 Cells , Histones/genetics , Histones/metabolism , Humans , Isocitrate Dehydrogenase/metabolism , Leukemia, Myeloid, Acute/genetics , Phenotype , Real-Time Polymerase Chain Reaction , Sequence Analysis, DNA
6.
PLoS One ; 7(4): e36211, 2012.
Article in English | MEDLINE | ID: mdl-22558385

ABSTRACT

Dysregulation of Otx2 is a hallmark of the pediatric brain tumor medulloblastoma, yet its functional significance in the establishment of these tumors is unknown. Here we have sought to determine the functional consequences of Otx2 overexpression in the mouse hindbrain to characterize its potential role in medulloblastoma tumorigenesis and identify the cell types responsive to this lineage-specific oncogene. Expression of Otx2 broadly in the mouse hindbrain resulted in the accumulation of proliferative clusters of cells in the cerebellar white matter and dorsal brainstem of postnatal mice. We found that brainstem ectopia were derived from neuronal progenitors of the rhombic lip and that cerebellar ectopia were derived from granule neuron precursors (GNPs) that had migrated inwards from the external granule layer (EGL). These hyperplasias exhibited various characteristics of medulloblastoma precursor cells identified in animal models of Shh or Wnt group tumors, including aberrant localization and altered spatiotemporal control of proliferation. However, ectopia induced by Otx2 differentiated and dispersed as the animals reached adulthood, indicating that factors restricting proliferative lifespan were a limiting factor to full transformation of these cells. These studies implicate a role for Otx2 in altering the dynamics of neuronal progenitor cell proliferation.


Subject(s)
Cell Movement , Gene Expression Regulation , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Neurons/cytology , Otx Transcription Factors/genetics , Rhombencephalon/cytology , Animals , Cell Differentiation , Cell Proliferation , Cerebellum/metabolism , Cerebellum/pathology , Female , Hedgehog Proteins/metabolism , Humans , Hyperplasia/genetics , Hyperplasia/pathology , Medulloblastoma/genetics , Medulloblastoma/pathology , Mice , Neural Stem Cells/pathology , Otx Transcription Factors/metabolism , Reproducibility of Results , Signal Transduction
7.
PLoS One ; 6(2): e16812, 2011 Feb 04.
Article in English | MEDLINE | ID: mdl-21326614

ABSTRACT

BACKGROUND: Gliomas frequently contain mutations in the cytoplasmic NADP(+)-dependent isocitrate dehydrogenase (IDH1) or the mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDH2). Several different amino acid substitutions recur at either IDH1 R132 or IDH2 R172 in glioma patients. Genetic evidence indicates that these mutations share a common gain of function, but it is unclear whether the shared function is dominant negative activity, neomorphic production of (R)-2-hydroxyglutarate (2HG), or both. METHODOLOGY/PRINCIPAL FINDINGS: We show by coprecipitation that five cancer-derived IDH1 R132 mutants bind IDH1-WT but that three cancer-derived IDH2 R172 mutants exert minimal binding to IDH2-WT. None of the mutants dominant-negatively lower isocitrate dehydrogenase activity at physiological (40 µM) isocitrate concentrations in mammalian cell lysates. In contrast to this, all of these mutants confer 10- to 100-fold higher 2HG production to cells, and glioma tissues containing IDH1 R132 or IDH2 R172 mutations contain high levels of 2HG compared to glioma tissues without IDH mutations (54.4 vs. 0.1 mg 2HG/g protein). CONCLUSIONS: Binding to, or dominant inhibition of, WT IDH1 or IDH2 is not a shared feature of the IDH1 and IDH2 mutations, and thus is not likely to be important in cancer. The fact that the gain of the enzymatic activity to produce 2HG is a shared feature of the IDH1 and IDH2 mutations suggests that this is an important function for these mutants in driving cancer pathogenesis.


Subject(s)
Brain Neoplasms/genetics , Glioma/genetics , Glutarates/metabolism , Isocitrate Dehydrogenase/genetics , Isocitrate Dehydrogenase/physiology , Binding, Competitive/genetics , Binding, Competitive/physiology , Brain Neoplasms/enzymology , Brain Neoplasms/metabolism , Cells, Cultured , Down-Regulation/genetics , Down-Regulation/physiology , Enzyme Activation/genetics , Genes, Dominant/physiology , Glioma/enzymology , Glioma/metabolism , Humans , Isocitrate Dehydrogenase/metabolism , Mutation, Missense/physiology
8.
Proc Natl Acad Sci U S A ; 108(8): 3270-5, 2011 Feb 22.
Article in English | MEDLINE | ID: mdl-21289278

ABSTRACT

Point mutations of the NADP(+)-dependent isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2) occur early in the pathogenesis of gliomas. When mutated, IDH1 and IDH2 gain the ability to produce the metabolite (R)-2-hydroxyglutarate (2HG), but the downstream effects of mutant IDH1 and IDH2 proteins or of 2HG on cellular metabolism are unknown. We profiled >200 metabolites in human oligodendroglioma (HOG) cells to determine the effects of expression of IDH1 and IDH2 mutants. Levels of amino acids, glutathione metabolites, choline derivatives, and tricarboxylic acid (TCA) cycle intermediates were altered in mutant IDH1- and IDH2-expressing cells. These changes were similar to those identified after treatment of the cells with 2HG. Remarkably, N-acetyl-aspartyl-glutamate (NAAG), a common dipeptide in brain, was 50-fold reduced in cells expressing IDH1 mutants and 8.3-fold reduced in cells expressing IDH2 mutants. NAAG also was significantly lower in human glioma tissues containing IDH mutations than in gliomas without such mutations. These metabolic changes provide clues to the pathogenesis of tumors associated with IDH gene mutations.


Subject(s)
Isocitrate Dehydrogenase/genetics , Metabolome/genetics , Mutation , Oligodendroglioma/genetics , Cell Line, Tumor , Dipeptides/analysis , Glioma/pathology , Glutarates/pharmacology , Humans , Oligodendroglioma/enzymology , Oligodendroglioma/pathology
9.
Am J Pathol ; 177(3): 1095-103, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20639454

ABSTRACT

Tumor-associated antigens, which induce the generation of autoantibodies, are useful as cancer biomarkers in early detection and prognostic prediction of cancer. To isolate a novel cancer marker, we used serum antibodies from colon cancer patients to screen a phage display peptide library. A positive peptide 249C (VPLYSNTLRYGF) that could specifically react with serum from colon cancer patients was isolated, and the corresponding antigen-human arrest defective 1 (ARD1A), which shares an identical LYSNTL motif with 249C, was identified. Both immunological assays and three-dimensional structure analysis showed that the LYSNTL region is an epitope of ARD1A. Using ELISA and immunohistochemistry, we found anti-ARD1A antibody levels in serum from patients with colon cancer were significantly higher than those in healthy volunteers (P < 0.001), and ARD1A expression was detected in 84.1% (227/270) of colon cancer tissues compared with 22.7% (55/242) of matched noncancerous tissues (P < 0.001) and 4.8% (2/42) of benign lesions (P < 0.001). Furthermore, multivariate analysis with Cox proportional hazards regression models revealed that ARD1A-positive patients had significantly shortened overall survival (OS) (HR, 1.91, P = 0.039) and borderline significantly shortened disease-free survival (DFS) (HR, 1.70; P = 0.068). Kaplan-Meier survival curves also showed that ARD1A expression was associated significantly with shortened DFS (P = 0.037) and OS (P = 0.019). These results indicate that ARD1A is a novel tumor-associated antigen and a potential prognostic factor for colon cancer.


Subject(s)
Acetyltransferases/blood , Antigens, Neoplasm/blood , Autoantibodies/blood , Colonic Neoplasms/blood , Colonic Neoplasms/mortality , Acetyltransferases/isolation & purification , Adult , Aged , Aged, 80 and over , Antigens, Neoplasm/isolation & purification , Biomarkers, Tumor/blood , Biomarkers, Tumor/isolation & purification , Blotting, Western , Cell Line, Tumor , Disease-Free Survival , Enzyme-Linked Immunosorbent Assay , Epitopes/isolation & purification , Humans , Kaplan-Meier Estimate , Middle Aged , N-Terminal Acetyltransferase A , N-Terminal Acetyltransferase E , Prognosis , Proportional Hazards Models
10.
Neuro Oncol ; 12(9): 956-66, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20472715

ABSTRACT

Glioblastoma multiforme (GBM) is one of the deadliest tumors afflicting humans, and the mechanisms of its onset and progression remain largely undefined. Our attempts to elucidate its molecular pathogenesis through DNA copy-number analysis by genome-wide digital karyotyping and single nucleotide polymorphism arrays identified a dramatic focal amplification on chromosome 1q32 in 4 of 57 GBM tumors. Quantitative real-time PCR measurements revealed that HDMX is the most commonly amplified and overexpressed gene in the 1q32 locus. Further genetic screening of 284 low- and high-grade gliomas revealed that HDMX amplifications occur solely in pediatric and adult GBMs and that they are mutually exclusive of TP53 mutations and MDM2 amplifications. Here, we demonstrate that HDMX regulates p53 to promote GBM growth and attenuates tumor response to chemotherapy. In GBM cells, HDMX overexpression inhibits p53-mediated transcriptional activation of p21, releases cells from G0 to G1 phase, and enhances cellular proliferation. HDMX overexpression does not affect the expression of PUMA and BAX proapoptotic genes. While in GBM cells treated with the chemotherapeutic agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), HDMX appears to stabilize p53 and promote phosphorylation of the DNA double-stranded break repair protein H2AX, up-regulate the DNA repair gene VPX, stimulate DNA repair, and confer resistance to BCNU. In summary, HDMX exhibits bona fide oncogenic properties and offers a promising molecular target for GBM therapeutic intervention.


Subject(s)
Brain Neoplasms/genetics , Gene Expression Regulation/genetics , Glioblastoma/genetics , Nuclear Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Tumor Suppressor Protein p53/metabolism , Adult , Animals , Antineoplastic Agents/pharmacology , Brain Neoplasms/drug therapy , Brain Neoplasms/metabolism , Carmustine/pharmacology , Cell Cycle Proteins , Child , Drug Resistance, Neoplasm/genetics , Gene Amplification , Gene Expression , Genome-Wide Association Study , Glioblastoma/drug therapy , Glioblastoma/metabolism , Humans , Immunoblotting , In Situ Hybridization, Fluorescence , Mice , Nuclear Proteins/genetics , Polymorphism, Single Nucleotide , Proto-Oncogene Proteins/genetics , RNA, Small Interfering , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Xenograft Model Antitumor Assays
11.
Biochem Biophys Res Commun ; 390(3): 547-51, 2009 Dec 18.
Article in English | MEDLINE | ID: mdl-19818334

ABSTRACT

IDH1 (isocitrate dehydrogenase 1) mutations have been identified as early and frequent genetic alterations in astrocytomas, oligodendrogliomas, and oligoastrocytomas as well as secondary glioblastomas. In contrast, primary glioblastomas very rarely contain IDH1 mutations, although primary and secondary glioblastomas are histologically indistinguishable. The IDH1 mutations are remarkably specific to a single codon in the conserved and functionally important Arg132 in IDH1. In gliomas, the most frequent IDH1 mutations (>90%) were G395A (R132H). In this study, we immunized mice with R132H-containing IDH1 (IDH1(R132H)) peptide. After cell fusion using Sendai virus envelope, the monoclonal antibodies (mAbs), which specifically reacted with IDH1(R132H), were screened in ELISA. One of the mAbs, IMab-1 reacted with the IDH1(R132H) peptide, but not with wild type IDH1 (IDH1(wt)) peptide in ELISA. In Western-blot analysis, IMab-1 reacted with only the IDH1(R132H) protein, not IDH1(wt) protein or the other IDH1 mutants, indicating that IMab-1 is IDH1(R132H)-specific. Furthermore, IMab-1 specifically stained the IDH1(R132H)-expressing cells in astrocytomas in immunohistochemistry, whereas it did not react with IDH1(R132H)-negative primary glioblastoma sections. In conclusion, we established an anti-IDH1(R132H)-specific monoclonal antibody IMab-1, which should be significantly useful for diagnosis and biological evaluation of mutation-bearing gliomas.


Subject(s)
Antibodies, Monoclonal/immunology , Glioma/enzymology , Isocitrate Dehydrogenase/immunology , Mutant Proteins/immunology , Animals , Arginine/genetics , Cell Line, Tumor , Glioma/diagnosis , Glioma/genetics , Histidine/genetics , Humans , Hybridomas , Immunohistochemistry , Isocitrate Dehydrogenase/analysis , Isocitrate Dehydrogenase/genetics , Mice , Mice, Inbred BALB C , Mutant Proteins/analysis , Mutant Proteins/genetics
12.
N Engl J Med ; 360(8): 765-73, 2009 Feb 19.
Article in English | MEDLINE | ID: mdl-19228619

ABSTRACT

BACKGROUND: A recent genomewide mutational analysis of glioblastomas (World Health Organization [WHO] grade IV glioma) revealed somatic mutations of the isocitrate dehydrogenase 1 gene (IDH1) in a fraction of such tumors, most frequently in tumors that were known to have evolved from lower-grade gliomas (secondary glioblastomas). METHODS: We determined the sequence of the IDH1 gene and the related IDH2 gene in 445 central nervous system (CNS) tumors and 494 non-CNS tumors. The enzymatic activity of the proteins that were produced from normal and mutant IDH1 and IDH2 genes was determined in cultured glioma cells that were transfected with these genes. RESULTS: We identified mutations that affected amino acid 132 of IDH1 in more than 70% of WHO grade II and III astrocytomas and oligodendrogliomas and in glioblastomas that developed from these lower-grade lesions. Tumors without mutations in IDH1 often had mutations affecting the analogous amino acid (R172) of the IDH2 gene. Tumors with IDH1 or IDH2 mutations had distinctive genetic and clinical characteristics, and patients with such tumors had a better outcome than those with wild-type IDH genes. Each of four tested IDH1 and IDH2 mutations reduced the enzymatic activity of the encoded protein. CONCLUSIONS: Mutations of NADP(+)-dependent isocitrate dehydrogenases encoded by IDH1 and IDH2 occur in a majority of several types of malignant gliomas.


Subject(s)
Brain Neoplasms/genetics , Glioma/genetics , Isocitrate Dehydrogenase/genetics , Mutation , Adult , Astrocytoma/genetics , Brain Neoplasms/enzymology , DNA Mutational Analysis , Disease Progression , Glioblastoma/genetics , Glioma/enzymology , Humans , Isocitrate Dehydrogenase/metabolism , Kaplan-Meier Estimate , Neoplasms/genetics , Oligodendroglioma/genetics , Tumor Cells, Cultured , Young Adult
13.
Cancer Lett ; 264(1): 83-92, 2008 Jun 08.
Article in English | MEDLINE | ID: mdl-18325661

ABSTRACT

Arrest defective 1 (ARD1) is an acetyltransferase involved in cell cycle control in yeast. ARD1 interacts with human N-acetyltransferase (NATH) to form a functional N-terminal acetyltransferase complex. Recently it had been linked with proliferation and apoptosis in mammalian cells, but its function in cancer development remains unclear. To evaluate significance of ARD1 expression in human colorectal cancer, we generated a panel of monoclonal antibodies (mAbs) with high specificity and sensitivity against ARD1. All of the 10 different clones could be used in ELISA and Western blot, and clone 10C12, 13G2, and 4D10 can interact with ARD1 in eukaryotic cells by immunoprecipitation (IP). Clones of 14D4 and 10C12 were strongly reacted to ARD1 in immunocytochemistry (ICH) and immunohistochemistry (IHC). ARD1 expression was evaluated in human colorectal cancer and colitis tissues by immunohistochemical analysis with mAb 14D4. Forty-one were ARD1-positive in 50 colorectal cancer tissues and only 12 were weak positive in the 50 matched normal tissues (P < 0.001). Moreover, ARD1 expression was not detectable in 20 cases of colitis tissue (P < 0.001). Furthermore, all of the six human colorectal cancer cell lines we examined were also ARD1-positive at mRNA and protein levels. Taken together, the novel mAbs against ARD1 we generated could be good tools for both basic and clinical studies, and ARD1 could be a potential biomarker in colorectal cancer.


Subject(s)
Acetyltransferases/biosynthesis , Antibodies, Monoclonal/chemistry , Biomarkers, Tumor/biosynthesis , Colorectal Neoplasms/metabolism , Acetyltransferases/analysis , Acetyltransferases/genetics , Animals , Antibodies, Monoclonal/biosynthesis , Antibody Specificity , Biomarkers, Tumor/analysis , Biomarkers, Tumor/genetics , Blotting, Western , Cell Line, Tumor , Colorectal Neoplasms/pathology , Enzyme-Linked Immunosorbent Assay , Humans , Hybridomas/chemistry , Hybridomas/metabolism , Immunohistochemistry , Immunoprecipitation , Mice , Mice, Inbred BALB C , N-Terminal Acetyltransferase A , N-Terminal Acetyltransferase E , RNA, Messenger/analysis , RNA, Messenger/biosynthesis , Recombinant Proteins/analysis , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics
14.
Cancer Sci ; 98(1): 37-43, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17052262

ABSTRACT

Although gastric cancer is the second leading cause of cancer death worldwide, specific and sensitive biomarkers that can be used for its diagnosis are still unavailable. Attempting to improve on current approaches to the serological diagnosis of gastric cancer, we subjected serum samples from 245 individuals (including 127 gastric cancer patients, 100 age- and sex-matched healthy individuals, nine benign gastric lesion patients and nine colorectal cancer patients) for analysis by surface-enhanced laser desorption/ionization (SELDI) mass spectrometry. Peaks were detected with Ciphergen SELDI software version 3.1.1 and analyzed with Biomarker Patterns' software 5.0. We developed a classifier for separating the gastric cancer groups from the healthy groups. Three protein masses with 1468, 3935 and 7560 m/z were selected as a potential 'fingerprint' for the detection of gastric cancer. It was able to distinguish the gastric cancer patients from the health volunteers with a sensitivity of 95.6% and a specificity of 92.0% in the training set. In the blinding set, it was capable of differentiating the gastric cancer samples from the others with a specificity of 88.0%, a sensitivity of 85.3%, and an accuracy of 86.4%. These values were all higher than those achieved in a parallel analysis by measuring serum carcinoembryonic antigen (CEA) and carbohydrate antigen (CA)19-9 together. Therefore, the decision tree analysis of serum proteomic patterns has the potential to be used in gastric cancer diagnosis.


Subject(s)
Biomarkers, Tumor/blood , Decision Trees , Proteomics/methods , Stomach Neoplasms/blood , Stomach Neoplasms/diagnosis , Adolescent , Adult , Aged , Aged, 80 and over , CA-19-9 Antigen/blood , Carcinoembryonic Antigen/blood , Female , Humans , Male , Middle Aged , Sensitivity and Specificity , Software , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
15.
Hum Mol Genet ; 15(11): 1847-57, 2006 Jun 01.
Article in English | MEDLINE | ID: mdl-16632484

ABSTRACT

Centrosome- and cilia-associated proteins play crucial roles in establishing polarity and regulating intracellular transport in post-mitotic cells. Using genetic mapping and positional candidate strategy, we have identified an in-frame deletion in a novel centrosomal protein CEP290 (also called NPHP6), leading to early-onset retinal degeneration in a newly identified mouse mutant, rd16. We demonstrate that CEP290 localizes primarily to centrosomes of dividing cells and to the connecting cilium of retinal photoreceptors. We show that, in the retina, CEP290 associates with several microtubule-based transport proteins including RPGR, which is mutated in approximately 15% of patients with retinitis pigmentosa. A truncated CEP290 protein (DeltaCEP290) is detected in the rd16 retina, but in considerably reduced amounts; however, the mutant protein exhibits stronger association with specific RPGR isoform(s). Immunogold labeling studies demonstrate the redistribution of RPGR and of phototransduction proteins in the photoreceptors of rd16 retina. Our findings suggest a critical function for CEP290 in ciliary transport and provide insights into the mechanism of early-onset photoreceptor degeneration.


Subject(s)
Antigens, Neoplasm/chemistry , Antigens, Neoplasm/genetics , Carrier Proteins/metabolism , Centrosome/ultrastructure , Eye Proteins/genetics , Gene Deletion , Nuclear Proteins/genetics , Retinal Degeneration/genetics , Animals , Base Sequence , Carrier Proteins/genetics , Cell Cycle Proteins , Centrosome/metabolism , Cytoskeletal Proteins , Disease Models, Animal , Eye Proteins/metabolism , Humans , Mice , Models, Genetic , Molecular Sequence Data , Mutation , Nuclear Proteins/physiology , Protein Binding
16.
Biochem Biophys Res Commun ; 342(1): 179-83, 2006 Mar 31.
Article in English | MEDLINE | ID: mdl-16472776

ABSTRACT

PRL-3 is a newly identified protein tyrosine phosphatase associated with tumor metastasis. It is over-expressed in various cancers, such as colorectal cancer, gastric cancer, and ovarian cancer, and is correlated with the progression and survival of cancers. Although PRL-3 plays a causative role in promoting cancer cell invasion and metastasis, the molecular mechanism is unknown. To investigate PRL-3's roles in tumorigenesis and signal transduction pathway, we screened the human placenta brain cDNA library with the bait of PRL-3 in yeast two-hybrid system. Then we identified integrin alpha1 as a PRL-3-interacting protein for the first time, and verified this physical association with pull-down and co-immunoprecipitation assays. Furthermore, we found that PRL-3 could down-regulate the tyrosine-phosphorylation level of integrin beta1 and increased the phosphorylation level of Erk1/2. Our present discovery will provide new clues for elucidating the molecular mechanism of PRL-3 in promoting cancer invasion and metastasis.


Subject(s)
Immediate-Early Proteins/metabolism , Integrin alpha1/metabolism , Neoplasm Proteins/metabolism , Protein Tyrosine Phosphatases/metabolism , Amino Acid Sequence , Cell Line , Down-Regulation , Humans , Immediate-Early Proteins/genetics , Integrin alpha1/chemistry , Integrin alpha1/genetics , Integrin alpha1/isolation & purification , Integrin beta Chains/chemistry , Integrin beta Chains/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Molecular Sequence Data , Neoplasm Proteins/genetics , Phosphorylation , Phosphotyrosine/metabolism , Protein Binding , Protein Tyrosine Phosphatases/genetics
17.
Int J Cancer ; 118(8): 1922-9, 2006 Apr 15.
Article in English | MEDLINE | ID: mdl-16287078

ABSTRACT

Stem cell genetics research may be critical to our understanding of carcinogenesis, as both stem cells and cancer cells possess the ability to self-renew. Recent discoveries have indicated that the piwi family of genes plays an essential role in stem cell self-renewal in diverse organisms. The hiwi gene, the human homolog of the piwi family, participates in germ cell proliferation and its overexpression may cause the development of germ cell malignancy, but its expression and function in epithelial solid cancers have not been explored. In the present study, we investigated whether there was an association between hiwi expression and human gastric cancer and its potential mechanism. RT-PCR findings demonstrated that hiwi was expressed in different gastric cancer cell lines. To identify the HIWI protein in gastric cancer, we developed a specific monoclonal antibody against HIWI and immunohistochemistry was performed on various gastric tissues. We found that the expression ratio of hiwi in normal gastric tissues, atrophic gastritis, intestinal metaplasia and gastric cancers was 10% (5/50), 36% (18/50), 36% (18/50) and 76% (38/50), respectively, which was consistent with precancerous development. Notably, the expression pattern of hiwi in gastric cancer tissues was similar to that of Ki67, which was used as a marker of proliferation. Moreover, the suppression of hiwi by antisense or RNAi inhibited the growth of gastric cancer cells and induced cell cycle arrest in G2/M phase. These results suggest that hiwi may be involved in the development of gastric cancer and is a potential target for cancer therapy.


Subject(s)
Proteins/genetics , Stomach Neoplasms/genetics , Stomach Neoplasms/pathology , Antibodies, Monoclonal , Argonaute Proteins , Cell Cycle/physiology , Cell Proliferation , Gene Expression Profiling , Humans , Immunohistochemistry , Precancerous Conditions , Proteins/physiology , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured
18.
World J Gastroenterol ; 11(31): 4812-4, 2005 Aug 21.
Article in English | MEDLINE | ID: mdl-16097049

ABSTRACT

AIM: To identify the proteins interacting with nucleostemin (NS), thereby gaining an insight into the function of NS. METHODS: Yeast two-hybrid assay was performed to screen a human placenta cDNA library with the full length of NS as a bait. X-Gal assay and beta-galactosidase filter assay were subsequently conducted to check the positive clones and the gene was identified by DNA sequencing. To further confirm the interaction of two proteins, the DNA fragment coding NS and the DNA fragment isolated from the positive clone were inserted into the mammalian expression vector pcDNA3 and pcDNA3-myc, respectively. Then, two plasmids were cotransfected into the COS-7 cells by DEAE-dextron. The total protein from the cotransfected cells was extracted and coimmunoprecipitation and Western blot were performed with suitable antibodies sequentially. RESULTS: Two positive clones that interacted with NS were obtained from human placenta cDNA library. One was an alpha isoform of human protein phosphatase 2 regulatory subunit B (B56) (PPP2R5A) and the other was a novel gene being highly homologous to the gene associated with spondylo paralysis. The co-immunoprecipitation also showed that NS specifically interacted with PPP2R5A. CONCLUSION: NS and PPP2R5A interact in yeast and mammalian cells, respectively, which is helpful for addressing the function of NS in cancer development and progression.


Subject(s)
Carrier Proteins/metabolism , Nuclear Proteins/metabolism , Animals , COS Cells , Chlorocebus aethiops , Cloning, Molecular , Female , GTP-Binding Proteins , Humans , Placenta/metabolism , Plasmids , Pregnancy , Recombinant Proteins/metabolism
19.
Beijing Da Xue Xue Bao Yi Xue Ban ; 37(3): 297-301, 2005 Jun 18.
Article in Chinese | MEDLINE | ID: mdl-15968324

ABSTRACT

OBJECTIVE: To understand the mechanism of cancer and embryo expression protein 65 (CEP65) in cancer development. METHODS: CEP65 was used as a bait protein to isolate the partners of CEP65 from a human placenta cDNA library with yeast two hybrid system. The DNA fragments from positive clone were expressed prokaryotic and mammalian cells respectively, the GST pull-down experiment was performed to ascertain the binding activity. RESULTS: After screening a human placenta cDNA library, the low density lipoprotein receptor-related protein-associated protein 1 (RAP) was isolated and shown to interact with CEP65. GST pull-down assay results indicated that His-CEP65 and GST-RAP expressed by prokaryotic system were immunoprecipitated, and so were Myc-RAP and GST-P65 expressed by mammalian cells. CONCLUSION: RAP can interact with CEP65 and RAP may be a candidate to mediate the function of CEP65.


Subject(s)
Antigens, Neoplasm/metabolism , Low Density Lipoprotein Receptor-Related Protein-1/metabolism , Neoplasm Proteins/metabolism , Neoplasms/metabolism , Transfection , Animals , COS Cells , Cell Cycle Proteins , Chlorocebus aethiops , Cytoskeletal Proteins , Humans , Plasmids , Protein Binding , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Two-Hybrid System Techniques
20.
Ai Zheng ; 24(5): 573-6, 2005 May.
Article in Chinese | MEDLINE | ID: mdl-15890100

ABSTRACT

BACKGROUND & OBJECTIVE: Previous studies show that cancer and embryo expression protein 65 (CEP65) only expresses in various cancer cells and embryo cells. This study was to detect intracellular distribution of CEP65, and make the foundation for further CEP65 function study. METHODS: Two kinds of eukaryotic expression plasmids, which can separately express full-length CEP65-GST and fluorescent fusion protein CEP65-Red, were constructed, and transfected into COS7 cells by DEAE-Dextran method. Plasmosin and nucleoprotein of COS7 cells were separated. Intracellular distribution of CEP65 was detected by Western blot, and observed under fluorescent microscope. RESULTS: Western blot confirmed that CEP65 distributed in cytoplasm and nuclei. Fluorescent protein localization showed diffuse distribution of fluorescence in the cells transfected with pcDNA3-Red, and collective fluorescent articles in cytoplasm and nuclei of the cells transfected with pcDNA3-Red-Cep65. CONCLUSIONS: CEP65 can express efficiently in COS7 cells, and locates in cytoplasm and nuclei. It may be a kind of nucleus-associated protein.


Subject(s)
Antigens, Neoplasm/metabolism , Cell Nucleus/metabolism , Cytoplasm/metabolism , Neoplasm Proteins/metabolism , Transfection , Animals , Antigens, Neoplasm/genetics , COS Cells , Cell Cycle Proteins , Chlorocebus aethiops , Cytoskeletal Proteins , Neoplasm Proteins/genetics , Plasmids , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...