ABSTRACT
Runt-related transcription factor 3 (RUNX3) is a well-documented tumour suppressor that is frequently inactivated in gastric cancer. Here, we define a novel mechanism by which RUNX3 exerts its tumour suppressor activity involving the TEAD-YAP complex, a potent positive regulator of proliferative genes. We report that the TEAD-YAP complex is not only frequently hyperactivated in liver and breast cancer, but also confers a strong oncogenic activity in gastric epithelial cells. The increased expression of TEAD-YAP in tumour tissues significantly correlates with poorer overall survival of gastric cancer patients. Strikingly, RUNX3 physically interacts with the N-terminal region of TEAD through its Runt domain. This interaction markedly reduces the DNA-binding ability of TEAD that attenuates the downstream signalling of TEAD-YAP complex. Mutation of RUNX3 at Arginine 122 to Cysteine, which was previously identified in gastric cancer, impairs the interaction between RUNX3 and TEAD. Our data reveal that RUNX3 acts as a tumour suppressor by negatively regulating the TEAD-YAP oncogenic complex in gastric carcinogenesis.
Subject(s)
Core Binding Factor Alpha 3 Subunit/metabolism , Stomach Neoplasms/pathology , Transcription Factors/metabolism , Amino Acid Sequence , Carcinogenesis , Cell Line, Tumor , Core Binding Factor Alpha 3 Subunit/chemistry , Core Binding Factor Alpha 3 Subunit/genetics , DNA/metabolism , Epithelial Cells/metabolism , Humans , Mutation , Protein Conformation, alpha-Helical , Protein Domains , Stomach Neoplasms/metabolism , Transcription Factors/chemistryABSTRACT
The study of RUNX3 in tumor pathogenesis is a rapidly expanding area of cancer research. Functional inactivation of RUNX3-through mutation, epigenetic silencing, or cytoplasmic mislocalization-is frequently observed in solid tumors of diverse origins. This alone indicates that RUNX3 inactivation is a major risk factor in tumorigenesis and that it occurs early during progression to malignancy. Conversely, RUNX3 has also been described to have an oncogenic function in a subset of tumors. Although the mechanism of how RUNX3 switches from tumor suppressive to oncogenic activity is unclear, this is of clinical relevance with implications for cancer detection and prognosis. Recent developments have significantly contributed to our understanding of the pleiotropic tumor suppressive properties of RUNX3 that regulate major signaling pathways. This review summarizes the important findings that link RUNX3 to tumor suppression.
Subject(s)
Core Binding Factor Alpha 3 Subunit/physiology , Neoplasms/etiology , Animals , Cellular Senescence , Core Binding Factor Alpha 3 Subunit/antagonists & inhibitors , Core Binding Factor Alpha 3 Subunit/genetics , Cytoplasm/metabolism , DNA Methylation , DNA Repair , Gene Deletion , Gene Silencing , Humans , Mutagenesis, Insertional , Protein Transport , Signal TransductionABSTRACT
DNA lesions that halt RNA polymerase during transcription are preferentially repaired by the nucleotide excision repair pathway. This transcription-coupled repair is initiated by the arrested RNA polymerase at the DNA lesion. However, the mutagenic O6-methylguanine (6MG) lesion which is bypassed by RNA polymerase is also preferentially repaired at the transcriptionally active DNA. We report here a plausible explanation for this observation: the human 6MG repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) is present as speckles concentrated at active transcription sites (as revealed by polyclonal antibodies specific for its N and C termini). Upon treatment of cells with low dosages of N-methylnitrosourea, which produces 6MG lesions in the DNA, these speckles rapidly disappear, accompanied by the formation of active-site methylated MGMT (the repair product of 6MG by MGMT). The ability of MGMT to target itself to active transcription sites, thus providing an effective means of repairing 6MG lesions, possibly at transcriptionally active DNA, indicates its crucial role in human cancer and chemotherapy by alkylating agents.
Subject(s)
DNA Damage , DNA Repair , Guanine/analogs & derivatives , Mutagenesis , O(6)-Methylguanine-DNA Methyltransferase/metabolism , Transcription, Genetic , Alkylating Agents/pharmacology , Alkylation , Animals , Antibodies, Monoclonal , Binding Sites , Blotting, Western , Cell Cycle , Cell Line , Cell Nucleus/metabolism , Deoxyribonuclease I/metabolism , Epitope Mapping , Epitopes, B-Lymphocyte , HeLa Cells , Humans , Methylnitrosourea/pharmacology , RabbitsABSTRACT
DNA-(cytosine-5) methyltransferase (MCMT) methylates newly replicated mammalian DNA, but the factors regulating this activity are unknown. Here, MCMT is shown to bind proliferating cell nuclear antigen (PCNA), an auxiliary factor for DNA replication and repair. Binding of PCNA requires amino acids 163 to 174 of MCMT, occurs in intact cells at foci of newly replicated DNA, and does not alter MCMT activity. A peptide derived from the cell cycle regulator p21(WAF1) can disrupt the MCMT-PCNA interaction, which suggests that p21(WAF1) may regulate methylation by blocking access of MCMT to PCNA. MCMT and p21(WAF1) may be linked in a regulatory pathway, because the extents of their expression are inversely related in both SV40-transformed and nontransformed cells.
Subject(s)
Cyclins/metabolism , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA Methylation , Proliferating Cell Nuclear Antigen/metabolism , Amino Acid Sequence , Cell Line, Transformed , Cell Nucleus/metabolism , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/chemistry , DNA Damage , DNA Repair , DNA Replication , Humans , Molecular Sequence Data , Peptides/pharmacology , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/pharmacology , Tumor Cells, CulturedABSTRACT
We report here a detailed mapping and characterisation of a DNA-binding domain at the N terminus of human DNA-(cytosine-5) methyltransferase. A small region, B1 (codon 202 to 369), was first identified by its Zn- and gross DNA-binding properties. Further fine-mapping using deletion and point mutation analysis shows that the DNA- and Zn-binding domains involve separate peptide motifs, KRRKTTPKEPTEKK (codons 202 to 215) for a bipartite DNA-binding oligopeptide (DB1) and CX2CX13HX2D(X)23EX2EX13CX3H (codons 232 to 297) for possibly two contiguous Zn-binding domains (AZn), which can function independently. However, B1 (containing DB1 and AZn) differs from DB1 because it does not bind to a 30 base-pair duplex. Interestingly, H3 codons 202 to 974, which encloses B1 and B2 (containing the Zn-binding CX2CX2CX4CX2CX2C motif from codon 533 to 550) binds preferentially to 0.8 kb duplexes, as compared with 0.4 to 0.6 kb duplexes. As the homologous murine B1, which targets the murine methylase to replication foci, also binds to DNA and Zn, it is possible that the N terminus of mammalian methylase may be involved in sensing the appropriate length of newly synthesized DNA before methylation by its C terminus. This may enable a time delay for the transient existence of hemi-methylation sites for their unknown biological functions in mammals.
Subject(s)
DNA (Cytosine-5-)-Methyltransferases/chemistry , DNA/metabolism , Zinc Fingers , Zinc/metabolism , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Base Sequence , Binding Sites , Binding, Competitive , Cloning, Molecular , Conserved Sequence , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA (Cytosine-5-)-Methyltransferases/metabolism , Electrophoresis, Polyacrylamide Gel , Gene Expression , Humans , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Point Mutation , Protein Structure, Secondary , Zinc Fingers/geneticsABSTRACT
Elementary form of nitrogen (protein), phosphorus, potassium and magnesium contained in 180 different kinds of Chinese medicines are analyzed by means of 14-MeV neutron activation technique. The percent contents of these elements in the Chinese medicines range from 0 to 34.41% (average: 11.73%) for protein, from 0.03 to 37.42% (average: 1.72%) for phosphorus, from 0.12 to 33.22% (average: 2.94%) for potassium and 0.03 to 5.62% (average: 0.43%) for magnesium. Comparison of the present results with the previous measurements for another 66 kinds of Chinese medicines is made.
Subject(s)
Activation Analysis/methods , Magnesium/analysis , Medicine, Chinese Traditional , Medicine, East Asian Traditional , Neutron Activation Analysis/methods , Phosphorus/analysis , Plants, Medicinal/analysis , Potassium/analysis , Proteins/analysisABSTRACT
Elementary form of nitrogen (protein), phosphorus, potassium and magnesium contained in sixty-six different kinds of Chinese medicines were analyzed by means of 14-MeV neutron activation technique. The % contents of these elements in Chinese medicines range from 4.1 to 38.6% (average: 12.7%) for protein, 0.1 to 10.5% (average: 1.3%) for phosphorus, 1.6 to 13.8% (average 4.5%) for potassium and 0.1 to 2.5% (average: 0.5%) for magnesium. The procedure proves to be quite precise and potentially quite accurate, while only requiring about 20 minutes per sample. The methods of determining the elements are described.
