ABSTRACT
Increasing epidemiological and experimental evidence implicates non-steroidal anti-inflammatory drugs (NSAIDs) as anti-tumorigenic agents. The precise mechanisms whereby NSAIDs exert their anti-neoplastic effects remain poorly understood. Studies from hereditary and sporadic colorectal cancer (CRC) patients suggest that NSAIDs may interfere with initiating steps of carcinogenesis, i.e. disturbances within the beta-catenin signaling pathway. We therefore investigated beta-catenin/TCF signaling in response to aspirin or indomethacin, respectively, in four CRC cell lines (SW948, SW480, HCT116, LoVo). Both, aspirin and indomethacin inhibited transcription of a beta-catenin/TCF-responsive reporter gene in a dose dependent manner. In addition, the beta-catenin/TCF transcriptional target cyclin D1 was downregulated by both drugs. Endogenous beta-catenin levels remained unaffected by either drug. Moreover, indirect immunofluorescence studies revealed no significant changes of subcellular beta-catenin localization in either cell line after NSAID treatment. Likewise, binding of the beta-catenin/TCF complex to its specific DNA-binding sites was not altered, as demonstrated by electrophoretic mobility shift assay (EMSA) of nuclear extracts derived from NSAID treated cells. These results strongly suggest that aspirin and indomethacin attenuate the transcription of beta-catenin/TCF-responsive genes, by modulating TCF activity without disrupting beta-catenin/TCF complex formation.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Aspirin/pharmacology , Cytoskeletal Proteins/physiology , Indomethacin/pharmacology , Signal Transduction/drug effects , Trans-Activators , Transcription Factors/physiology , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Cyclin D1/biosynthesis , Cyclin D1/genetics , Cyclooxygenase 2 , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , DNA, Neoplasm/genetics , DNA, Neoplasm/metabolism , Down-Regulation/drug effects , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Membrane Proteins , Prostaglandin-Endoperoxide Synthases/metabolism , Response Elements , Subcellular Fractions/metabolism , TCF Transcription Factors , Transcription Factor 7-Like 2 Protein , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic/drug effects , Tumor Cells, Cultured , beta CateninABSTRACT
We report the cloning and characterization of the human eukaryotic protein translation initiation factor EIF2C1 gene. The human EIF2C1 gene consists of 19 exons and 18 introns that span a region of almost 50 kb. It is located on the short arm of chromosome 1 in the region 1p34-p35. This genomic region is frequently lost in human cancers such as Wilms tumors, neuroblastoma, and carcinomas of the breast, liver, and colon. The human EIF2C1 gene is ubiquitously expressed at low to medium levels. Differential polyadenylation and splicing result in a complex transcriptional pattern. The cDNA sequence is 7478 bp long and contains an extremely large 3' untranslated region of 4799 bp with multiple, short repeated segments composed of mono-, tri-, or quattronucleotides interspersed throughout. The human EIF2C1 gene belongs to a multigene family in human. It is highly conserved during evolution, sharing about 90% identity with rabbit eIF2C and 70% identity with plant AGO1 at the amino acid level. These facts suggest that human EIF2C1 might play an important physiological role.
Subject(s)
Chromosomes, Human, Pair 1 , Eukaryotic Initiation Factors , Peptide Initiation Factors/genetics , Adult , Amino Acid Sequence , Argonaute Proteins , Base Sequence , Blotting, Northern , Chromosome Banding , Chromosome Mapping , Cloning, Molecular , Conserved Sequence , Female , Fetus/metabolism , Humans , In Situ Hybridization, Fluorescence , Molecular Sequence Data , Peptide Initiation Factors/metabolism , RNA, Messenger/metabolism , Sequence Alignment , Transcription, GeneticABSTRACT
Inactivation of the adenomatous polyposis coli (APC) gene product initiates colorectal tumorigenesis. Patients with familial APC (FAP) carry germ-line mutations in the APC gene and develop multiple colorectal adenomas and subsequent carcinomas early in life. The severity of the disease correlates with the position of the inherited APC mutation (genotype-phenotype correlation). Together with the fact that both germ-line and sporadic APC mutations cluster in the central region of the APC gene, this points to a dominant negative effect of certain APC mutants. Loss of APC function was recently shown to result in enhanced beta-catenin-/Tcf-mediated transcription in colon epithelial cells. Here, we provide experimental evidence for a dominant negative effect of APC gene products associated with severe polyposis. Wild-type APC activity in beta-catenin-/Tcf-mediated transcription was strongly inhibited by a mutant APC that is truncated at codon 1309. In contrast, mutant APC gene products that are associated with attenuated polyposis (codon 386 or 1465) interfered only weakly with wild-type APC activity. These results suggest a molecular explanation for the genotype-phenotype correlation in FAP patients and support the idea that colorectal tumor growth might be, in part, driven by selection for a mutation in the mutation cluster region.
Subject(s)
Adenomatous Polyposis Coli/genetics , Cytoskeletal Proteins/genetics , Adenomatous Polyposis Coli Protein , Colorectal Neoplasms/genetics , Genes, Reporter , Genotype , Humans , Mutation , PhenotypeABSTRACT
We have analyzed soft-tissue sarcomas (STS) molecularly for mutations in the tumor-suppressor gene p53 and immunohisto-chemically for expression of p53 and mdm2 proteins. In this study, tumor samples from 3 groups of soft-tissue sarcomas, i.e., fibrosarcomas, myogenic sarcomas and malignant neural tumors (MNT), were investigated. The methods applied encompass immunohistochemistry on 198 tumor samples using p53 antibodies (DO-1 and DO-7) and an mdm2 antibody (IF-2). Out of these, 100 samples were subjected to non-radioactive PCR-SSCP-sequencing analysis. Immunohistochemical detection rate for p53 (range of 57% to 67%) and for mdm2 proteins (range of 19 to 44%) was similar in all 3 groups. In higher tumor grades, an increased rate of immunopositivity was found for p53 but not for mdm2. Investigation of p53 mutational status revealed 6 mutations in myogenic sarcomas but none in malignant neural tumors or fibrosarcomas, suggesting different roles of p53 in the 3 STS groups. Interestingly, a G-->A transition in codon 245 (a CpG site) was found in 3 myogenic sarcomas. Our results and those of others suggest p53 codon 245 as a mutational hotspot in sarcomas, as recognized in carcinomas.
Subject(s)
Fibrosarcoma/genetics , Genes, p53/genetics , Leiomyosarcoma/genetics , Mutation , Neoplasms, Nerve Tissue/genetics , Rhabdomyosarcoma/genetics , Base Sequence , Codon , Humans , Molecular Sequence Data , Polymerase Chain Reaction , Polymorphism, Single-Stranded ConformationalABSTRACT
Toxic shock syndrome, caused by an exotoxin of staphylococcus aureus is very rare in children. On admission, beside the shock, abdominal problems as vomiting, diarrhoea and a developing adynamic ileus were outstanding in our patient. Not before additional symptoms as staphylococcal pneumonia with bacteriemia occurred and later desquamation of palms and feet, diagnosis of toxic shock syndrome could be confirmed.
