ABSTRACT
BACKGROUND: Thromboembolic events are a major complication in ovarian cancer patients. Tissue factor (TF) is frequently overexpressed in ovarian cancer tissue and correlates with intravascular thrombosis. TF binds to coagulation factor VII (fVII), changing it to its active form, fVIIa. This leads to activation of the extrinsic coagulation cascade. fVII is produced by the liver and believed to be supplied from blood plasma at the site of coagulation. However, we recently showed that ovarian cancer cells express fVII transcripts under normoxia and that this transcription is inducible under hypoxia. These findings led us to hypothesise that ovarian cancer cells are intrinsically associated with TF-fVIIa coagulation activity, which could result in thrombosis. METHODS: In this study, we examined whether ectopically expressed fVII could cause thrombosis by means of immunohistochemistry, RT-PCR, western blotting and flow cytometry. RESULTS: Ectopic fVII expression occurs frequently in ovarian cancers, particularly in clear cell carcinoma. We further showed that ovarian cancer cells express TF-fVIIa on the cell surface under normoxia and that this procoagulant activity is enhanced by hypoxic stimuli. Moreover, we showed that ovarian cancer cells secrete microparticles (MPs) with TF-fVIIa activity. Production of this procoagulant secretion is enhanced under hypoxia. CONCLUSION: These results raise the possibility that cancer cell-derived TF-fVIIa could cause thrombotic events in ovarian cancer patients.
Subject(s)
Factor VII/metabolism , Ovarian Neoplasms/metabolism , Thromboplastin/metabolism , Venous Thromboembolism/etiology , Cell Hypoxia , Cell Line, Tumor , Cell-Derived Microparticles/metabolism , Female , Humans , Neoplasms, Glandular and Epithelial/chemistry , Ovarian Neoplasms/complications , Ovarian Neoplasms/pathologyABSTRACT
Pelizaeus-Merzbacher disease (PMD) is a rare X-linked leukodystrophy caused by proteolipid protein 1 (PLP1) gene mutations. Previous studies indicated that proteolipid proteins (PLPs) with disease-associated mutations are misfolded and trapped in the endoplasmic reticulum (ER) during transportation to the cell surface, which eventually leads to oligodendrocyte cell death in PMD. Here we report a PMD patient with a very mild phenotype carrying a novel mutation (485G-->T) in exon 4 of the PLP1 gene that causes a Trp(162)Leu substitution in the protein. We also investigated intracellular trafficking of this mutant PLP in COS-7 cells. Transiently transfected mutant PLP(W162L) fused to an enhanced green fluorescent protein (EGFP) or a short peptide tag was not carried to the plasma membrane. However, in contrast to previous studies, this mutant PLP was not retained in the ER, indicating an escape of the newly translated protein from the quality control machinery. We also found that the mutant PLP accumulated in the nuclear envelope (NE) in a time-dependent manner. This mutant PLP, with its distribution outside the ER and a very mild phenotype, supports the idea that accumulation of misfolded mutant protein in the ER causes the severe phenotype of PMD.
Subject(s)
Membrane Proteins/metabolism , Myelin Proteolipid Protein/metabolism , Pelizaeus-Merzbacher Disease/metabolism , Animals , COS Cells , Child, Preschool , Chlorocebus aethiops , Cloning, Molecular/methods , Exons , Fluorescent Antibody Technique/methods , Gene Expression/genetics , Green Fluorescent Proteins/metabolism , Humans , Leucine/genetics , Magnetic Resonance Imaging/methods , Male , Membrane Proteins/genetics , Mutagenesis/physiology , Mutation/genetics , Myelin Proteolipid Protein/genetics , Pelizaeus-Merzbacher Disease/genetics , Pelizaeus-Merzbacher Disease/pathology , Protein Transport/physiology , Receptors, Peptide/metabolism , Subcellular Fractions/metabolism , Time Factors , Transfection/methods , Tryptophan/geneticsABSTRACT
We found that single-stranded DNA oligomers containing a 7, 8-dihydro-8-oxoguanine (8-oxo-G) residue have high reactivity toward KMnO4; the oxidation of 8-oxo-G induces damage to the neighboring nucleotide residues. This paper describes the novel reaction in detail, including experiments that demonstrate the mechanism involved in the induction of DNA damage. The results using DNAs of various base compositions indicated that damaged G, T and C (but not A) sites caused strand scissions after hot piperidine treatment and that the damage around the 8-oxo-G occurred at G sites in both single and double strands with high frequency. The latter substrates were less sensitive to damage. Further, kinetic studies of the KMnO4reaction of single-stranded oligomers suggested that thereactivity of the DNA bases at the site 5'-adjacent to the 8-oxo-G was in the order G >A >T, C. This preference correlates with the electron donating abilities of the bases. In addition, we found that the DNA damage at the G site, which was connected with the 8-oxo-G by a long abasic chain, was inhibited in the above order by the addition of dG, dA or dC. On the other hand, the damage reactions proceeded even after the addition of scavengers for active oxygen species. This study suggests the involvement of a redox process in the unique DNA damage initiated by the oxidation of the 8-oxo-G.
Subject(s)
DNA Damage , Guanine/analogs & derivatives , Manganese Compounds/metabolism , Oxidants/metabolism , Oxides/metabolism , Base Composition , DNA, Single-Stranded , Guanine/metabolism , Kinetics , Manganese Compounds/pharmacology , Nucleosides , Oxidants/pharmacology , Oxidation-Reduction , Oxides/pharmacology , Oxygen , Phosphates , RiboseABSTRACT
New types of DNA substrates containing an 8-oxoguanine residue (8-oxo-G) were prepared in order to examine the mechanisms for the neighboring base damage initiated by KMnO4 oxidation of the 8-oxo-G. The results obtained from the reactions suggested that the damage at remote sites in the single strands can be explained by an electronic interaction (redox reaction) between an oxidized 8-oxo-G species and the base (to be damaged), which are close each other in a loop structure. For the inefficient damage observed in duplex substrates, electron transfer through stacked bases might be involved.
Subject(s)
DNA Damage , DNA/chemistry , Guanine/analogs & derivatives , Oligodeoxyribonucleotides/chemistry , Potassium Permanganate , Base Sequence , Hydrolysis , Nucleic Acid Conformation , Oligodeoxyribonucleotides/chemical synthesis , Oxidation-ReductionABSTRACT
We previously reported that 8-hydroxyguanine (7,8-dihydro-8-oxoguanine) at the second position of codon 12 of the c-Ha-ras gene induces many types of mutations in NIH3T3 cells. In this study we incorporated the modified base into the first and second positions of codon 12 in the coding strand and into the first position of codon 61 in the non-coding strand of the gene using a new 8-hydroxyguanine phosphoramidite as a building block during oligonucleotide synthesis. The ras genes with 8-hydroxyguanine were transfected into NIH3T3 cells and the mutations induced were analyzed. 8-Hydroxyguanine residues at the first positions of codons 12 and 61 induced mutations to T at the modified sites almost exclusively. On the other hand, the DNA lesion at the second position of codon 12 induced a G-->A transition in addition to a G-->T transversion, confirming our previous results. Mutations in 5'-flanking sites were observed with 8-hydroxyguanine at the second position of codon 12 or the first position of codon 61. These results indicate that 8-hydroxyguanine in mammalian cells mainly induces a G-->T transversion at the modified site, but that other types of mutations are also elicited.
