Malignant ovarian ascites are rich in cellular components, membranebound vesicles, and soluble proteins. This study focused on the structure of membrane-bound vesicles and their ability to promote invasion in cultured malignant ovarian epithelium. Membrane vesicles were derived from women with stage I-IV malignant ovarian ascites and from nonmalignant gynecologic ascites. Isolated vesicles were characterized by immunofluorescence and Western blot analysis. Using gel zymography for matrix metalloproteinase (MMP) detection and a colorimetric assay for urokinase-type plasminogen activator (uPA) analysis, we analyzed the proteinase activities of MMP-2, MMP-9, and uPA from the prepared vesicles, whole cells isolated from ascites, and the cell-free ultracentrifuged supernatant. The invasiveness of established cultured malignant ovarian epithelium on addition of ascites-derived vesicles was tested using a Matrigel-based invasion assay. Fractionation of malignant ascites revealed that extracellular matrix-degrading proteinases including MMPs and uPA are localized preferentially in membrane vesicles. All malignant vesicles tested, regardless of cancer stage, stimulated invasion. Furthermore, the combination of ovarian cancer cells and membrane vesicles resulted in greater uPA activation than that of cells or vesicles alone. Membrane vesicles from malignant ascites were also found to contain activated MMP-2, MMP-9, and uPA. Our data suggest that vesiclestimulated proteinase activation leads to increased extracellular matrix degradation, which may facilitate tumor cell invasion and metastasis.
The Caenorhabditis elegans sex determination gene, tra-2, is translationally regulated by elements in the 3Ј-untranslated region called TGEs. TGEs govern the translation of mRNAs in both invertebrates and vertebrates, indicating that this is a highly conserved mechanism for controlling gene activity. A factor called DRF, found in worm extracts binds the TGEs and may be a repressor of translation. Using the yeast threehybrid screen and RNA gel shift analysis, we have found that the protein GLD-1, a germline-specific protein and a member of the STAR family of RNAbinding proteins, specifically binds to the TGEs. GLD-1 is essential for oogenesis, and is also necessary for spermatogenesis and inhibition of germ cell proliferation. Several lines of evidence demonstrate that GLD-1 is a translational repressor acting through the TGEs to repress tra-2 translation. GLD-1 can repress the translation of reporter RNAs via the TGEs both in vitro and in vivo, and is required to maintain low TRA-2A protein levels in the germline. Genetic analysis indicates that GLD-1 acts upstream of the TGE control. Finally, we show that endogenous GLD-1 is a component of DRF. The conservation of the TGE control and the STAR family suggests that at least a subset of STAR proteins may work through the TGEs to control translation.
TRA-1, a member of the GLI family of transcription factors, is required for C. elegans female development. We find that TRA-1 has a sex-specific distribution consistent with its role in female development: nuclear TRA-1 is higher in hermaphrodite intestines and in specific germline regions than in males. TRA-1 patterns rely on nuclear export since treatment with leptomycin B, a CRM1-dependent export inhibitor, increases nuclearTRA-1 in males. TRA-1 export requires TRA-1 binding to the tra-2 3' untranslated region (3' UTR), as disruption of binding increases nuclear TRA-1 and female development. Our data are consistent with coexport of a TRA-1/tra-2 mRNA complex reducing TRA-1 nuclear activity, and identify an interesting RNA-based mechanism for controlling transcriptional activity and cell fate determination.
The GLI protein family is involved in several key developmental processes in both vertebrates and invertebrates. The Drosophila GLI protein, Cubitus interuptus (Ci), regulates segment polarity and wing and leg development. In vertebrates, the GLI proteins control neural, lung, bone and gut development. In the nematode Caenorhabditis elegans, the GLI family member TRA-1 is necessary for normal sexual development. GLI, Ci and TRA-1 each contain five zinc-finger domains and bind the identical DNA sequence. Previous analyses are consistent with these proteins being transcription factors. Here we show that TRA-1 can act posttranscriptionally to govern gene activity. Our results indicate that the binding of TRA-1 to the 3' untranslated region of tra-2 regulates the export of tra-2 messenger RNA from the nucleus. The fact that TRA-1 is part of a conserved family of proteins raises the possibility that GLI family members are both transcriptional and post-transcriptional regulators of gene expression.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.